CA2327548A1 - Nt2lp, novel g-protein coupled receptors having homology to neurotensin-2 receptors - Google Patents

Abstract

The present invention is based on the identification of a novel G-protein coupled receptor (GPCR) that is expressed predominantly in the brain and nucleic acid molecules that encoded the GPCR, referred to herein as the NT2LP protein and NT2LP gene respectively. The NT2LP protein has high sequence homology to the NT family of receptors, particularly members of the NT2 subfamily. Based on this identification, the present invention provides: 1) isolated NT2LP protein, 2) isolated nucleic acid molecules that encode a NT2LP protein, 3) antibodies that selectively bind to the NT2LP protein, 4) methods of isolating allelic variants of the NT2LP protein and gene, 5) methods of identifying cells and tissues that express the NT2LP protein/gene, 6) methods of identifying agents and cellular compounds that bind to the NT2LP protein, 7) methods of identifying agents that modulate the expression of the NT2LP gene, 8) methods of modulating the activity of the NT2LP protein in a cell or organism and 9) a method for treating diseases, pathologies, or biological processes, for example, pain, comprising administering an agent that binds to and/or modulates NT2LP proteins.

Description

NT2LP, NOVEL G-PROTEIN COUPLED RECEPTORS HAVING

Cross Reference to Related Applications This application is a continuation-in-part of U.S.
Application Serial No. 09/076,313, filed May 11, 1998.
Backcrround of the Invention G-protein coupled receptors (GPCRs) are one of the major class of proteins that are responsible for transducing signals within cells. GPCRs are proteins that have seven transmembrane domains. Upon binding of a ligand to an extracellular portion of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property of the cell.
G protein-coupled receptors (GPCRs), along with G-proteins and effectors (intracellular enzymes and channels which are modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs. These genes and gene-products are potential causative agents of disease (Spiegel et al, J.
Clin. Invest. 92:1119-1125 (1993); McKusick and Amberger, J. Med. Genet. 30:1-26 (1993)). Specific defects in the rodopsin gene and the V2 vasopressin receptor gene have been shown to cause various forms of autosomal dominant and autosomal recessive retinitis pigmentosa (see Nathans et al., Annu. Rev. Genet. 26:403-424(1992)), nephrogenic diabetes insipidus (Holtzman et al., Hum. Mol. Genet.

2:1201-1204 (1993) and references therein). These receptors are of critical importance to both the central nervous system and peripheral physiological processes.
Evolutionary analyses suggest that the ancestor of these proteins originally developed in concert with complex body plans and nervous systems.
G protein-coupled receptors regulate many important physiological processes and this is one of the reasons that G protein-coupled receptors are common drug targets. For example, there are at least three major classes of opioid G protein-coupled receptors that regulate pain, mu, kappa, and delta. The endogenous ligands of these receptors include the enkephalins, dynorphins, and endorphins. Opioid analgesic drugs that agonize the opioid receptors and are used for treating pain include morphine, levorphanol, and fentanyl (for more information, see Reisine and Pasternak (1996), in "Goodman and Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition" pages 521-555.).
The GPCR protein superfamily now contains over 250 types of paralogues, receptors that represent variants generated by gene duplications (or other processes), as opposed to orthologues, the same receptor from different species and homologues, different forms of a receptor isolated from a single organism. The superfamily can be broken down into five families: Family I, receptors typified by rhodopsin and the beta2-adrenergic receptor and currently represented by over 200 unique members (reviewed by Dohlman et al., Annu. Rev. Biochem.
60:653-688 (1991) and references therein); Family II, the recently characterized parathyroid hormone/calcitonin/secretin receptor family (Juppner et al., Science 254:1024-1026 (1991); Lin et al., Science 254:1022-1024 (1991)); Family III, the metabotropic glutamate receptor family in mammals, including NT
receptors (Nakanishi Science 258 597:603 (1992)); Family IV, the cAMP receptor family, important in the chemotaxis and development of D. discoideum (Klein et al., Science 241:1467-1472 (1988)); and Family V, the fungal mating pheromone receptors such as STE2 (reviewed by Kurjan, Annu. Rev. Biochem. 61:1097-1129 (1992)).
In addition to these groups of GPCRs, there are a small number of other proteins which present seven putative hydrophobic segments and appear to be unrelated to GPCRs; however, they have not been shown to couple to G-proteins. Drosophila express a photoreceptor-specific protein bride of sevenless (boss), a seven-transmembrane-segment protein which has been extensively studied and does not show evidence of being a GPCR (Hart et al., Proc. Natl. Acad. Sci. USA
90:5047-5051 (1993)). The gene frizzled (fz) in Drosophila is also thought to be a protein with seven transmembrane segments. Like boss, fz has not been shown to couple to G-proteins (Vinson et al., Nature 338:263-264 (1989)).
G proteins represent a family of heterotrimeric proteins composed of a, (3, and 'y-subunits, which bind guanine nucleotides. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane domains, such as the family of NT receptors.
Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the a-subunit to exchange a bound GDP molecule for a GTP
molecule and to dissociate from the a~y-subunits. The GTP-bound form of the a-subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cyclic AMP (e. g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates. Greater than 20 different types of a-subunits are known in man, which associate with a smaller pool of ~i and 'y-subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish H. et al. Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference.
GPCRs are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown GPCRs. One class of GPCRs that are of particular pharmaceutical interest are the Neurotensin receptor proteins (NTRs).
Neurotensin (NT), is a tridecapeptide that is distributed in both the nervous system and the peripheral tissues. NT is involved in mediating hypotensin, hyperglycemia, hypothermia, increased vascular permeability and analgesia: serving as both a neurotransmitter and neuromodulator in the brain and as a cellular mediator and hormone in the peripheral tissues.
In particular, NT modulates dopamine transmission in the nitrostrial and mesolimbic pathways.
Previous studies have indicated that NT binds to two distinct receptors. Representatives of NT1 have been identified from rat and human (Tanaka et al., Neuron 4:847-854 (1990) and Vita et al., FEBS Ltr. 317:139-142 (1993) and representatives of NT2 have been identified from rat and mice (Chalon et al., FEBS Ltrs. 366:91-94 (1996) and Mazella et al., J. Neuro. 16:5613-5620 (1996)).
Recently, nonpeptide inhibitors of NT1 have been identified (Labbe-Jullie, et al., Mol. Pharm.
47:1050-1056 (1995). The identification of a nonpeptide inhibitor of a member of the NTR family of proteins makes the identification of other family members important in the field of drug development.
The present invention advances the state of the art by providing a previously unidentified GPCR that is expressed predominantly in the brain, with low levels of expression detected in the ovaries, the NT2LP protein.

Human or monkey forms of the NT2LP protein show high sequence homology to the family of NT receptors, particularly members of the NT2 subfamily of receptors.
Summarv of the Invention The present invention is based on the identification of novel G-protein coupled receptors (GPCR) that are expressed predominantly in the brain and nucleic acid molecules that encode the GPCR, referred to herein as the NT2LP protein and the NT2LP gene respectively. The NT2LP proteins are human or monkey proteins that have high sequence similarity to the NT
receptor family, particularly members of the NT2 subfamily of receptors. Based on this identification, the present invention provides; 1) isolated NT2LP
proteins and fragments, 2) isolated nucleic acid molecules that encode NT2LP proteins and fragments, 3}
antibodies that selectively bind to the NT2LP proteins, 4) methods of isolating allelic variants of the NT2LP
proteins and genes, 5) methods of identifying cells and tissues that express the NT2LP proteins/genes, 6) methods of identifying agents and cellular compounds that bind to the NT2LP proteins, 7) methods of identifying agents that modulate the expression of the NT2LP genes, 8) methods of modulating the activity of the NT2LP proteins in a cell or organism and 9) a method for treating diseases, pathologies, or biological processes, for example pain, comprising administering an agent that binds to and/or modulates NT2LP proteins.
Brief Description of the Drawincts Figure 1 depicts monkey NT2LP nucleotide (SEQ ID
NO:1) and the NT2LP amino acid (SEQ ID N0:2) sequence.
The start and stop codons are identified in bold.

Figure 2 depicts the human NT2LP nucleotide (SEQ
ID NOs:3 and 5) of two splice variants. The start codons are identified in bold and underlines.
Figure 3 depicts the human NT2LP amino acid (SEQ
ID N0:4) sequence.
Figure 4 is a hydropathy plot of huamn NT2LP. The location of the predicted transmembrane (TM), cytoplasmic (IN), and extracellular (OUT) domains are indicated as are the position of cysteines (cys; vertical,bars immediately below the plot). Relative hydrophobicity is shown above the dotted line, and relative hydrophilicity is shown below the dotted line.
Figure 5 is a sequence comparison between a portion of human NT2LP and a seven transmembrane receptor consensus sequence derived from a hidden Markov Model.
Figure 6 is a set of alignments between portions of human NT2LP (sbjct) and portions of rat neurotensin receptor tpe 2 (query; Q63384).
Figure 7 is a set of alignments between portions of NT2LP (sbjct) and portions of mouse neurotensin receptor type 2 (query; P70310) Description of the Preferred Embodiments The present invention is based on the discovery of a family of novel G-protein coupled receptors (GPCR) molecules that are expressed predominantly in the brain, the NT2LP proteins, and nucleic acid molecules that encode the NT2LP proteins, the NT2LP genes or NT2LP
nucleic acid molecules. The NT2LP proteins are GPCRs that have high sequence homology to the NT family of receptors, particularly members of the NT2 subfamily of receptors.
Specifically, an EST was first identified, in a public database, that had low homology to G-protein coupled receptors. PCR primers were then designed based on this sequence and a cDNA was identified by screening a monkey fetal cDNA library (See Example 1). Positive clones were sequenced and contigs were assembled.
Analysis of the assembled sequence revealed that the cloned cDNA molecule encoded a GPCR, denoted herein as the NT2LP protein that had significant homology to the NT
family of receptors, particularly members of the NT2 subfamily of receptors.
The monkey NT2LP nucleic acid sequence was used to screen a variety of human cDNA libraries. Several clones were identified and sequenced. Two contigs representing two different splice forms of the entire human NT2LP gene were assembled and represented full length clones (SEQ ID
Nos:3 and 5).
The NT2LP proteins are GPCRs and play a role in and function in signaling pathways within cells that express the NT2LP protein, particularly brain cells.
Various aspects of the invention are described in further detail in the following subsections:
I. Isolated NT2LP Protein The present invention provides isolated human and monkey NT2LP protein as well as peptide fragments of the NT2LP proteins.
As used herein, a protein is said to be "isolated"
or "purified" when it is substantially free of cellular material or when it is isolated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals or when it is chemically synthesized.
The language "substantially free of cellular material"
includes preparations of NT2LP protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced.
In one embodiment, the language "substantially free of cellular material" includes preparations of a NT2LP

