WO2005121755A1

WO2005121755A1 - Cell free g-protein coupled receptor and ligand assay

Info

Publication number: WO2005121755A1
Application number: PCT/AU2005/000845
Authority: WO
Inventors: Edward John Mcmurchie; Wayne Richard Leifert
Original assignee: Commonwealth Scientific And Industrial Research Organisation
Priority date: 2004-06-14
Filing date: 2005-06-14
Publication date: 2005-12-22

Abstract

The present invention relates to compositions and methods for identifying G-protein-coupled receptors (GPCRs), ligands thereof and compounds that modulate G-protein signal transduction. More specifically, the invention relates to an assay system which involves the formation of a functional cell free complex comprising one or more G-protein subunits labeled with an energy donor or an energy acceptor and optionally a GPCR.

Description

Cell free G-protein coupled receptor (GPCR) and ligand assay

Field of the Invention

The present invention relates to compositions and methods for identifying G-protein- coupled receptors (GPCRs), ligands thereof and compounds that modulate G-protein signal transduction. More specifically, the invention relates to an assay system which involves the formation of a functional cell free complex comprising one or more G- protein subunits labeled with an energy donor or an energy acceptor and optionally a GPCR.

Background of the Invention

The actions of many extracellular signals are mediated by the interaction of a GPCR with its cognate guanine nucleotide-binding regulatory protein (G-protein). G-protein- mediated signaling systems have been identified in many divergent organisms, such as mammals and yeast. GPCRs respond to, among other extracellular signals, neurotransmitters, hormones, odorants and light (Watson, S. and Arkinstall, S., The G- protein Linked receptor facts Book, Academic Press, London, 1994).

The family of GPCRs has been estimated to include up to a thousand members, fully more than 1.5% of all the proteins encoded in the human genome. The GPCR family members play roles in regulation of biological phenomena involving virtually every cell in the body. Although the ligands and functions of many of these GPCRs are known, a significant portion of these identified receptors are without known ligands. These latter GPCRs, known as "orphan receptors", also generally have unknown physiological roles. Several GPCRs have been characterised in detail and sequence analysis reveals that they are structurally similar, possessing a number of highly conserved amino acid residues. Accordingly, GPCRs collectively form a large "superfamily" of receptor proteins capable of associating with the plasma membrane such that the N-terminal portion is localized in the extracellular space, the C-terminus is cytoplasmic, with there being seven transmembrane domains (i.e. 7-TM) in the GPCR polypeptide (see Figure Individual GPCR types activate particular signal transduction pathways. At least ten different signal transduction pathways are known to be activated via GPCR polypeptides. For example, the secretin receptor sub-family of GPCR polypeptides are activated by a ligand selected from the group consisting of: secretin, glucagon, calcitonin, glucagon-like peptide 1, parathyroid hormone, parathyroid-related peptide, corticotropin-releasing factor (CRF), growth hormone-releasing hormone (GHRH), gastric inhibitory polypeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and insect diuretic hormone (DHR). Known human chemokine receptors include CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR1, CXCR2, CXCR3, and CXCR4.

GPCRs mediate signal transduction across a cell membrane upon the binding of a ligand to an intra-membranous portion or an extracellular portion of a GPCR. Another intracellular portion of a GPCR interacts with G-proteins to modulate signal transduction from outside to inside a cell. A GPCR is therefore said to be "coupled" to G-proteins. G-proteins are composed of three polypeptide subunits: an alpha subunit, which binds and hydrolyzes guanine triphosphate (GTP), and a dimeric beta plus gamma subunit. In the basal, inactive state, the G-proteins exist as a heterotrimer of the alpha, beta and gamma subunits. When the GPCR is inactive (ie. ligand inactivation has not occurred), guanosine diphosphate (GDP) is associated with a part of the alpha subunit protein. When a ligand binds to a receptor and subsequently causes its activation, there is a resultant decrease in the affinity of the alpha subunit for GDP. In its active state, the alpha subunit exchanges GDP for GTP and active alpha subunit is reported to disassociate from both the receptor and the beta-gamma dimer. The disassociated, active alpha, and beta and gamma subunits transduce signals to various effectors that are "downstream" in the G-protein signalling pathway within the cell. Eventually, the G alpha's endogenous GTPase activity returns active alpha subunit to its inactive state, in which it is associated with GDP and reassociation with the beta- gamma dimer occurs.

Many available therapeutic drugs in use today target GPCRs, as they mediate vital physiological responses, including vasodilation, heart rate, bronchodilation, endocrine secretion, and gut peristalsis. See, eg., Lefkowitz et al., Ann. Rev. Biochem. 52:159 (1983); Gilman, A. G. (1987) Annu. Rev. Biochem 56: 615-649; Hamm, H. E. (1998) JBC 273: 669-672; Ji ,T. H. (1998) JBC 273: 17229-17302. Kanakin, T. (1996) Pharmacological review, 48:413-463; Gudermann T. and Schultz, G. (1997), Annu. Rev. Neurosci., 20: 399-427. In fact, it has been estimated that more than 50% of the drugs in use clinically in humans at the present time are directed at GPCRs, including the adrenergic receptors (ARs). For example ligands to beta ARs are used in the treatment of anaphylaxis, shock, hypertension, hypotension, asthma and other conditions.

Although in general GPCRs require agonist binding for activation, agonist-independent signaling activity has been well documented in the native form of a variety of GPCRs. This spontaneous activation of GPCRs occurs, in both normal and pathological processes, where a GPCR cellular response is generated in the absence of a ligand. For example, native dopamine DIB and prostaglandin EPlb receptors have been found to possess constitutive activity. In addition, a number of GPCRs, for example, receptors for thyroid-stimulating hormone have mutants that exhibit agonist-independent activity and cause disease in humans.