_ g _ protein having less than about 30% (by dry weight) of non-NT2LP protein (also referred to herein as a ''contaminating protein"), more preferably less than about 20% of non-NT2LP protein, still more preferably less than about 10% of non-NT2LP protein, and most preferably less than about 5% non-NT2LP protein. When the NT2LP protein or fragment thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of NT2LP
protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of NT2LP protein having less than about 30% (by dry weight) of chemical precursors or non-NT2LP chemicals, more preferably less than about 20% chemical precursors or non-NT2LP
chemicals, still more preferably less than about 10%
chemical precursors or non-NT2LP chemicals, and most preferably less than about 5% chemical precursors or non-NT2LP chemicals. In preferred embodiments, isolated proteins or fragments thereof lack contaminating proteins from the same animal from which the NT2LP protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a human or monkey NT2LP protein in a non-human or non-monkey cell.
As used herein, a NT2LP protein is defined as a protein that comprises: 1) the amino acid sequence shown in SEQ ID NOs:2 or 4; 2) functional and non-functional naturally occurring allelic variants of human or monkey NT2LP protein; 3) recombinantly produced variants of _ g _ human or monkey NT2LP protein; and 4) NT2LP protein isolated from organisms other than human or monkeys (orthologues of human or monkey NT2LP protein.) As used herein, an allelic variant of a human or monkey NT2LP protein is defined as: 1) a protein isolated from human or monkey cells or tissues; 2) a protein encoded by the same genetic locus as that encoding the human or monkey NT2LP protein; and 3) a protein has substantial sequence similarity to a human or monkey NT2LP protein.
As used herein, two proteins are substantially similar when the amino acid sequence of the two protein (or a region of the proteins) are at least about 60-65%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-955 or more identical to each other. To determine the percent homology of two amino acid sequences (e. g., SEQ ID NOs:2 or 4 and an allelic variant thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
When a position in one sequence (e.g., SEQ ID NOs:2 or 4) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e. g., an allelic variant of the human or monkey NT2LP
protein), then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100).
Allelic variants of human or monkey NT2LP protein include both functional and non-functional NT2LP

proteins. Functional allelic variants are naturally occurring amino acid sequence variants of a human or monkey NT2LP protein that maintain the ability to bind ligand (such as NT with NT receptors) and transduce a signal within a cell, preferably a nerve cell.
Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NOs:2 or 4 (allelic variants of NT2LP) or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.
Non-functional allelic variants are naturally occurring amino acid sequence variants of a human or monkey NT2LP protein that do not have the ability to either bind ligand (such as NT for NT receptors) or/and transduce a signal within a cell. Non-functional allelic variants will typically contain one or more non-conservative amino acid substitutions, deletions, insertions or premature truncation of the amino acid sequence of SEQ ID NOs:2 or 4 (allelic variants of NT2LP) or a substitution, insertion or deletion in critical residues or critical regions.
The present invention further provides non-human or non-monkey and non-human orthologues of the human or monkey NT2LP proteins of the present invention. An orthologue of a human or monkey NT2LP protein is protein isolated from a non-human or non-human or non-monkey organism and possesses the same ligand binding (such as NT for NT receptors) and signaling capabilities of the NT2LP protein. Orthologues of the NT2LP protein can readily be identified as comprising an amino acid sequence that is substantially homologous of SEQ ID NOs:2 or 4 (orthologues of NT2LP). The present invention does not however encompass the known rat and mouse NT2 proteins (see Background section).

The NT2LP protein is a GPCR that participates in signaling pathways within cells that express NT2LP, particularly brain cells. As used herein, a signaling pathway refers to the modulation (e.g., stimulation or inhibition) of a cellular function/activity upon the binding of a ligand to the GPCR (NT2LP protein), similar to NT binding to NT receptors. Examples of such functions include mobilization of intracellular molecules that participate in a signal transduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5-triphosphate (IP3) or adenylate cyclase;
polarization of the plasma membrane; production or secretion of molecules; alteration in the structure of a cellular component; cell proliferation, e.g., synthesis of DNA; cell migration; cell differentiation; cell survival; and transduction of a pain signal. Since the NT2LP protein is expressed substantially in the brain, examples of cells participating in a NT2LP signaling pathway include neural cells, e.g., peripheral nervous system and central nervous system cells such as brain cells, e.g., limbic system cells, hypothalamus cells, hippocampus cells, substantia nigra cells, cortex cells, brain stem cells, neocortex cells, basal ganglion cells, caudate putamen cells, olfactory tubercle cells, dorsal root ganglion cells, trigeminal ganglion cells, sensory ganglion cells, nociceptive neuronal cells, or superior colliculi cells.
Depending on the type of cell, the response mediated by the NT2LP protein may be different. For example, in some cells, binding of a ligand to a NT2LP
protein may stimulate an activity such as release of compounds, gating of a channel, cellular adhesion, migration, differentiation, etc., through phosphatidylinositol or cyclic AMP metabolism and turnover while in other cells, the binding of a ligand to the NT2LP protein will produce a different result.
Regardless of the cellular activity/response modulated by the NT2LP protein, it is universal that the NT2LP protein is a GPCR and interact with "G proteins~~ to produce one or more secondary signals, in a variety of intracellular signal transduction pathways, e.g., through phosphatidylinositol or cyclic AMP metabolism and turnover, calcium channeling, etc., in a cell. G
proteins represent a family of heterotrimeric proteins composed of a, ~i and 'y subunits, which bind guanine nucleotides. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane domains, such as the NT receptors.
Following ligand binding to the receptor, a conformational change is transmitted to the G protein, which causes the a-subunit to exchange a bound GDP molecule for a GTP
molecule and to dissociate from the ~iy-subunits. The GTP-bound form of the a-subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cyclic AMP (e. g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates, or calcium. Greater than 20 different types of a-subunits are known in man, which associate with a smaller pool of ~i and 'y subunits. Examples of mammalian G
proteins include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish H. et al. Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference.
As used herein, ~~phosphatidylinositol turnover and metabolism~~ refers to the molecules involved in the turnover and metabolism of phosphatidylinositol 4,5-bisphosphate (PIP2) as well as to the activities of these molecules. PIP2 is a phospholipid found in the cytosolic leaflet of the plasma membrane. Binding of ligand to the NT2LP may activate, in some cells, the WO 99/58641 PC'T/US99/10311 plasma-membrane enzyme phospholipase C that in turn can hydrolyze PIP2 to produce 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Once formed IP3 can diffuse to the endoplasmic reticulum surface where it can bind an IP3 receptor, e.g., a calcium channel protein containing an IP3 binding site. IP3 binding can induce opening of the channel, allowing calcium ions to be released into the cytoplasm. IP3 can also be phosphorylated by a specific kinase to form inositol 1,3,4,5-tetraphosphate (IP4), a molecule which can cause calcium entry into the cytoplasm from the extracellular medium. IP3 and IP4 can subsequently be hydrolyzed very rapidly to the inactive products inositol 1,4-biphosphate (IP2) and inositol 1,3,4-triphosphate, respectively.
These inactive products can be recycled by the cell to synthesize PIP2. The other second messenger produced by the hydrolysis of PIP2, namely 1,2-diacylglycerol (DAG), remains in the cell membrane where it can serve to activate the enzyme protein kinase C. Protein kinase C is usually found soluble in the cytoplasm of the cell, but upon an increase in the intracellular calcium concentration, this enzyme can move to the plasma membrane where it can be activated by DAG. The activation of protein kinase C in different cells results in various cellular responses such as the phosphorylation of glycogen synthase, or the phosphorylation of various transcription factors, e.g., NF-kB. The language ~~phosphatidylinositol activity~~, as used herein, refers to an activity of PIP2 or one of its metabolites.
Another signaling pathway the NT2LP protein may participate in is the CAMP turnover pathway. As used herein, ~~cyclic AMP turnover and metabolism~~ refers to the molecules involved in the turnover and metabolism of cyclic AMP (CAMP) as well as to the activities of these molecules. Cyclic AMP is a second messenger produced in response to ligand induced stimulation of certain G