Increased spontaneous activity and/or basal activity of GPCRs can be decreased by inverse agonism of the GPCRs. Such methods are therapeutically important where diseases cause an increase in spontaneous GPCR activity, or where it is desirable to decrease the basal activity of GPCR. Thus, a technology for identifying inverse agonists of native and mutated GPCRs has important pharmaceutical applications. Furthermore, because certain constitutively active GPCRs can be tumorigenic, the identification of inverse agonists for these GPCRs can lead to the development of anti- tumor and/or anti-cell proliferation drugs. These compounds have become increasingly important, especially for the treatment of psychological disorders such as depression and bipolar disorder. Conventional assays are not particularly suited to reliably identify inverse agonists as the activity of the GPCRs in response to an inverse agonist cannot be directly measured.

Since GPCRs and G-protein signaling pathways are critical targets for therapeutics, there is a need for fast, effective and reproducible methods for identifying agonists, antagonists and inverse agonists that modulate G-protein signaling, and in particular compounds that regulate this signaling through a GPCR (such as the recently reported family of RGS (regulators of G-protein signalling) and AGS (activators of G-protein signalling) proteins (see Wieland and Mittmann (2003) Pharmacology and Therapeutics 97:95-115)). US 6,004,808 (Negulescu et al) and US 6,448,377 (Kobilka et al) describe assays for identifying agonists and antagonists that modulate GPCR activity. However, these assays require generation of particular downstream effectors (which vary depending on the specific GPCR of interest) in cell based systems. Typically mammalian (or insect) cells are used in these assay systems for convenience. Such cells are relatively fragile, and there are numerous ways in which toxic compounds can interfere with signalling, giving rise to false positives or false negatives in ligand screening.

Fang et al (2002) ChemBio Chem 3 :987-991 and Neumann et al (2002) ChemBio Chem 3:993-998 describe attempts to assay GPCR in cell free systems, however, screening is achieved by detection of ligand binding to displayed receptors rather than by signal transduction. These types of agonist/antagonist screens generally rely on displacement of radio-actively or fluorescently labeled ligands by the test compound. The screens therefore tend to involve numerous additions and washing steps, require radioactive counting, are expensive in consumables and generate considerable waste.

Summary of the Invention

The present inventors have now found that reconstitution of a GPCR/G-protein complex in a cell free environment gives rise to a functional complex in which the G- proteins remain active when the receptor is stimulated by its cognate ligand. The finding that reconstituted GPCR/G-protein complexes can function in this manner has led to the development of a sensitive and adaptable cell-free assay system for detecting GPCR activity.

Stimulation of the receptor by its cognate ligand results in a series of sequential events which are referred to herein as "primary", secondary" and "tertiary" events. By "primary" event we mean binding of the ligand to the receptor, which in the case of an agonist or inverse agonist, leads to some form of change in the receptor moiety. By "secondary" event we mean the exchange of GDP for GTP which occurs on the G- alpha subunit. By "tertiary event" we mean any rearrangement or change in conformation of one or more G-protein subunits within the complex. For example, tertiary events include dissociation of the G-alpha subunit from the G-beta-gamma heterodimer; changes in the proximity of any one of the G-protein subunits to another G-protein subunit or to the GPCR, and any interaction that occurs between an RGS or AGS protein and a G-protein subunit.

In one particular embodiment the cell free assay system of the present invention enables detection of the tertiary events that occur as a result of stimulation of the GPCR which may occur in the presence or absence of a cognate ligand. Importantly, detection of these tertiary events can be achieved in a homogeneous assay system. This assay system is therefore highly suitable for use in point of care diagnostic applications and for high-throughput drug screening using, for example, microarray, suspension bead, nanoparticle or biochip formats.

Accordingly, the present invention provides a method for determining the activity or function of a GPCR, the method comprising providing a cell free complex comprising a GPCR coupled to at least one G- protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor; and detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex.

The nature of the GPCR is not critical to the present invention and accordingly, any type of GPCR may be used. For example, the GPCR may be a receptor for: Acetylcholine 1 -» 5, IL-8, Adrenoceptor α 1, 2; β 1, 2, 3, Vasopressin, Dopamine 1 - 4, Oxytocin, Serotonin 1 -=► 7, Prostaglandin E, F, Histamine 1, 2, Prostacyclin, Angiotensin 1, 2, TXB₂, Opioid δ, K, μ, Adenosine 1 -» 3, Rhodopsin 1 - 4, Platelet Activating Factor, Olfactory 1 → 11, Cholecystokinin A, B, Substance P, K, or Endothelin. In one embodiment, the GPCR is an adrenergic receptor (for example, an alpha adrenergic or beta adrenergic receptor) or a muscarinic receptor (for example, an Ml, M2, M3 or M4 muscarinic receptor). Alternatively, the GPCR may be an "orphan" GPCR. The GPCR may be derived from any species including animals, plants, and microorganisms.

Similarly, any G-protein subunit or combination of G-protein subunits may be used. In one embodiment the G-protein coupled receptor/G-protein subunit complex comprises more than one type of G-protein subunit. Preferably, the complex comprises at least a G-alpha subunit, more preferably at least two G-protein subunits. Preferably, the complex comprises a G-alpha subunit, a G-beta subunit and a G-gamma subunit or any combination thereof. In a more preferred embodiment, the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta-gamma dimer.

In a further preferred embodiment of the invention the tertiary event is a change in proximity of the at least one G-protein subunit with respect to another G-protein subunit.

In yet a further preferred embodiment of the invention the tertiary event is dissociation of the G-alpha subunit from the G-beta-gamma heterodimer.

In yet a further preferred embodiment, the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa. In one particular example, the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor. In another embodiment, the G-beta subunit or G-gamma subunit is labeled with an energy donor and the G-alpha subunit is labeled with an energy acceptor.

In yet another embodiment, two or more different energy donor/acceptor pairs can be used so that different changes in G-protein subunits can be detected simultaneously.

In a further preferred embodiment of the invention the tertiary event is an interaction that occurs between the at least one G-protein subunit and a protein that normally interacts with a G-protein subunit.

A protein that normally interacts with a G-protein subunit may be, for example, an RGS or AGS. Representative RGS proteins include but are not limited to RGS1, RGS2, RGS4 and RGS 16. Indeed, any of the over 20 RGS proteins expressed in mammals can be employed in a system of the present invention. See Zeng et al., (1998) J. Biol. Chem. 273(52):34687-34690; Xu et al., (1999) J. Biol. Chem. 274(6):3549-3556; and Mukhopadhyay et al, (1999) Proc. Natl. Acad. Sci. USA ^" 96:9539-9544.

In one preferred embodiment the G-protein subunit is labeled with an energy donor and the protein that normally interacts with a G-protein is labeled with an energy acceptor or vice versa. A variety of energy donor and acceptor moieties can be used in the context of the present invention, differing in the physical nature of the detectable signal produced by the interaction of the donor and acceptor moieties. For example, the detectable signal may be a fluorescent or electrochemical signal, or may involve nuclear magnetic resonance (NMR) or electron paramagnetic resonance (EPR).

In a preferred embodiment, the detectable signal is a detectable spectral change. The detectable spectral change may be a change in fluorescent decay time (determined by time domain or frequency domain measurement), fluorescent intensity, fluorescent quenching, fluorescent anisotropy or polarization; fluorescent correlation spectroscopy, a spectral shift of the emission spectrum; a change in time-resolved anisotropy decay (determined by time domain or frequency domain measurement), and the like.

Preferred energy donor and acceptors are those which display resonance energy transfer such as fluorescence resonance energy transfer (FRET), luminescence resonance energy transfer (LRET), and bioluminescence resonance energy transfer (BRET).

In a preferred embodiment, the energy donor and acceptor moieties are fluorescent or luminescent donor and acceptor moieties. Preferred energy donor and acceptor are Terbium/Alexa546, Terbium/fluorescein, Terbium/GFP,

Terbiurn/tetramethylrhodarnine (TMR), Terbium/Cy3, Terbium/QSY-7, Terbium/R phycoerythrin, Europium/Cy5, Europium/Allophycocyanin (APC), Europium/Alexa 633, Alexa 488/AIexa 555, Alexa 568/Alexa 647, Alexa 594/Alexa 647, Alexa 647/Alexa 594, Cy3/Cy5, BODIPY FL/BODIPY FL, Fluorescein/TMR, IEDANS/fluorescein, and fluorescein fluorescein.

Still another class of useful energy donor and acceptor pairs include fluorophore- quencher pairs in which the second group is a quencher which decreases the fluorescence intensity of the fluorescent group. Suitable quenchers include acrylamide groups, heavy atoms such as iodide and bromate, nitroxide spin labels such as TEMPO, and the like. For example, a suitable quencher may be selected from the group consisting of DABCYL, DABSYL, QSY 7, QSY 9, QSY 21, and QSY 35.

It will be appreciated that any suitable method may be used to label the G-protein subunit or the protein that normally interacts with a G-protein subunit with an energy donor and/or acceptor moiety. In one example, the energy donor and/or acceptor moiety is attached by way of a fusion protein or amino acid motif present within the G- protein subunit or the protein that normally interacts with a G-protein. The amino acid motif may be, for example, a lanthanide binding tag motif. This particular embodiment is described in detail in Example 5.

In a further preferred embodiment of the present invention the cell free complex is tethered to a support. The complex may be tethered to the support via the GPCR or any one of the G-protein subunits. In a preferred embodiment, the complex is tethered to the support via one of the G-protein subunits. The G-protein subunit tethered to the support may be the same as or different to the G-protein subunit which is associated with the detectable signal. In one preferred embodiment of the invention the G-alpha subunit is tethered to the support.

The G-protein subunit or GPCR may be tethered to the support by any suitable means. For example, the G-protein subunit or GPCR may be labeled with a polyhistidine tag and exposed to a nickel-coated support. Alternatively, other forms of attachment, such as the streptavidin-biotin system, the digoxin-antidigoxin system, myc, haemaglutin, GST or FLAG tags may be used.

Any suitable support may be used in the present invention. For example, the support may be a support matrix, a solid support such as a dish, tray, plate, multi-well plate, microtiter plate, bead, dendrimer, nanoparticle, nanobead, nano-tube, micro-chip, micro-well or arrays (including addressable arrays and micro-arrays). Alternatively, the support may be a bibulous material including silica gel, hydrogel or other useful gels, cellulosic beads, glass and glass fibres, filter paper or natural and synthetic membranes such as nitrocellulose and nylon membranes. The support may comprise glass, gold, mica, plastic or other polymeric material or gel elements fixed on a solid support.

In one embodiment, the solid support is part of a biosensor device. A biosensor may be defined as an analytical device which comprises a biological molecular recognition component, which device typically produces an electronic or other addressable signal dependent on the presence and/or concentration of an analyte interacting with the biological recognition component. Such biosensor devices are well-known and are described, for example, in EP 0 341 927, EP 0 416 730 and EP 0453 224. In a further embodiment of the present invention the cell free complex is stimulated prior to detection of the signal. The cell free complex may be stimulated in any manner. For example, the complex may be stimulated by exposing the complex to a sample comprising a cognate ligand of the GPCR or by exposing the GPCR to an odor or taste. Alternatively, spontaneous stimulation of the GPCR may occur in the absence of a cognate ligand.

The present invention also provides a method for determining the amount of a GPCR ligand in a sample, the method comprising providing a cell free complex comprising a GPCR coupled to at least one G- protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor; detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex; and determining the amount of ligand in the sample based on the level of signal detected.

In the context of this aspect, the ligand may be an agonist, antagonist or inverse agonist of the GPCR.

In a another embodiment of these aspects, the GPCR is a known receptor. In another embodiment, the GPCR is an "orphan" GPCR.

The present invention also provides a method of screening for an agent that modulates G-protein interactions, the method comprising exposing a candidate agent to a cell free complex comprising at least one G- protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor; and detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex.