protein coupled receptors. In the cAMP signaling pathway, binding of a ligand to a GPCR, such as NT to NT receptor, can lead to the activation of the enzyme adenylate cyclase, which catalyzes the synthesis of cAMP. The newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase. This activated kinase can phosphorylate a voltage-gated potassium channel protein, or an associated protein, and lead to the inability of the potassium channel to open during an action potential. The inability of the potassium channel to open results in a decrease in the outward flow of potassium, which normally repolarizes the membrane of a neuron, leading to prolonged membrane depolarization.
The present invention further provides fragments of NT2LP protein. As used herein, a fragment comprises at least 8 contiguous amino acids from a NT2LP protein.
Preferred fragments are fragments that possess one or more of the biological activities of the NT2LP protein, for example the ability to bind to a G-protein or ligand, as well as fragments that can be used as an immunogen to generate anti-NT2LP antibodies. The most preferred fragments are those unique to NT2LP, not being present in any other known protein.
Biologically active fragments of the NT2LP protein include peptides comprising amino acid sequences derived from the amino acid sequence of a NT2LP protein, e.g., the amino acid sequence shown in SEQ ID N0:2 or the amino acid sequence of a protein homologous to the NT2LP protein, which include less amino acids than the full length NT2LP
protein or the full length protein which is homologous to the NT2LP protein, and exhibit at least one activity of the NT2LP protein. Typically, biologically active fragments (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif, e.g., a transmembrane domain, multiple extracellular domains or G-protein binding domain.
Preferred fragments include, but are not limited to: 1) soluble peptides of SEQ ID NOs:2 or 4; and 2) peptides comprising the G-protein binding site of a NT2LP
protein.
The isolated NT2LP protein can be purified from cells that naturally express the protein, purified from cells that have been altered to express the NT2LP protein, or synthesized using known protein synthesis methods.
Preferably, as described below, the isolated NT2LP protein is produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector, the expression vector is introduced into a host cell and the NT2LP protein is expressed in the host cell. The NT2LP protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, the NT2LP protein or fragment can be synthesized chemically using standard peptide synthesis techniques. Lastly, native NT2LP protein can be isolated from cells that naturally express the NT2LP protein (e. g., hippocampal cells, or substantia nigra cells.
The present invention further provides NT2LP
chimeric or fusion protein. As used herein, a NT2LP
"chimeric protein" or "fusion protein" comprises a NT2LP
protein operatively linked to a non-NT2LP protein. An "NT2LP protein" refers to a protein having an amino acid sequence corresponding to a NT2LP protein, whereas a "non-NT2LP protein" refers to a heterologous protein having an amino acid sequence corresponding to a protein which is not substantially homologous to the NT2LP
protein, e.g., a protein which is different from the NT2LP
protein. Within the context of fusion proteins, the term "operatively linked" is intended to indicate that the NT2LP protein and the non-NT2LP protein is fused in-frame WO 99/58641 PCf/US99/i0311 to each other. The non-NT2LP protein can be fused to the N-terminus or C-terminus of the NT2LP protein. For example, in one embodiment the fusion protein is a GST-NT2LP fusion protein in which the NT2LP sequences are fused to the C-terminus of the GST sequences. Other types of fusion proteins include, but are not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant NT2LP protein. In another embodiment, the fusion protein is a NT2LP protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e. g., mammalian host cells), expression and/or secretion of a NT2LP protein can be increased by using a heterologous signal sequence.
Preferably, a NT2LP chimeric or fusion protein is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds.
Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e. g., a GST protein). A NT2LP
-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NT2LP protein.
The present invention also provides altered forms of NT2LP protein that has been generated using recombinant DNA or mutagenic methods/agents. Altered forms of a NT2LP
protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the NT2LP protein and recombinant DNA method that are well known in the art.
II. Antibodies That Bind To A NT2LP Protein The present invention further provides antibodies that selectively bind to a NT2LP protein. As used herein, an antibody is said to selectively bind to ~a NT2LP protein when the antibody binds to NT2LP protein and does not substantially bind to unrelated proteins. A skilled artisan will readily recognize that an antibody may be considered to substantially bind a NT2LP protein even if it binds to proteins that share homology with a fragment or domain of the NT2LP protein.
The term ~~antibody~~ as used herein refers to immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as a NT2LP
protein. Examples of immunologically active fragments of immunoglobulin molecules include Flab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind a NT2LP
protein. The term ~~monoclonal antibody~~ or ~~monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a NT2LP

WO 99/58641 PCf/US99/10311 protein. A monoclonal antibody composition thus typically displays a single binding affinity for a particular NT2LP
protein with which it immunoreacts.
To generate anti-NT2LP antibodies, an isolated NT2LP protein, or a fragment thereof, is used as an immunogen to generate antibodies that bind NT2LP using standard techniques for polyclonal and monoclonal antibody preparation. The full-length NT2LP protein can be used or, alternatively, an antigenic peptide fragment of NT2LP
can be used as an immunogen. An antigenic fragment of the NT2LP protein will typically comprises at least 8 contiguous amino acid residues of a NT2LP protein, e.g. 8 contiguous amino acids from SEQ ID NOs:2 or 4.
Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues of a NT2LP protein. Preferred fragments for generating anti-NT2LP antibodies are regions of NT2LP that are located on the surface of the protein, e.g., hydrophilic regions, and are identified in the antigenicity plot provided in Figure 3.
A NT2LP immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e. g., rabbit, goat, mouse or other mammal) with the immunogen.
An appropriate immunogenic preparation can contain, for example, recombinantly expressed NT2LP protein or a chemically synthesized NT2LP peptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
Immunization of a suitable subject with an immunogenic NT2LP preparation induces a polyclonal anti-NT2LP antibody response.
Polyclonal anti-NT2LP antibodies can be prepared as described above by immunizing a suitable subject with a NT2LP peptide immunogen. The anti-NT2LP antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA.) using an immobilized NT2LP
protein . If desired, the antibody molecules directed against the NT2LP protein can be isolated from the mammal (e. g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-NT2LP antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.
127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72). the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387 402; M. L. Gefter et al.
(1977) Somatic Cell Genet. 3:231 36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a NT2LP immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds NT2LP.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-NT2LP

monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC.
Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind to a NT2LP protein, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-NT2LP
antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a NT2LP protein to thereby isolate immunoglobulin library members that bind NT2LP . Kits for generating and screening phage display libraries are commercially available (e. g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-O1;
and the Stratagene SurfZAP~ Phage Display Kit, Catalog No.
240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT
International Publication No. WO 92/18619; Dower et al.
PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication No. WO 92/20791;
Markland et al. PCT International Publication No. WO
92/15679; Breitling et al. PCT International Publication No. WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT
International Publication No. WO 92/09690; Ladner et al.
PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J
12:725-734; Hawkins et al. (1992) J. Mol. Biol.
226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc.
Acid Res. 19:4133-4137; Barbas et al. (1991) PNAS
88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.
Additionally, recombinant anti-NT2LP antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human fragments, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. PCT
International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., WO 99/58641 PCTlUS99/10311 European Patent Application 171,496; Morrison et al.
European Patent Application 173,494; Neuberger et al. PCT
International Publication No. WO 86/01533; Cabilly et al.
U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Canc. Res.
47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559);
Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.
(1986) BioTechniques 4:214; Winter U.S. Patent 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J.
Immunol. 141:4053-4060.
Completely human antibodies are particularly desirable for therapeutic treatment of human patients.
Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which are capable of expressing human heavy and light chain genes.
Such transgenic mice can be immunized in the normal fashion with a selected antigen, e.g., all or a portion of NT2LP. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such mice, it is possible to produce therapeutically useful human IgG, IgA and IgE
antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S.

Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
First, a non-human monoclonal antibody which binds a selected antigen (epitope), e.g., an antibody which inhibits NT2LP activity, is identified. The heavy chain and the light chain of the non-human antibody are cloned and used to create phage display Fab fragments. For example, the heavy chain gene can be cloned into a plasmid vector so that the heavy chain can be secreted from bacteris. The light chain gene can be cloned into a phage coat protein gene so that the light chain can be expressed on the surface of the phage. A repertoire (random collection) of human light chains fused to phage is used to infect the bacteria which express the non-human heavy chain. The resulting progeny phage display hybrid antibodies (human light chain/non-human heavy chain). The selected antigen is used in a panning screen to select phage which bind the selected antigen. Several rounds of selection may be required to identify such phage. Next, human light chain genes are isolated from the selected phage which bind the selected antigen. These selected human light chain genes are then used to guide the selection of human heavy chain genes as follows. The selected human light chain genes are inserted into vectors for expression by bacteria. Bacteria expressing the selected human light chains are infected with a repertoire of human heavy chains fused to phage. The resulting progeny phage display human antibodies (human light chain/human heavy chain). Next, the selected antigen is used in a panning screen to select phage which bind the selected antigen. The phage selected in this step display completely human antibody which recognize the same epitope recognized by the original selected, non-human monoclonal antibody. The genes encoding both the heavy and light chains are readily isolated and can be further manipulated for production of human antibodies. This technology is described by Jespers et al. (1994, Biotechnology 12:899-903 ) .
An anti-NT2LP antibody (e. g., monoclonal antibody) can be used to isolate a NT2LP protein by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-NT2LP antibody can facilitate the purification of a natural NT2LP protein from cells and recombinantly produced NT2LP protein expressed in host cells. Moreover, an anti-NT2LP
antibody can be used to detect NT2LP protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the NT2LP
protein. Importantly, the detection of circulating fragments of a NT2LP protein can be used to identify NT2LP
protein turnover in a subject. Anti-NT2LP antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, (-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include l2sl, ~3~I, 358 or 3H .
III. Isolated NT2LP Nucleic Acid Molecules The present invention further provides isolated nucleic acid molecules that encode a NT2LP protein, hereinafter the NT2LP gene or NT2LP nucleic acid molecule, as well as fragments of a NT2LP gene.
As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e. g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
As used herein, an "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NT2LP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e. g., a substantia nigra cell). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the NT2LP nucleic acid molecule can be fused to other protein encoding or regulatory sequences and still be considered isolated.
The isolated nucleic acid molecules of the present invention encode a NT2LP protein. As described above, a NT2LP protein is defined as a protein comprising the amino acid sequence depicted in SEQ ID N0:2 (monkey NT2LP
protein) or SEQ ID N0:4 (human NT2LP protein), allelic variants of human or monkey NT2LP protein, and orthologues of the human or monkey NT2LP protein. A preferred NT2LP
nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NOs:l or 3. The sequence of SEQ ID N0:1 corresponds to the monkey NT2LP cDNA. The sequence of SEQ
ID N0:3 corresponds to the human NT2LP cDNA. This cDNA
comprises sequences encoding the human NT2LP protein (i.e., "the coding region", start and stop codons depicted in Figures 1 and 2), as well as 5' untranslated sequences and 3' untranslated sequences (see Figures 1 and 2).
Two forms of the human NT2LP cDNA have been found, although both have the same predicted coding region. The difference between the two forms is a result of differential splicing in the 5' UTR. The human NT2LP
amino acid sequence is shown in Figure 2. The human NT2LP
protein is expressed primarily in the brain and ovaries.
One form of a partial monkey NT2LP cDNA has been found. The monkey NT2LP amino acid sequence is shown in Figure 1. The monkey NT2LP protein is expressed primarily in the brain and ovaries.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NOs:l and 3 (NT2LP) (and fragments thereof) due to degeneracy of the genetic code and thus encode the same NT2LP protein as that encoded by the nucleotide sequence shown in SEQ ID NOs:l and 3.
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NOs:1 or 3 or a fragment of either of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NOs:i or 3 is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID
NOs:l or 3 such that it can hybridize to the nucleotide sequence shown in SEQ ID NOs:l or 3, thereby forming a stable duplex.
Orthologues and allelic variants of the human or monkey NT2LP gene can readily be identified using methods well known in the art. Allelic variants and orthologues of the human or monkey NT2LP gene will comprise a nucleotide sequence that is at least about 60-65%, typically at least about 70-75%, more. typically at least about 80-85%, and most typically at least about 90-95% or more homologous to the nucleotide sequence shown in SEQ ID
NOs:l or 3 or a fragment of these nucleotide sequences.
Such nucleic acid molecules can readily be identified as being able to hybridize, preferably under stringent conditions, to the nucleotide sequence shown in SEQ ID
NOs:1 or 3 or a fragment of either of these nucleotide sequences.
Moreover, the nucleic acid molecule of the invention can comprise only a fragment of the coding region of an NT2LP gene, such as a fragment of SEQ ID
NOs:l or 3. The nucleotide sequence determined from the cloning of the human or monkey NT2LP gene allows the generation of probes and primers designed for use in identifying and/or cloning NT2LP gene homologues from other cell types, e.g., from other tissues, as well as NT2LP gene orthologues from other mammals. A probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of SEQ ID NOs:l or 3 sense, an anti-sense sequence of SEQ ID NOs:l or 3, or naturally occurring mutants thereof. Primers based on the nucleotide sequence in SEQ ID NOs:l or 3 can be used in PCR reactions to clone NT2LP gene homologues. Probes based on the NT2LP
nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a NT2LP protein, such as by measuring a level of a NT2LP -encoding nucleic acid in a sample of cells from a subject e.g., detecting NT2LP or NT2LP mRNA levels or determining whether a genomic NT2LP
gene has been mutated or deleted.
In addition to the NT2LP nucleotide sequence shown in SEQ ID NOs:l or 3, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of a NT2LP
protein may exist within a population (e.g., the human or monkey population). Such genetic polymorphism in the NT2LP gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms ~~gene~~ and ~~recombinant gene~~ refer to nucleic acid molecules comprising an open reading frame encoding a NT2LP protein, preferably a mammalian NT2LP protein. Such natural allelic variations can typically result in 1-5~
variance in the nucleotide sequence of the NT2LP gene.
Any and all such nucleotide variations and resulting amino acid polymorphisms in a NT2LP gene that are the result of natural allelic variation are intended to be within the scope of the invention. Such allelic variation includes both active allelic variants as well as non-active or reduced activity allelic variants, the later two types typically giving rise to a pathological disorder.
Moreover, nucleic acid molecules encoding NT2LP protein from other species, and thus which have a nucleotide sequence which differs from the human or monkey sequence of SEQ ID NOs:l or 3, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and non-human or non-monkey orthologues of the human or monkey NT2LP cDNA of the invention can be isolated based on their homology to the human or monkey NT2LP nucleic acid disclosed herein using the human or monkey cDNAs, or a fragment thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs:l or 3. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length. In another preferred embodiment, the fragment of the NT2LP gene encodes the extracellular domain, one or more of the transmembrane domains or one or more of the intracellular domains of an NT2LP protein. As used herein, the term ~~hybridizes under stringent conditions~~ is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% identical to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A

preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOs:l or 3 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural human or monkey NT2LP protein.
In addition to naturally-occurring allelic variants of the NT2LP nucleic acid sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NOs:l or 3, thereby leading to changes in the amino acid sequence of the encoded NT2LP
protein, without altering the functional ability of the NT2LP protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential"
amino acid residues can be made in the sequence of SEQ ID
N0:2. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a NT2LP
protein (e. g., the sequence of SEQ ID N0:2) without altering the activity of NT2LP, whereas an "essential"
amino acid residue is required for NT2LP protein activity.
For example, conserved amino acid residues, e.g., aspartates, prolines, threonines, and tyrosines, in the transmembrane domains of the NT2LP protein is most likely important for binding to ligand and are thus essential residues of the NT2LP protein. Other amino acid residues, however, (e. g., those that are not conserved or only semi-conserved in the transmembrane domain) may not be essential for activity and thus are likely to be amenable to alteration without altering NT2LP protein activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding NT2LP protein that contain changes in amino acid residues that are not essential for NT2LP activity. Such NT2LP protein differ in amino acid sequence from SEQ ID NOs:2 or 4 yet retain at least one of the NT2LP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 30-35%, preferably at least about 40-45%, more preferably at least about 50-55%, even more preferably at least about 60-65%, yet more preferably at least about 70-75%, still more preferably at least about 80-85%, and most preferably at least about 90-95% or more homologous to the amino acid sequence of SEQ ID NOs:2 or 4.
An isolated nucleic acid molecule encoding a NT2LP
protein homologous to the protein of SEQ ID NOs:2 or 4 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOs:l or 3, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NOs:l or 3 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e. g., lysine, arginine, histidine), acidic side chains (e. g., aspartic acid, glutamic acid), uncharged polar side chains (e. g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e. g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e. g., thr-eonine, valine, isoleucine) and aromatic side chains (e. g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, a predicted nonessential amino acid residue in NT2LP
is preferably replaced with another amino acid residue from the same side chain fatriily. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NT2LP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a NT2LP activity described herein to identify mutants that retain NT2LP activity. Following mutagenesis of SEQ ID NOs:l or 3, the encoded protein can be expressed recombinantly (e.g., as described in Examples 3 and 4) and the activity of the protein can be determined using, for example, assays described herein.
In addition to the nucleic acid molecules encoding NT2LP protein described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
Accordingly, an antisense nucleic acid molecule can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire NT2LP
coding strand, or to only a fragment thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a NT2LP protein.
The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues, e.g., the entire coding region of SEQ ID NOs:1 or 3 shown in Figures 1 and 2. In another embodiment, the antisense nucleic acid molecule is antisense to a ~~noncoding region~~ of the coding strand of a nucleotide sequence encoding a NT2LP
protein. The term ~~noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequence encoding the NT2LP
protein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NT2LP mRNA, but more preferably is an oligonucleotide which is antisense to only a fragment of the coding or noncoding region of NT2LP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NT2LP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e. g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NT2LP protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of an antisense nucleic acid molecule of the invention includes direct injection at a tissue site. Alternatively, an antisense nucleic acid molecule can be modified to target selected cells and then administered systemically. For example, for systemic administration, an antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an (-anomeric nucleic acid molecule. An (-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Let.
215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e. g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave NT2LP
mRNA transcripts to thereby inhibit translation of NT2LP
mRNA. A ri.bozyme having specificity for a NT2LP -encoding nucleic acid can be designed based upon the nucleotide sequence of a NT2LP cDNA disclosed herein (i.e., SEQ ID
NOs:i or 3). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NT2LP -encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742. Alternatively, NT2LP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
Alternatively, NT2LP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NT2LP gene (e.g., the NT2LP gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the NT2LP
gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.
IV. Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a NT2LP protein (or a fragment thereof). As used herein, the term ~~vector~~ refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid'~, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e. g., replication defective retroviruses, adenoviruses and adeno-associated viruses), that serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operabiy linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences) in a manner which allows for expression of the nucleotide sequence (e. g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e. g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). ~ Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e. g., a NT2LP protein, altexed forms of a NT2LP protein, fusion proteins, and the like).
The recombinant expression vectors of the~invention can be designed for expression of a NT2LP protein, or fragment thereof, in prokaryotic or eukaryotic cells. For example, a NT2LP protein can be expressed in bacterial cells such as E. coli, insect cells (e. g., using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerise.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson; K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the NT2LP gene is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-NT2LP
protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin.
Recombinant NT2LP protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET lld (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET lld vector relies on transcription from a T7 gnl0-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons far each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the NT2LP gene expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
Alternatively, a NT2LP gene can be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e. g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDMB (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.
6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.
1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e. g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e. g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for example the marine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the a-fetoprotein promoter (Camper and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule encoding a NT2LP protein cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to NT2LP