Any significant change in the detected signal in the presence of the candidate agent when compared to the absence of the candidate agent is an indication that the candidate agent modulates G-protein interactions. In the context of this method, the candidate agent may be an agonist, inverse agonist or antagonist of a ligand of a GPCR. Alternatively, the candidate agent may be a G- protein regulator.

Preferably, the complex comprises at least a G-alpha subunit, more preferably at least two G-protein subunits. Preferably, the complex comprises a G-alpha subunit, a G-beta subunit and a G-gamma subunit or any combination thereof. In a more preferred embodiment, the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta-gamma dimer.

In yet a further preferred embodiment, the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa. In one particular example, the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor. In another embodiment, the G-beta subunit or G-gamma subunit is labeled with an energy donor and the G-alpha subunit is labeled with an energy acceptor. In another embodiment, two or more different energy donor/acceptor pairs can be used so that different changes in G-protein subunits can be detected simultaneously.

A protein that normally interacts with a G-protein subunit may be, for example, an RGS or AGS.

In one preferred embodiment the G-protein subunit is labeled with an energy donor and the protein that normally interacts with a G-protein is labeled with an energy acceptor or vice versa. In a further embodiment of this method, the cell-free complex further comprises a GPCR coupled to the at least one G-protein subunit. The GPCR may be a known receptor or an "orphan" GPCR. In this embodiment, the candidate agent tested in the method may be a ligand of the GPCR. Alternatively, the candidate agent may be an agonist, inverse agonist or antagonist of a ligand of a GPCR and the method may involve contacting the candidate agent with the complex in the presence of a known ligand of the GPCR.

The present invention also provides a method for detecting a GPCR in a sample, the method comprising exposing a sample to a cell free G-protein complex comprising at least one G- protein subunit which is labeled with an energy donor or an energy acceptor, wherein an associated GPCR present in the sample is capable of coupling to the G-protein complex such that stimulation of the GPCR leads to generation of a detectable signal associated with a tertiary event involving at least one G-protein subunit in the complex, and detecting a signal associated with a change in at least one G-protein subunit in the complex.

In a preferred embodiment of this method, stimulation of the GPCR is achieved by the presence in the sample of a cognate ligand of the GPCR.

In preferred embodiments of the methods of the present invention, additional components are included in the sample in order to enhance or improve the assay system. For example, RGS or AGS proteins may be exposed to the complex during the screening process.

In the context of the methods of the present invention, generation of a detectable signal is preferably associated with a tertiary event following stimulation of the GPCR/G- protein complex. An advantage of this approach is that it allows detection of a generic signal that will be common to a broad range of GPCRs. In contrast, detection systems that are based on primary events, or on down-stream signalling of G-proteins, generally need to be adapted to the specific GPCR of interest. In a particularly preferred embodiment, the detectable change involves dissociation of a G-alpha subunit from the G-beta and/or G-gamma subunits. This dissociation may be detected by, for example, attaching energy donor and acceptor labels separately to either the G-alpha subunit or the G-beta-gamma heterodimer. In another preferred embodiment, the detectable change involves a change in proximity of one of the G- protein subunits to another G-protein subunit or to the GPCR. This may be detected for example, by attaching energy donor and acceptor labels separately to the GPCR and one of the G-protein subunits or to two G-protein subunits. In another preferred embodiment, the detectable change involves an interaction between an RGS or AGS protein and a G-protein subunit.

The present invention also provides a cell free complex comprising a GPCR coupled to at least one G-protein subunit, wherein the at least one G-protein is labeled with an energy donor or an energy acceptor; and wherein stimulation of the GPCR leads to a detectable tertiary event involving the at least one G-protein subunit in the complex..

In a preferred embodiment of this cell free complex, the at least one G-protein subunit is a G-alpha subunit.

Preferably, the complex comprises at least two G-protein subunits. More preferably, the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta-gamma dimer. In a further preferred embodiment the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa.

In a further preferred embodiment the cell free complex is tethered to a support. Preferably, the cell free complex is tethered to the support via a G-alpha subunit. The support may be selected from the group consisting of a dish, tray, plate, multi-well plate, microtiter plate, bead, dendrimer, nanoparticle, nanobead, nano-tube, micro-chip, micro- well, microarray, silica gel, hydrogel, cellulosic bead, glass, filter paper, nitrocellulose and nylon membrane. In one particular example the solid support is part of a biosensor device.

The present invention also provides a composition comprising a plurality of cell free G- protein coupled receptor protein subunit complexes according to the present invention. The present invention also provides a microarray comprising a plurality of cell free G- protein coupled receptor protein subunit complexes according to the present invention.

The present invention also provides a biosensor or bio-diagnostic device comprising at least one cell free G-protein coupled receptor protein subunit complex according to the first aspect and a means for detecting a signal associated with a change in at least one of the G-protein subunits in the complex.

In a further aspect the present invention provides a kit for screening for an agent that modulates the activity of a GPCR, the kit comprising a cell free complex comprising at least one G-protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor.

In a preferred embodiment of this kit the cell free complex comprises a heterotrimeric G-protein complex comprising a G-alpha subunit and a G-beta-gamma dimer. In a further preferred embodiment the cell free complex further comprises a GPCR coupled to the at least on G-protein subunit. Preferably, the cell free complex is tethered to a support.

Preferably, at least two components of the cell free complex are labeled, one with an energy donor and one with an energy acceptor. Preferably the labels are attached to the at least two G-protein subunits. Preferably, the labels are fluorescent or luminescent probes.

As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to other aspects of the invention.

Brief Description of the Figures

Figure 1 : Diagram showing the mode of action of GPCRs (adapted from Marinissen and Gutkind (2001) Trends in Pharmacological Sciences 22:368-375)

Figure 2: Schematic representation of a preferred embodiment of the invention showing a cell free GPCR/G-protein complex tethered to a support, wherein binding of a cognate ligand to the GPCR/G-protein complex leads to generation of a detectable signal.

Figure 3: Schematic representation showing use of a cell free complex of the present invention to assay for compounds that modulate GPCR signalling.

Figure 4: Schematic representation of a preferred method for identifying unknown GPCRs in a sample.

Figure 5: Schematic representation of a preferred method for identifying modulators of G-protein signaling, (i) unknown modulators of G-subunit interactions, (ii) RGS moieties that interfere with G subunits, (iii) AGS moieties that interfere with receptor and G protein interactions.

Figure 6: Reconstitution of c2A-AR/G-protein complex (transductosome) - dependence on G-protein subunits. Sf9 membranes extracted with 7 M urea (M) expressing the α2A-AR were reconstituted with 50 nM Gail (filled squares), 50 nM G il and 50 nM βlγ2 (filled circles) or no G-proteins (open and filled triangles) as indicated. α2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the partial α2A-AR agonist, clonidine (100 μM). ³⁵S-GTP (0.2 nM) binding was assayed at the indicated time points at 26°C. Data are the means of duplicate determinations.

Figure 7: Concentration dependence of GDP on ³⁵S-GTP binding in the a2A-AR/G- protein complex. Sf9 membranes extracted with 7 M urea expressing the α2A-AR were reconstituted with 52 nM Gail and 3 nM βlγ2. α2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the partial α2A-AR agonist, clonidine (100 μM), with increasing concentrations of GDP as indicated. S- GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means (± SEM) of triplicate determinations. Numbers in parentheses represent fold stimulation.

Figure 8: Effect of the a2A-AR agonist on ³⁵S-GTP binding in the a2A-AR/G-protein complex. Sf9 membranes extracted with 7 M urea expressing the α2A-AR were reconstituted with 52 nM Gail and 3.1 nM βlγ2. The reconstituted α2A-AR membranes (0.4 mg/ml) were incubated with various concentrations of the alpha 2A- AR agonist UK 14303 ("UK") (filled symbols) as indicated + the α2A-AR antagonist rauwolscine (500 μM) (open symbols). ³⁵S-GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means (± SEM) of triplicate determinations. At some points, the SEMs are too small to be seen (as in other figures).

Figure 9: Concentration dependence of βlγl on ³ⁱ 'S-GTP binding in the cι2A-ARJG- protein complex. Sf9 membranes extracted with 7 M urea expressing the α2A-AR were reconstituted with 52 nM Gail and increasing concentrations of βlγ2 as indicated. α2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the α2A-AR agonist, UK (10 μM). ³⁵S-GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means (± SEM) of triplicate determinations.

Figure 10: Concentration dependence of Goal on ³⁵S-GTP binding in the 2A-AR/G- protein complex. Sf9 membranes extracted with 7 M urea expressing the α2A-AR were reconstituted with 3.1 nM βlγ2 and increasing concentrations of Gail as indicated. α2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the α2A-AR agonist, UK (10 μM). ³⁵S-GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means (± SEM) of triplicate determinations. Numbers in parentheses represent fold stimulation.

Figure 11 : Concentration dependence ofhis-tagged Goal on S-GTP binding in the ct2A~AR/G-protein complex. Sf9 membranes extracted with 7 M urea expressing the α2A-AR were reconstituted with 20 nM βlγ2 and increasing concentrations of His6- tagged Gail (Gail (his)) as indicated. α2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the α2A-AR agonist, UK (100 μM). ³⁵S-GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means (± SEM) of triplicate determinations.

Figure 12: Dependence of G-protein subunits on UK-stimulated 35S-GTP binding in the cc2A-AR/G-protein complex. Sf9 membranes extracted with 7 M urea expressing the α2A-AR were reconstituted with combinations of His6-tagged, non Hisό-tagged G- protein subunits, or no addition of G-proteins as indicated. The final concentrations were as follows: βlγ2 = 20 nM, βlγ2(his) = 20 nM, G il = 52 nM, Gail (his) = 10 nM. a2A-AR membranes (0.4 mg/ml) were incubated in the absence (open bars)or presence (solid bars) of the α2A-AR agonist, UK (100 μM) as indicated ("UK"). ³⁵S- GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means (+ SEM) of triplicate determinations. Figure 13: Ni ⁺ specificity for capture of His6-tagged G-protein/ a2A-AR transductosome complex. Sf9 membranes extracted with 7 M urea expressing the α2A- AR were reconstituted with 10 nM Gail (his) and 20 nM βlγ2 (final concentrations in assay). 50 μl of Ni-NTA beads was added and further incubated for 60 minutes prior to the binding assay. The Ni-NTA beads with captured α2A-AR transductosomes were incubated with (solid bars) or without (open bars) the α2A-AR agonist, UK (100 μM) and ³⁵S-GTP (0.2 nM) binding was assayed at 60 minutes by filtration through Whatman filter paper.

Figure 14: Ni specific capture of His6-tagged G-protein/ a2A-AR transductosome. Sf9 membranes extracted with 7 M urea expressing the α2A-AR were reconstituted with 10 nM G il (his) and 20 nM βlγ2 (final concentrations in assay). 0 - 50 μl of Ni- NTA beads were added and further incubated for 60 minutes prior to the binding assay. The Ni-NTA beads with captured α2A-AR transductosomes was incubated with (solid symbols) or without (open symbols) the α2A-AR agonist, UK (100 μM) and ³⁵S-GTP (0.2 nM) binding was assayed at 60 minutes by filtration over Whatman filter paper. Data are the means of duplicate determinations.

Figure 15: Saturation binding of [³ HJ-Scopolamine. Specific binding of the muscarinic receptor antagonist [³H]-Scopolamine to Sf9 cell membranes overexpressing M₂-muscarinic receptors. The assay was carried out with triplicate determinations using 5μg membrane protein at 28°C for 60min in the absence or presence of lOOμM atropine. Non-specific binding was less than 5%.