WO 99/58b41 PCT/US99/10311 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol.
1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein.
It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NT2LP protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e. g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual.
2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the NT2LP protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e. g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NT2LP protein. Accordingly, the invention further provides methods for producing NT2LP protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a WO 99/58641 PCT/tfS99/10311 NT2LP protein has been introduced? in a suitable medium until the NT2LP protein is produced. In another embodiment, the method further comprises isolating the NT2LP protein from the medium or the host cell.
The host cells of the invention can also be used to produce non-human transgenic animals. The non-human transgenic animals can be used in screening assays designed to identify agents or compounds, e.g., drugs, pharmaceuticals, etc., which are capable of ameliorating detrimental symptoms of selected disorders or biological processes such as nervous system disorders, e.g., psychiatric disorders, disorders affecting circadian rhythms and the sleep-wake cycle, or pain. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NT2LP protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NT2LP gene sequences have been introduced into their genome or homologous recombinant animals in which endogenous NT2LP gene sequences have been altered. Such animals are useful for studying the function and/or activity of a NT2LP protein and for identifying and/or evaluating modulators of NT2LP protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal include a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A
transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NT2LP gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing NT2LP protein encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The human or monkey NT2LP cDNA sequence of SEQ ID
NOs:l or 3 can be introduced as a transgene into the genome of a non-human animal. Moreover, a non-human or non-monkey homologue of the human or monkey NT2LP gene, such as a mouse NT2LP gene, can be isolated based on hybridization to the human or monkey NT2LP cDNA (described further above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequences) can be operably linked to the NT2LP transgene to direct expression of a NT2LP protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos.
4,736,866 and 4,870,009, both by Leder et al., U.S.
Patent No. 4,873,191 by Wagner et al. arid in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NT2LP transgene in its genome and/or expression of NT2LP mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a NT2LP protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a fragment of a NT2LP
gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NT2LP gene. The NT2LP gene can be a human gene (e.g., from a human genomic clone isolated from a human genomic library screened with the cDNA of SEQ ID
NOs:l or 3), but more preferably is a non-human homologue of a human NT2LP gene. For example, a mouse NT2LP gene can be isolated from a mouse genomic DNA library using the NT2LP cDNA of SEQ ID NOs:l or 3 as a probe. The mouse NT2LP gene then can be used to construct a homologous recombination vector suitable for altering an endogenous NT2LP gene in the mouse genome. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous NT2LP gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out"
vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NT2LP
gene is mutated or otherwise altered but still encodes functional protein (e. g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NT2LP protein). In the homologous recombination vector, the altered fragment of the NT2LP
gene is flanked at its 5' and 3' ends by additional nucleic acid of the NT2LP gene to allow for homologous recombination to occur between the exogenous NT2LP gene carried by the vector and an endogenous NT2LP gene in an embryonic stem cell. The additional flanking NT2LP
nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see for example, Thomas and Capecchi (1987) Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NT2LP gene has homologously recombined with the endogenous NT2LP gene is selected (see e.g., Li et al.
(1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e. g., a mouse) to form aggregation chimeras (see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos. WO
90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP
recombinase system, see, e.g., Lakso et al. (1992) PNAS
89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of ~~double~~ transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO
97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase.
The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, modulators, and antibodies described herein can be used in one or more of the following methods: a) drug screening assays; b) diagnostic assays particularly in disease identification, allelic screening and pharmocogenetic testing; c) methods of treatment; d) pharmacogenomics; and e) monitoring of effects during clinical trials. A NT2LP protein of the invention can be used as a drug target for developing agents to modulate the activity of the NT2LP protein (a NT receptor). The isolated nucleic acid molecules of the invention can be used to express NT2LP protein (e. g., via a recombinant expression vector in a host cell or in gene therapy applications), to detect NT2LP mRNA (e. g., in a biological sample) or a naturally occurring or recombinantly generated genetic mutation in a NT2LP gene, and to modulate NT2LP protein activity, as described further below. In addition, the NT2LP protein can be used to screen drugs or compounds which modulate NT2LP protein activity. Moreover, the anti-NT2LP antibodies of the invention can be used to detect and isolate a NT2LP
protein, particularly fragments of a NT2LP protein present in a biological sample, and to modulate NT2LP protein activity.
a. Drug Screening Assays.
The invention provides methods for identifying compounds or agents that can be used to treat disorders characterized by (or associated with) aberrant or abnormal or normal NT2LP nucleic acid expression and/or NT2LP
protein activity, for example, pain. These methods are also referred to herein as drug screening assays and typically include the step of screening a candidate/test compound or agent to identify compounds that are an agonist or antagonist of a NT2LP protein or fragment thereof, and specifically for the ability to interact with (e.g., bind to) a NT2LP protein, to modulate the interaction of a NT2LP protein and a target molecule (such as a ligand), and/or to modulate NT2LP nucleic acid expression and/or NT2LP protein activity. Candidate/test compounds or agents which have one or more of these abilities can be used as drugs to treat disorders characterized by aberrant or abnormal or normal NT2LP
nucleic acid expression and/or NT2LP protein activity, e.g., pain. Candidate/test compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84;
Houghten et al. (1991) Nature 354:84-86) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e. g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al.
(1993) Cell 72:767-778); 3) antibodies (e. g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')2, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e. g., molecules obtained from combinatorial and natural product libraries).
In one embodiment, the invention provides assays for screening candidate/test compounds which interact with (e. g., bind to) a NT2LP protein, or fragment thereof.
Typically, the assays are recombinant cell based or cell-free assays which include the steps of combining a cell expressing a NT2LP protein or a fragment thereof, or an isolated NT2LP protein or fragment thereof, and a candidate/test compound, e.g., under conditions which allow for interaction of (e.g., binding of) the candidate/test compound to the NT2LP protein or fragment thereof to form a complex, and detecting the formation of a complex, in which the ability of the candidate compound to interact with (e.g., bind to) the NT2LP protein or fragment thereof is indicated by the presence of the candidate compound in the complex. Formation of complexes between the NT2LP protein and the candidate compound can be detected using competition binding assays, and can be quantitated, for example, using standard immunoassays.
In another embodiment, the invention provides screening assays to identify candidate/test compounds which modulate (e.g., stimulate or inhibit) the interaction (and most likely NT2LP protein activity as well) between a NT2LP protein and a molecule (target molecule) with which the NT2LP protein normally interacts.
Examples of such target molecules include ligands (such as NT for NT receptors) and proteins in the same signaling path as the NT2LP protein, e.g., proteins which may WO 99/58641 PCT/US99/i0311 function upstream (including both stimulators and inhibitors of activity) or downstream of the NT2LP protein in, for example, a cognitive function signaling pathway or in a pathway involving NT2LP protein activity, e.g., a G
protein or other interactor involved in cAMP or phosphatidylinositol turnover, and/or adenylate cyclase or phospholipase C activation. Typically, the assays are recombinant cell based assays which include the steps of combining a cell expressing a NT2LP protein, or a fragment thereof, a NT2LP protein target molecule (e.g., ligand or a NT2LP binding signaling partner) and a candidate/test compound, e.g., under conditions wherein but for the presence of the candidate compound, the NT2LP protein or biologically active fragment thereof interacts with (e. g., binds to) the target molecule, and detecting the formation of a complex which includes the NT2LP protein and the target molecule or detecting the interaction/reaction of the NT2LP protein and the target molecule. Detection of complex formation can include direct quantitation of the complex by, for example, measuring inductive effects of the NT2LP protein. A statistically significant change, such as a decrease, in the interaction of the NT2LP
protein and target molecule (e.g., in the formation of a complex between the NT2LP protein and the target molecule) in the presence of a candidate compound (relative to what is detected in the absence of the candidate compound) is indicative of a modulation (e.g., stimulation or inhibition) of the interaction between the NT2LP protein and the target molecule. Modulation of the formation of complexes between the NT2LP protein and the target molecule can be quantitated using, for example, an immunoassay.
To perform cell free drug screening assays, it is desirable to immobilize either the NT2LP protein, or fragment, or its target molecule to facilitate separation of complexes~from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Interaction (e. g., binding of') of the NT2LP
protein to a target molecule, in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/NT2LP fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., 35S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of NT2LP -binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
Other techniques for immobilizing proteins on matrices can also be used in the drug screening assays of the invention. For example, either the NT2LP protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NT2LP protein molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art (e. g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with a NT2LP protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and NT2LP protein trapped in the wells by antibody conjugation. As described above, preparations of a NT2LP
-binding protein and a candidate compound are incubated in the NT2LP protein-presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NT2LP protein target molecule, or which are reactive with NT2LP protein and compete with the target molecule; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
In yet another embodiment, the invention provides a method for identifying a compound (e. g., a screening assay) capable of use in the treatment of a disorder characterized by (or associated with) aberrant or abnormal or normal NT2LP nucleic acid expression or NT2LP protein activity, e.g., pain. This method typically includes the step of assaying the ability of the compound or agent to modulate the expression of the NT2LP nucleic acid or the activity of the NT2LP protein thereby identifying a compound for treating a disorder characterized by aberrant or abnormal or normal NT2LP nucleic acid expression or NT2LP protein activity. Methods for assaying the ability of the compound or agent to modulate the expression of the NT2LP nucleic acid or activity of the NT2LP protein is typically cell-based assays. For example, cells that transduce signals via a pathway involving a NT2LP protein can be induced to overexpress a NT2LP protein in the presence and absence of a candidate compound. Candidate compounds which produce a statistically significant change in NT2LP protein-dependent responses (either stimulation or inhibition) can be identified. In one embodiment, expression of the NT2LP nucleic acid or activity of a NT2LP protein is modulated in cells and the effects of candidate compounds on the readout of interest (such as CAMP or phosphatidylinositol turnover) are measured. For example, the expression of genes which are up- or down-regulated in response to a NT2LP protein-dependent signal cascade can be assayed. In preferred embodiments, the regulatory regions of such genes, e.g., the 5' flanking promoter and enhancer regions, are operably linked to a detectable marker (such as luciferase) which encodes a gene product that can be readily detected.
Phosphorylation of a NT2LP protein or NT2LP protein target molecules can also be measured, for example, by immunoblotting.
Alternatively, modulators of NT2LP gene expression (e.g., compounds which can be used to treat a disorder or biological process characterized by aberrant or abnormal or normal NT2LP nucleic acid expression or NT2LP protein activity, for example, pain) can be identified in a method wherein a cell is contacted with a candidate compound and the expression of NT2LP mRNA or protein in the cell is determined. The level of expression of NT2LP mRNA or protein in the presence of the candidate compound is compared to the level of expression of NT2LP mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NT2LP nucleic acid expression based on this comparison and be used to treat a disorder characterized by aberrant NT2LP nucleic acid expression. For example, when expression of NT2LP mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NT2LP nucleic acid expression. Alternatively, when NT2LP nucleic acid expression is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NT2LP nucleic acid expression. The level of NT2LP nucleic acid expression in the cells can be determined by methods described herein for detecting NT2LP
mRNA or protein.
In yet another aspect of the invention, the NT2LP
protein, or fragments thereof, can be used as "bait proteins" in a two-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;
Madura et al. (1993) J. Biol: Chem. 268:12046-12054;
Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO 94/10300), to identify other proteins, which bind to or interact with the NT2LP protein ("NT2LP -binding proteins" or "NT2LP
-by") and modulate NT2LP protein activity. Such NT2LP
-binding proteins are also likely to be involved in the propagation of signals by the NT2LP protein as, for example, upstream or downstream elements of the NT2LP
protein pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Bartel et al. "Using the Two-Hybrid System to Detect Protein-Protein Interactions" in Cellular Interactions in Development: A
Practical Approach, Hartley, D.A. ed. (Oxford University Press, Oxford, 1993) pp. 153-179. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that encode a NT2LP protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA
sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to interact, in vivo, forming a NT2LP
-protein dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close WO 99/58641 PCT/US99/i0311 proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the NT2LP protein.
Modulators of NT2LP protein activity and/or NT2LP
nucleic acid expression identified according to these drug screening assays can be used to treat, for example, nervous system disorders and processes or pain. These methods of treatment include the steps of administering the modulators of NT2LP protein activity and/or nucleic acid expression, e.g., in a pharmaceutical composition as described in subsection IV above, to a subject in need of such treatment, e.g., a subject with a disorder or biological process described herein.
b. Diagnostic Assays.
The invention further provides a method for detecting the presence of a NT2LP protein or NT2LP nucleic acid molecule, or fragment thereof, in a biological sample. The method involves contacting the biological sample with a compound or an agent capable of detecting NT2LP protein or mRNA such that the presence of NT2LP
protein/encoding nucleic acid molecule is detected in the biological sample. A preferred agent for detecting NT2LP
mRNA is a labeled or labelable nucleic acid probe capable of hybridizing to NT2LP mRNA. The nucleic acid probe can be, for example, the full-length NT2LP cDNA of SEQ ID
NOs:l or 3, or a fragment thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NT2LP mRNA. A
preferred agent for detecting NT2LP protein is a labeled or labelable antibody capable of binding to NT2LP protein.