Figure 16: Competition of H-Scopolamine binding. Inhibition of specific H- Scopolamine binding in urea-treated Sf9 cell membranes overexpressing M₂- muscarinic receptors by the non-specific muscarinic receptor antagonist atropine, Mϊ- muscarinic receptor antagonist pirenzepine or the muscarinic receptor agonist carbachol. The assay was carried out with triplicate determinations using 5μg membrane protein at 28°C for 60min with lnM ³H-scopolamine.

Figure 17: Reconstitution of M₂-muscarinic receptor /G-protein complex

(transductosome) - time course of carbachol stimulation. Sf9 membranes extracted with 7 M urea (M) overexpressing M₂-muscarinic receptors were reconstituted with 5 nM Gαπ_(hj_S), 5 nM βrγ₂ and incubated with (solid symbols) or without (open symbols) the M₂-muscarinic receptor agonist carbachol (25mM). ³⁵S-GTP (0.2 nM) binding was assayed at the indicated time points at 28°C in the presence of 25μM GDP, 1 OμM AMP and 5ug membrane protein per assay (25μl). Data are the means of duplicate determinations.

Figure 18: Effect of the M∑-muscarinic receptor agonist carbachol on ³⁵ S-GTP binding in the M R/G-protein complex with 25 μM GDP. Sf9 membranes extracted with 7 M urea overexpressing M₂-muscarinic receptors (M₂-R) were reconstituted with 5 nM Gocii(his) and 5 nM β₁γ₂. The reconstituted M₂R membranes were incubated with various concentrations of carbachol (filled symbols) as indicated + the non-specific muscarinic receptor antagonist atropine (500 μM) (open symbol). ³⁵S-GTP (0.2 nM) binding was assayed at 90 min (28°C) in the presence of 25 μM GDP, lOμM AMP and 5ug membrane protein per assay (25μl). Data are the means (± SEM) of triplicate determinations. The number in parentheses represents the fold stimulation above basal. Appropriate blanks have been subtracted (ie membranes with no G-proteins).

Figure 19: Effect of the M₂-muscarinic receptor agonist carbachol on ^3t 'S-GTP binding in the M₂R/G-protein complex attached to Ni²⁺-NTA coated beads with 25 μM GDP. Sf9 membranes extracted with 7 M urea overexpressing M₂-muscarinic receptors (M₂- R) were reconstituted with 5 nM Gαu(his) and 5 nM β₁γ₂. The reconstituted M₂R membranes were incubated with lOμl of Ni-NTA beads and various concentrations of carbachol (filled symbols) as indicated ± the non-specific muscarinic receptor antagonist atropine (500 μM) (open symbols) ³⁵S-GTP (0.2 nM) binding was assayed at 90 min (28°C) in the presence of 25μM GDP, lOμM AMP and 20ug membrane protein per assay (lOOμl). Samples were filtered over Whatman #1 paper filters. The number in parentheses represents the fold stimulation above basal. Appropriate blanks have been subtracted.

Figure 20: Effect of the M₂-muscarinic receptor agonist carbachol on ³ 'S-GTP binding in the M₂R G-protein complex with IμM GDP. Sf9 membranes extracted with 7 M urea overexpressing M₂-muscarinic receptors (M₂R) were reconstituted with 5 nM Gαπ₍ i_s) and 5 nM βιγ₂. The reconstituted M₂R membranes were incubated with various concentrations of carbachol (filled symbols) as indicated ± the non-specific muscarinic receptor antagonist atropine (500 μM) (open symbol). S-GTP (0.2 nM) binding was assayed at 90 min (28°C) in the presence of lμM GDP, lOμM AMP and 5ug membrane protein per assay (25μl). Data are the means (± SEM) of triplicate determinations. The number in parentheses represents the fold stimulation above basal.

Figure 21 : Effect of the M₂-muscarinic receptor agonist carbachol on ³ 'S-GTP binding in the M₂R/G-protein complex attached to Ni²⁺-NTA coated beads with IμM GDP. Sf9 membranes extracted with 7 M urea overexpressing M₂-muscarinic receptors (M₂- R) were reconstituted with 5 nM

and 5 nM βrγ₂. The reconstituted M₂R membranes were incubated with lOμl of Ni-NTA beads and various concentrations of carbachol (filled symbols) as indicated + the non-specific muscarinic receptor antagonist atropine (500 μM) (open symbol) ³⁵S-GTP (0.2 nM) binding was assayed at 90 min (28°C) in the presence of lμM GDP, lOμM AMP and 20ug membrane protein per assay (lOOμl). Samples were filtered over Whatman #1 paper filters. The number in parentheses represents the fold stimulation above basal. Appropriate blanks have been subtracted.

Figure 22. Agonist (UK14304) affinity and Ni²⁺-bead capture of ci_2A-AR-activated ³⁵SγGTP:Gθiihis. Various concentrations of UK14304 were incubated with 0.2 mg/ml α_2A-AR containing reconstituted Gα_ϋhis (50 nM) and βιγ₂ (50 nM), 5 μM GDP; 10 μM AMPNP and 0.2 nM ³⁵SγGTP in the absence (D) or presence (■) of 10 μl Ni(NTA) agarose beads. The mix was incubated for 90 minutes at 27°C with shaking. Final volume was 100 μl and the entire reaction was filtered over a Whatman #1 filter and washed with 3 x 4 ml with ice-cold TMN buffer. The EC₅₀ values were 12 nM and 30 nM in the absence and presence of Ni(NTA) agarose beads, respectively. A representative experiment is shown.

Figure 23. Antagonist dose efficacy at the 2A-AR using the adrenergic receptor subtype-specific antagonists: rauwolscine ( ), yohimbine (O), prazosin ( ) or propranolol (O) as indicated. Antagonist dose efficacy of Ni2+-bead captured, α2A- AR stimulated 35SγGTP:Gαilhis binding was carried out using 20 nM of both Gαilhis and Gβlγ2 combined with 0.1 mg/ml of α2A-AR membranes, 5 μM GDP and 10 μM AMP-PNP ("reconstitution mix"), 0.2 nM 35SγGTP in the presence of various concentrations of the different adrenergic receptor subtype-specific antagonists as indicated and 10 μl Ni(NTA) agarose beads (n = 3, mean ± S.E.M.). UK14304 (1 μM final concentration) was added to start the reactions and the mix was incubated for 90 minutes at 27°C with shaking. Final volume was 100 μl and the entire reaction was filtered over a Whatman #1 filter and washed with 3 x 4 ml with ice-cold TMN buffer. The IC50 values for each of the antagonists was determined as 0.051 μM (rauwolscine: selective α2-AR antagonist); 0.080 μM (yohimbine: selective α2-AR antagonist); 8.3 μM (prazosin: selective αl-AR antagonist) and 86.9 μM (propranolol: β-AR antagonist).