WO 99/58b41 PCT/US99/10311 Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab~)2) can be used. The term °labeled or labelable~~, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking} a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
The term °biological sample~~ is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NT2LP mRNA or protein in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NT2LP mRNA
include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NT2LP protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. Alternatively, NT2LP protein can be detected in vivo in a subject by introducing into the subject a labeled anti-NT2LP antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods which detect the allelic variant of a NT2LP
protein expressed in a subject and methods which detect fragments of a NT2LP protein in a sample.
The invention also encompasses kits for detecting the presence of a NT2LP protein in a biological sample.
For example, the kit can comprise reagents such as a labeled or labelable compound or agent capable of detecting NT2LP protein or mRNA in a biological sample;
means for determining the amount of NT2LP protein in the sample; and means for comparing the amount of NT2LP
protein in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NT2LP mRNA or protein.
The methods of the invention can also be used to detect naturally occurring genetic mutations in a NT2LP
gene, thereby determining if a subject with the mutated gene is at risk for a disorder characterized by aberrant or abnormal NT2LP nucleic acid expression or NT2LP protein activity, for example, pain, as described herein. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic mutation characterized by at least one of an alteration affecting the integrity of a gene encoding a NT2LP protein, or the misexpression of the NT2LP gene.
For example, such genetic mutations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a NT2LP gene; 2) an addition of one or more nucleotides to a NT2LP gene; 3) a substitution of one or more nucleotides of a NT2LP gene, 4) a chromosomal rearrangement of a NT2LP gene; 5) an alteration in the level of a messenger RNA transcript of a NT2LP gene, 6) aberrant modification of a NT2LP gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a NT2LP gene, 8) a non-wild type level of a NT2LP -protein, 9) allelic loss of a NT2LP
gene, and 10) inappropriate post-translational modification of a NT2LP -protein. As described herein, there are a large number of assay techniques known in the art that can be used for detecting mutations in a NT2LP
gene.

In certain embodiments, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which can be particularly useful for detecting point mutations in the NT2LP-gene (see Abravaya et al. (1995) Nucleic Acids Res .23:675-682). This method can include the steps of collecting a sample of cells from a patient;
isolating nucleic acid (e. g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a NT2LP gene under conditions such that hybridization and amplification of the NT2LP -gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
In an alternative embodiment, mutations in a NT2LP
gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NT2LP gene and detect mutations by comparing the sequence of the sample NT2LP gene with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on technigues developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463). A variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT
International Publication No: WO 94/16101; Cohen et al.
(1996) Adv. Chromatogr. 36:127-162; and Griffin et al.
(1993) Appl. Biochem. Biotechnol. 38:147-159).
Other methods for detecting mutations in the NT2LP
gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985) Science 230:1242);
Cotton et al. (1988) PNAS 85:4397; Saleeba et al. (1992}
Meth. Enzymol. 217:286-295), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al. (1989) PNAS 86:2766; Cotton (1993) Mutat. Res.
285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al. (1985) Nature 313:495}. Examples of other techniques for detecting point mutations include, selective oligonucleotide hybridization, selective amplification, and selective primer extension.
c. Methods of Treatment.
Another aspect of the invention pertains to methods for treating a subject, e.g., a human, having a disease, disorder, or biological process characterized by (or associated with) aberrant or abnormal or normal NT2LP
nucleic acid expression and/or normal or abnormal NT2LP
protein activity, for example, pain. These methods include the step of administering a NT2LP protein/gene modulator (agonist or antagonist) to the subject such that treatment occurs. The language "aberrant or abnormal NT2LP protein expression" refers to expression of a non-wild-type NT2LP protein or a non-wild-type level of expression of a NT2LP protein. Aberrant or abnormal NT2LP
protein activity refers to a non-wild-type NT2LP protein activity or a non-wild-type level of NT2LP protein activity. As the NT2LP protein is involved in a pathway involving signaling within cells, aberrant or abnormal NT2LP protein activity or expression interferes with the normal regulation of functions mediated by NT2LP protein signaling, and in particular brain cells.
The terms "treating" or "treatment", as used herein, refer to reduction or alleviation of at least one adverse effect or symptom of a disorder, disease, or biological process e.g., a disorder, disease, or biological process characterized by or associated with abnormal or aberrant or normal NT2LP protein activity or NT2LP nucleic acid expression. Particularly useful is the treatment of disorders mediated by abnormal or normal NT2LP receptor interaction/signaling, for example, pain.
The terms "treating" or "treatment", as used herein, also refer to reduction or alleviation of at least one adverse effect or symptom of a disorder, disease, or biological process characterized by its ability to be assuaged by modulating the activity or expression of a normal NT2LP
nucleic acid or protein.
As used herein, a NT2LP protein/gene modulator is a molecule which can modulate NT2LP nucleic acid expression and/or NT2LP protein activity. For example, a NT2LP gene or protein modulator can modulate, e.g., upregulate (activate/agonize) or downregulate (suppress/antagonize), NT2LP nucleic acid expression. In another example, a NT2LP protein/gene modulator can modulate (e. g., stimulate/agonize or inhibit/antagonize) NT2LP protein activity. If it is desirable to treat a disorder or disease or biological process characterized by (or associated with) aberrant or abnormal (non-wild-type) or normal NT2LP nucleic acid expression and/or NT2LP protein activity by inhibiting NT2LP nucleic acid expression, a NT2LP modulator can be an antisense molecule, e.g., a ribozyme, as described herein. Examples of antisense molecules which can be used to inhibit NT2LP nucleic acid expression include antisense molecules which are complementary to a fragment of the 5' untranslated region of SEQ ID NOs:l or 3 which also includes the start codon and antisense molecules which are complementary to a fragment of the 3' untranslated region of SEQ ID NOs:l or 3. An example of an antisense molecule which is complementary to a fragment of the 5' untranslated region of SEQ ID NOs:l or 3 and which also includes the start codon is a nucleic acid molecule which includes nucleotides which are complementary to nucleotides l to 616 of SEQ ID NOs:l or 3.
A NT2LP modulator that inhibits NT2LP nucleic acid expression can also be a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which inhibits NT2LP
nucleic acid expression. If it is desirable to treat a disease; disorder, or biological process characterized by (or associated with) aberrant or abnormal (non-wild-type) or normal NT2LP nucleic acid expression and/or abnormal or normal NT2LP protein activity by stimulating NT2LP nucleic acid expression, for example, pain, a NT2LP modulator can be, for example, a nucleic acid molecule encoding a NT2LP
protein (e.g., a nucleic acid molecule comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NOs:l or 3) or a small molecule or other drug, e.g., a small molecule (peptide) or drug identified using the screening assays described herein, which stimulates NT2LP nucleic acid expression.
Alternatively, if it is desirable to treat a disease, disorder, or biological process characterized by (or associated with) aberrant or abnormal (non-wild-type) or normal NT2LP nucleic acid expression and/or abnormal or normal NT2LP protein activity, e.g., pain, by inhibiting NT2LP protein activity a NT2LP modulator can be an anti-NT2LP antibody, a small molecule or other drug, or fragment of an NT2LP protein (e. g. the extracellular domain) e.g., a small molecule or drug identified using the screening assays described herein, which inhibits NT2LP protein activity. If it is desirable to treat a disease or disorder or biological process characterized by (or associated with) aberrant or abnormal (non-wild-type) or normal NT2LP nucleic acid expression and/or normal or abnormal NT2LP protein activity, for example, pain, by stimulating NT2LP protein activity, a NT2LP modulator can be an active NT2LP protein or fragment thereof (e.g., a NT2LP protein or fragment thereof having an amino acid sequence which is homologous to the amino acid sequence of SEQ ID NOs:2 or 4 or a fragment thereof) or a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which stimulates NT2LP protein activity.
Other aspects of the invention pertain to methods for modulating a NT2LP protein mediated cell activity.
These methods include contacting the cell with an agent (or a composition which includes an effective amount of an agent) which modulates NT2LP protein activity~or NT2LP
nucleic acid expression such that a NT2LP protein mediated cell activity is altered relative to normal levels (for example, CAMP or phosphatidylinositol metabolism). As used herein, "a NT2LP protein mediated cell activity"
refers to a normal or abnormal activity or function of a cell. Examples of NT2LP protein mediated cell activities include phosphatidylinositol turnover, production or secretion of molecules, such as proteins, contraction, proliferation, migration, differentiation, cell survival, and participation in a pain pathway. In a preferred embodiment, the cell is a brain cell, e.g., a hippocampal cell. The term "altered" as used herein refers to a change, e.g., an increase or decrease, of a cell associated activity particularly cAMP or phosphatidylinositol turnover, and adenylate cyclase or phospholipase C activation. In one embodiment, the agent stimulates NT2LP protein activity or NT2LP nucleic acid expression. In another embodiment, the agent inhibits NT2LP protein activity or NT2LP nucleic acid expression.
These modulatory methods can be performed in vitro (e. g., by culturing the cell with the agent) or, alternatively, in vivo (e. g., by administering the agent to a subject).
In a preferred embodiment, the modulatory methods are performed in vivo, i.e., the cell is present within a subject, e.g., a mammal, e.g., a human, and the subject has a disorder or disease or biological process characterized by or associated with abnormal or aberrant or normal NT2LP protein activity or NT2LP nucleic acid expression.
A nucleic acid molecule, a protein, a NT2LP
modulator, a compound etc. used in the methods of treatment can be incorporated into an appropriate pharmaceutical composition described below and administered to the subject through a route which allows the molecule, protein, modulator, or compound etc. to perform its intended function. d. Pharmacogenomics.
Test/candidate compounds, or modulators which have a stimulatory or inhibitory effect on NT2LP protein activity (e.g., NT2LP gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders or biological processes (e.g., CNS disorders and pain) associated with aberrant NT2LP protein activity. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permit the selection of effective compounds (e. g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens.
Accordingly, the activity of NT2LP protein, expression of NT2LP nucleic acid, or mutation content of NT2LP gene in an individual can be determined to thereby select appropriate compounds) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (1996) Clin. Exp.
Pharmacol. Physiol. 23(10-11) :983-985 and Linder, M.W.
(1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated.
Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e. g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizes (EM) and poor metabolizes (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of NT2LP protein, expression of NT2LP nucleic acid, or mutation content of NT2LP gene in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of a subject. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of a subject's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NT2LP modulator, such as a modulator identified by one of the exemplary screening assays described herein.
e. Monitoring of Effects During Clinical Trials.
Monitoring the influence of compounds (e. g., drugs) on the expression or activity of NT2LP protein/gene can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay, as described herein, to increase NT2LP gene expression, protein levels, or up-regulate NT2LP activity, can be monitored in clinical trials of subjects exhibiting decreased NT2LP
gene expression, protein levels, or down-regulated NT2LP
protein activity. Alternatively, the effectiveness of an agent, determined by a screening assay, to decrease NT2LP
gene expression, protein levels, or down-regulate NT2LP
protein activity, can be monitored in clinical trials of subjects exhibiting increased NT2LP gene expression, protein levels, or up-regulated NT2LP protein activity.
In such clinical trials, the expression or activity of a NT2LP protein and, preferably, other genes which have been implicated in, for example, a nervous system related disorder can be used as a "read out" or markers of the particular cell.
For example, and not by way of limitation, genes, including a NT2LP gene, which are modulated in cells by treatment with a compound (e. g., drug or small molecule) which modulates NT2LP protein/gene activity (e. g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of compounds on CNS disorders or processes or pain, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of a NT2LP gene and other genes implicated in the disorder or biological process. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods described herein, or by measuring the levels of activity of a NT2LP protein or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the compound. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the compound.
In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with a compound (e. g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the compound; (ii) detecting the level of expression of a NT2LP protein, mRNA, or genomic DNA in the preadministration sample;
(iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NT2LP protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NT2LP protein, mRNA, or genomic DNA in the pre-administration sample with the NT2LP protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the compound to the subject accordingly.
For example, increased administration of the compound may be desirable to increase the expression or activity of a NT2LP protein/gene to higher levels than detected, i.e., to increase the effectiveness of the agent.
Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NT2LP
to lower levels than detected, i.e. to decrease the effectiveness of the compound.

VI. Pharmaceutical Compositions The NT2LP nucleic acid molecules, NT2LP protein (particularly fragments of NT2LP such as the extracellular domain), modulators of a NT2LP protein, and anti-NT2LP
antibodies (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. Such compositions typically comprise the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e. g., inhalation), transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, WO 99/58641 PCT/US99/1031 i citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose, pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL~ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays WO 99/58641 PC'f/US99/10311 absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e. g., a NT2LP protein or anti-NT2LP antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Far administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e. g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.
4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) PNAS 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

VII. Uses of Partial NT2LP Sectuences Fragments or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to:
(a) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (b) identify an individual from a minute biological sample (tissue typing); and (c) aid in forensic identification of a biological sample. These applications are described in the subsections below.
a. Chromosome Mapping.
Once the sequence (or a fragment of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, fragments of a NT2LP nucleic acid sequences can be used to map the location of the NT2LP gene, respectively, on a chromosome. The mapping of the NT2LP sequence to chromosomes is an important first step in correlating these sequence with genes associated with disease.
Briefly, the NT2LP gene can be mapped to a chromosome by preparing PCR primers (preferably 15-25 by in length) from the NT2LP gene sequence. Computer analysis of the NT2LP gene sequence can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NT2LP gene sequence will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e. g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D~Eustachio et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NT2LP gene sequence to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a NT2LP gene sequence to its chromosome include in situ hybridization (described in Fan et al. (1990) PNAS, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
Fluorescence in situ hybridization (FISH) of a DNA
sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes.
Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data (such data are found, for example, in V.
McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library).
The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al.
(1987) Nature, 325:783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NT2LP gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected _ 77 _ individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
b. Tissue Typing.
The NT2LP gene sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual s genomic DNA
is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of ~~Dog Tags~~ which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected fragments of an individual s genome. Thus, the NT2LP sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences.
These primers can then be used to amplify an individual s DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals prepared in this manner can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can WO 99/58641 PC'T/US99/10311 _ 78 _ be used to obtain such identification sequences from individuals and from tissue. The NT2LP gene sequences of the invention uniquely represent fragments of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers o~f polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequence of SEQ ID NOs:l or 3, can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If a predicted coding sequence, such as that shown in Figures 1 and 2 of SEQ ID NOs:l or 3, is used, a more appropriate number of primers for positive individual identification would be 500-2,000.
If a panel of reagents from the NT2LP gene sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.
c. Use of Partial NT2LP Geae Sequences is Forensic Biology.
DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to _ 79 _ amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As described above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
Sequences targeted to the noncoding region of SEQ ID NOs:l or 3 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the NT2LP sequences or fragments thereof, e.g., fragments derived from the noncoding region of SEQ ID NOs:l or 3, having a length of at least 20 bases, preferably at least 30 bases.
The NT2LP sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such NT2LP probes can be used to identify tissue by species and/or by organ type.
In a similar fashion, these reagents, e.g., NT2LP
primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patent applications, patents, and published patent applications cited throughout this application are hereby incorporated by reference.

EXAMPLES
EXAMPLE 1: IDENTIFICATION OF HUMAN AND MONKEY NT2LP cDNA
In this example, the human and monkey NT2LP nucleic acid molecule was identified. A non-annotated EST
(GenBank° Accession number T0062I) was first identified by analysis of an EST database (a GenBank~ search of the dbEST database) based on a search designed to identify sequence that showed low levels of homology to GPCRs.
Primers were then designed based on the EST sequence and used to screen a human or monkey fetal cDNA library.
Several positive clones were identified, sequenced, and the sequences were assembled (Figure 1 and SEQ ID Nos:l and 2). BLAST analysis of nucleic acid databases in the public domain showed homologies to members of the NT
family of receptors and glutamate receptors.
The monkey NT2LP DNA sequence was used to probe human sequences obtained from a variety of library sources. Several clones from a human brain cDNA library were identified as containing sequences that had high homology to the monkey NT2LP sequences. The sequences were assembled into contig groups, yielding the identification of two splice forms of human NT2LP. These two splice variants (Figure 2) have the same coding region and differ only in the 5' UTR.
EXAMPLE 2: NORTHERN BLOTTING ANALYSIS OF TISSUE

Human brain multiple tissue northern (MTN) blots, human MTN I, II, and III blots (Clontech, Palo Alto, CA), containing 2~Cg of poly A+ RNA per lane were probed with monkey NT2LP-specific probes. The filters were prehybridized in 10 ml of Express Hyb hybridization solution (Clontech; Palo Alto, CA) at 68°C for 1 hour, after which 100 ng of 32P labeled probe was added. The probe was generated using the Stratagene Prime-It kit, Catalog Number 300392 (Clontech, Palo Alto, CA).