Figure 24: Effect of conjugation of G-protein alpha subunits with TMR fluorescent probes on a2-AR signalling activity in a cell-free, reconstituted assay. Gail (his) and βlγ2 G-protein subunits were labelled with the fluorescent probes TMR- NCS or TMR-maleimide. α2A-AR membranes (0.05 mg/ml final concentration in assay) were reconstituted with the fluorescent labelled G-protein subunits at 20 nM each (final concentration). lOμL Ni-NTA beads were added and incubated for 10 minutes prior to the binding assay. The beads with captured transductosomes (receptor plus G-protein subunit) were incubated in the absence (basal condition, open bars) or presence (solid bars) of the α2A-AR agonist, UK (10 μM) or in the presence (stipled bars) of the α2A-AR antagonist yohimbine (lOOuM), ³⁵S-GTP (0.2 nM) binding was assayed at 90 minutes. Data are the means (± SEM) of triplicate determinations.

Figure 25: Effect of conjugation of G-protein alpha subunits with Terbium fluorescent probes on a2-AR signalling activity in a cell-free, reconstituted assay. Gail (his) g- protein subunits (derived from a single infection or a triple infection with β lγ2) were labelled with the fluorescent probes Tb-mal or Tb-NCS. The subunits were stored in buffer with or without glycerol as indicated. α2A-AR membranes (0.05 mg/ml final concentration in assay) were reconstituted with the fluorescent labelled Gail (his) and unlabelled βlγ2 G-protein subunits at 20 nM each (final concentration). The reconstituted membranes were incubated in the absence (basal condition, open bars) or presence (solid bars) of the α2A-AR agonist, UK (10 μM). Signalling activity measured by ³⁵S-GTP (0.2 nM) binding was assayed at 90 minutes. Data are the means of duplicate determinations.

Figure 26: Effect of conjugation of G-protein beta/gamma subunits with Terbium fluorescent probes on A_∑AR signalling activity in a cell-free, reconstituted assay βlγ2 G-protein subunits (derived from a triple infection with G il (his)) were labelled with the fluorescent probes Tb-Mal or Tb-NCS. The subunits were stored in buffer with or without glycerol as indicated. α2A-AR membranes (0.05 mg/ml final concentration in assay) were reconstituted with unlabelled Gail (his), Terbium labelled βlγ2, unlabelled βlγ2 and unlabelled β4γ2 G-protein subunits at 20 nM each (final concentration). The reconstituted membranes were incubated in the absence (basal condition, open bars) or presence (solid bars) of the α2A-AR agonist, UK (10 μM). Signalling activity measured by ³⁵S-GTP (0.2 nM) binding was assayed at 90 minutes. Data are the means of duplicate determinations.

Figure 27: Effect of conjugation of G-protein alpha and beta/gamma subunits with fluorescent probes on A2AR signalling activity in a cell-free, reconstituted assay. α2A- AR membranes (at 0.05mg/ml final assay concentration) were reconstituted with fluorescent labelled Gail (his) and βlγ2 G-protein subunits at a 20 nM each (final concentration). The reconstituted membranes were incubated in the absence (basal condition, open bars) or presence (solid bars) of the α2A-AR agonist, UK (10 μM). (A) Signalling activity following reconstitution with Gail (his) labelled with Tb-mal and βlγ2 labelled with TMR (maleimide form). (B) Signalling activity with Gail (his) labelled with TMR (maleimide form), and βlγ2 with Tb-mal. ³⁵S-GTP (0.2 nM) binding was assayed at 90 minutes. Data are the means of duplicate determinations.

Figure 28: Time resolved fluorescence resonance energy transfer (TR-FRET) between lOnM G ,n_ns labelled with Terbium chelate (Ga,ιι„_sTb) and WnM β4j2 labelled with Alexa-546 (β Alexa). A Victor3 was used to determine TR-FRET. The excitation and emission filters used were 340nm and 570nm, respectively. Following a 50μs delay, emission data were collected for a period of 1400μs. G-protein samples were diluted from 1-lOμM stocks in 50mM TRIS pH 8.0, lOmM MgCl₂, 50mM NaCl and ImM dithiothreitol to a final volume of lOOμl in black 96-well plates at 23°C. To demonstrate dissociation of subunits, 5μl unlabelled

was added (200nM final) as indicated by the vertical line. Background fluorescence at 570nm due to Gα,ihιsTb alone is shown (o). There was no significant background fluorescence of β₄γ₂ labelled with Alexa-546 under time-resolved conditions (data not shown).

Figure 29: Time resolved fluorescence resonance energy transfer (TR-FRET) between 3nM Go_ti_fus labelled with Terbium chelate (G ,ιι,_ιsTb) and 8nM β₄f₂ labelled with Alexa-546 γiAlexa) (•). A Victor3 was used to determine TR-FRET. The excitation and emission filters used were 340nm and 570nm, respectively. Following a 50μs delay, emission data were collected for a period of 1400μs. G-protein samples were diluted from 1-1 OμM stocks in 50mM TRIS pH 8.0, lOmM MgCl₂, 50mM NaCl and ImM dithiothreitol to a final volume of lOOμl in black 96-well plates at 23°C. To demonstrate dissociation of subunits, 5μl unlabelled β γ₂ was added (200nM final) as indicated by the vertical line. Background fluorescence at 570nm due to Gαα_hi_sTb alone is shown (o). There was no significant background fluorescence of β₄γ₂ labelled with Alexa-546 under time-resolved conditions (data not shown).