Hybridization was allowed to proceed at 68°C for approximately 2 hours. The filters were washed in a 0.05%
SDS/2X SSC solution for 15 minutes at room temperature and then twice with a 0.1% SDS/O.1X SSC solution for 20 minutes at 50°C and then exposed to autoradiography film overnight at -80°C with one screen. The human tissues tested included: heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus, small intestine, colon (mucosal lining), and peripheral blood leukocyte.
There was a strong hybridization to human whole brain and a weaker signal in the ovaries. No signal was found in placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus, small intestine, colon (mucosal lining), and peripheral blood leukocyte. Within the brain, hybridization was seen in all subregions.
EXAMPLE 3: EXPRESSION OF RECOMBINANT NT2LP PROTEIN IN
BACTERIAL CELLS
In this example, NT2LP is expressed as a recombinant glutathione-S-transferase (GST) fusion protein in E. coli and the fusion protein is isolated and characterized. Specifically, NT2LP is fused to GST and this fusion protein is expressed in E. coli, e.g., strain PEB199. Expression of the GST-NT2LP fusion protein in PEB199 is induced with IPTG. The recombinant fusion protein is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the protein purified from the bacterial lysates, the molecular weight of the resultant fusion protein is determined.

EXAMPLE 4: EXPRESSION OF RECOMBINANT NT2LP PROTEIN _IN
COS CELLS
To express the NT2LP gene in COS cells, the pcDNA/Amp vector by Invitrogen Corporation (San Diego, CA) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E, coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire NT2LP protein and a HA tag (Wilson et al. (1984) Cell 37:767) fused in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
To construct the plasmid, the NT2LP DNA sequence is amplified by PCR using two primers. The 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the NT2LP coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag and the last 20 nucleotides of the NT2LP coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CLAP
enzyme (New England Biolabs; Beverly, MA). Preferably the two restriction sites chosen are different so that the NT2LP gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DHSa, SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

COS cells are subsequently transfected with the NT2LP-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook et al.; Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The expression of the NT2LP
protein is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells are labelled for 8 hours with 35S-methionine (or 'SS-cysteine) . The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated proteins are then analyzed by SDS-PAGE.
Alternatively, DNA containing the NT2LP coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites.
The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the NT2LP
protein is detected by radiolabelling and immunoprecipitation using a NT2LP specific monoclonal antibody EXAMPLE 5: CHARACTERIZATION OF THE HUMAN NT2LP PROTEIN
In this example, the amino acid sequence of human NT2LP protein was compared to amino acid sequences of known proteins and various motifs were identified.

Hydrophobicity analysis indicated that the human NT2LP protein contains seven transmembrane domains (amino acids 34-58, 72-96, 113-131, 154-175, 212-236, 298-314 and 339-358; Figure 4). As shown in Figure 5, human NT2LP has a region (amino acids 49-358) that has homology to a seven transmembrane receptor family consensus sequence derived from a hidden Markov Model (PF0001). For general information regarding PFAM identifiers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.htm 1. The nucleotide sequence of the human and monkey NT2LP
was used as a database query using the BLASTN program (BLASTN1.3MP, Altschul et al. (1990) J. Mol. Biol.
215:403). Figure 7 is a set of alignments between portions of NT2LP (sbjct) and portions of mouse neurotensin receptor type 2 (P70310).
EXAMPLE 6: TISSUE DISTRIBUTION OF NT2LP mRNA
For in situ hybridization analysis, ten-micrometer-thick sections of selected tissues were postfixed with 4%
formaldehyde in DEPC treated 1X phosphate-buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC 1X phosphate-buffered saline and once in 0.1 M triethanolamine-HC1 (pH 8.0). Following incubation in 0.25% acetic anhydride-0.1 M triethanolamine-HC1 for 10 minutes, sections were rinsed in DEPC 2X, PBS 1X. Tissues were then dehydrated through a series of ethanol washes, incubated in 100% chloroform for 5 minutes (twice), and then rinsed in 100% ethanol for 1 minute and 95% ethanol for 1 minute and allowed to air dry.
Hybridizations were performed with 35S-radiolabeled (5 X 107 cpm/ml) cRNA probes designed to specifically hybridize to NT2LP messenger RNA. Probes were incubated in the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1 X Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.25% sodium dodecyl sulfate (SDS), for 18 hours at 55°C.
After hybridization, slides were washed with 2 X
SSC. Sections and then sequentially incubated at RT in THE (a solution containing 10 mM Tris-HC1 (pH 7.6), 500 mM
NaCl, and 1 mM EDTA), for 10 minutes, in THE with 40 micrograms of RNase A per ml for 30 minutes, and finally in THE for l0 minutes. Slides were then rinsed with 2 X
SSC at room temperature, washed with 2 X SSC at 60°C for 30 min., washed with 0:2 X SSC at 65°C for 30 min., and 0.2 X SSC at 65°C for 30 min. Sections were then dehydrated rapidly through serial ethanol before being air dried and exposed to Kodak Biomax MR scientific imaging film for 24 to 48 hours and subsequently dipped in NTB-2 photoemulsion and exposed at room temperature for 14 days before being developed and counter stained.
This analysis revealed that human NT2LP is expressed in neurons of the central nervous system and the peripheral nervous system. NT2LP was detected in the dorsal root ganglion, the trigeminal ganglion, and the superior cervical ganglion.
EXAMPLE 7: GENOMIC MAPPING OF HUMAN NT2LP
The Genebridge 4 Radiation Hybrid Panel was used to map the human NT2LP gene. NT2LP maps to chromosome 2, 19.5 cR3ooo telomeric to Whitehead Institute framework marker WI-6565 and 90 cR3ooo centromeric to Whitehead Institute framework marker D2S359. NT2LP is located at cytogenic location 2p24pTEL, at the extreme telomeric end of the P arm of chromosome 2.
Eauivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (22)

What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule which encodes a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4;
b) a nucleic acid molecule which encodes a fragment of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NOs:2 or 4; and c) a nucleic acid molecule which encodes a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NOs:2 or 4 under stringent conditions.

2. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences.

3. The nucleic acid molecule of claim 1 further comprising nucleic acid sequences encoding a heterologous protein.

4. A host cell which contains the nucleic acid molecule of claim 1.

5. The host cell of claim 4 which is a mammalian host cell.

6. A non-human mammalian host cell containing the nucleic acid molecule of claim 1.

7. The isolated nucleic acid molecule of claim 1, which is selected from the group consisting of the coding region of SEQ ID NOs:1 or 3 and the extracellular domain encoded by SEQ ID NOs:1 or 3.

8. An isolated protein selected from the group consisting of:
a) a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4;
b) a fragment of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID
NOs:2 or 4; and c) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NOs:1 or 3 under stringent conditions.

9. The protein of claim 8 further comprising heterologous amino acid sequences.

10. An antibody which selectively binds to a protein of claim 8.

11. A method for producing a protein selected from the group consisting of:
a) a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4;
b) a fragment of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID
NOs:2 or 4; and c) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NOs:1 or 3 under stringent conditions;

the method comprising the step of culturing the host cell of claim 4 under conditions in which the nucleic acid molecule is expressed.

12. A method for detecting the presence of a protein selected from the group consisting of:
a) a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4;
b) a fragment of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID
NOs:2 or 4; and c) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NOs:1 or 3 under stringent conditions;
in a sample, the method comprising the steps of:
i) contacting the sample with a compound which selectively binds to the protein; and ii) determining whether the compound binds to the protein in the sample.

13. The method of claim 12, wherein the compound which binds to the protein is an antibody.

14. A kit comprising reagents used for the method of claim 12, wherein the reagents comprise a compound which selectively binds to a protein selected from the group consisting of:
a) a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4;
b) a peptide comprising at least 15 contiguous amino acids of SEQ ID NOs:2 or 4; and c) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NOs:1 or 3 under stringent conditions.

15. A method for detecting the presence of a nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule which encodes a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4;
b) a nucleic acid molecule which encodes a fragment of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NOs:2 or 4; and c) a nucleic acid molecule which encodes a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NOs:1 or 3 under stringent conditions;
in a sample, the method comprising the steps of:
i) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and ii) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.

16. The method of claim 15, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.

17. A kit comprising reagents used for the method of claim 15, wherein the reagents comprise a compound which selectively hybridizes to a nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule which encodes a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4;
b) a nucleic acid molecule which encodes a fragment of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NOs:2 or 4; and c) a nucleic acid molecule which encodes a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NOs:1 or 3 under stringent conditions.

18. A method for identifying a compound which binds to a protein selected from the group consisting of:
a) a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4;
b) a fragment of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID
NOs:2 or 4; and c) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NOs:1 or 3 under stringent conditions, the method comprising the steps of:
i) contacting the protein, or a cell expressing the protein with a test compound; and ii) determining whether the protein binds to the test compound.

19. The method of claim 18, wherein the binding of the test compound to the protein is detected by a method selected from the group consisting of:
a) detection of binding by direct detecting of test compound/protein binding;
b) detection of binding using a competition binding assay;
c) detection of binding using an assay for NT2LP
activity.

20. A method for modulating the activity of a protein selected from the group consisting of:
a) a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4; and b) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NOs:2 or 4, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NOs:1 or 3 under stringent conditions, the method comprising the steps of:
i) contacting a cell expressing the protein with a compound which binds to the protein in a sufficient concentration to modulate the activity of the protein.

21. The method of claim 20, wherein the activity is a phosphatidylinositol activity.

22. The method of claim 20, wherein the activity is a cAMP activity.