Figure 30: Time resolved fluorescence resonance energy transfer (TR-FRET) between 50nM Gauhi labelled with Terbium chelate (G ,i_hj_STb) and 50nM β4/2 labelled with Alexa-546 (β₄γ2Alexa) (•). A Victor3 was used to determine TR-FRET. The excitation and emission filters used were 340nm and 570nm, respectively. Following a 50μs delay, emission data were collected for a period of lOOμs. G-protein samples were diluted from l-10μM stocks in 50mM TRIS pH 8.0, lOmM MgCl₂, 50mM NaCl and ImM dithiothreitol to a final volume of lOOμl in black 96-well plates at 23°C. A1F₄ ^" (■) was added (as indicated by the vertical line) to dissociate subunits. Background fluorescence at 570nm due to Gααω_sTb (o) or Gαi_lhi_STb + A1F₄ ^" (D) is shown. There was no significant background fluorescence of β₄γ₂Alexa under time-resolved conditions (data not shown). Data are shown in duplicate.

Figure 31 : Time resolved fluorescence resonance energy transfer (TR-FRET) between 50nM Gau_hi_s labelled with Alexa-546 (G u_htsAlexa) and lOnM β4γ2 labelled with Terbium chelate (β₄γ_∑Tb) (•). A Victor3 was used to determine TR-FRET. The excitation and emission filters used were 340nm and 570nm, respectively. Following a 50μs delay, emission data were collected for a period of lOOμs. G-protein samples were diluted from l-10μM stocks in 50mM TRIS pH 8.0, lOmM MgCl₂, 50mM NaCl and ImM dithiothreitol to a final volume of lOOμl in black 96-well plates at 23 °C. A1F ^" was added to dissociate subunits, as indicated by the vertical line. Background fluorescence at 570nm due to β₄γ₂Tb (o) or β₄γ₂Tb + A1F₄ ^" (D) is shown. There was no significant background fluorescence of Gαπ_hi_sAlexa under time-resolved conditions (data not shown). Data are shown in duplicate.

Figure 32: Time Resolved Resonance Energy Transfer between GocuhisTb (the concentrations as indicated next to each plot) + β₄γ₂Alexa. K S (nM) for each concentration are shown in parentheses. The excitation and emission filters used were 340nm and 572nm, respectively. Following a 50μs delay, emission data were collected for a period of 900μs. TMND (50mM TRIS pH 8.0, lOmM MgCl₂, 50mM NaCl and ImM dithiothreitol) was dispensed to mix G-protein subunits to a final volume of 1 OOul. Experiments were carried out in triplicate. Figure 33: Time resolved fluorescence resonance energy transfer (TR-FRET) between 5 OnM G uhis labelled with Terbium chelate (Gau_hisTb) and l50nMRGS4 (his) labelled with Alexa546 (RGS4_hisAlexa) (•) or 50nM β₄γ labelled with Terbium (β Y₂Tb) and RGS4hisAlexa (A.). A Victor3 was used to determine TR-FRET. The excitation and emission filters used were 340nm and 570nm, respectively. Following a 50μs delay, emission data were collected for a period of 900μs. G-protein or RGS4_hi_S samples were diluted from l-10μM stocks in 50mM TRIS pH 8.0, lOmM MgCl₂, 50mM NaCl and ImM dithiothreitol to a final volume of lOOμl in black 96-well plates at 23°C. To demonstrate dissociation of labelled RGS4hi_s and Gauhis (■), 5μl unlabelled Gauhis was added (lOOOnM final) as indicated by the vertical line. Background fluorescence at 570nm due to Gαα_hisTb (o) or (β₄γ₂Tb) (Δ) is shown. There was no significant background fluorescence of RGS4_hi_SAlexa under time-resolved conditions (data not shown).

Figure 34: Time Resolved Fluorescent Resonance Energy Transfer (TR-FRET) between RGS4ι,i_sAlexa (concentrations as indicated next to each plot) + 50nM Gau_htsTb after a 5 minute incubation period. The excitation and emission filters used were 340nm and 572nm, respectively. Following a 50μs delay, emission data were collected for a period of 900μs. G-protein or RGS4hi_S samples were diluted from 1- lOμM stocks in 50mM TRIS pH 8.0, lOmM MgCl₂, 50mM NaCl and ImM dithiothreitol to a final volume of lOOμl in black 96-well plates at 23 °C. Experiments were carried out in triplicate.

Figure 35: Time resolved fluorescence resonance energy transfer (TR-FRET) between 70nM RGS4]_lisAlexa and 40nM Gau if b in the inactive GDP bound state (L), in the activated GTPyS bound state ( ) and in the transitional state mimicked in the presence of aluminium fluoride (a). A Victor3 was used to determine TR-FRET. The excitation and emission filters used were 340nm and 570nm, respectively. Following a 50μs delay, emission data were collected for a period of 900μs. TMND (50mM TRIS pH 8.0, lOmM MgCl₂, 50mM NaCl and ImM dithiothreitol) was dispensed to mix G- protein subunits to a final volume of lOOul. Experiments were carried out in triplicate.

Figure 36: Comparison of rate constants between G u_htsTb and 70nM RGS4hisAlexa in the in the presence of GTPγS. The rate constants of association between 40nM Gαπ_hi_sTb and 70nM RGS4_hi_S Alexa in the in the presence of GTPγS (active state) was

0.80 ± 0.07 min^"1, this was significantly slower than in the GDP-bound (inactive state) that had a rate of 1.36 ± 0.13 min^"1 (p<0.05). A1F₄ ^" (transition state) increased the rate of Gα:RGS4 binding to 2.67 ± 0.12 min^"1. This was significantly greater compared to when GTPγS or GDP was present. (p<0.05). Experiments were carried out in triplicate.

Detailed Description of the Preferred Embodiments

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in spectroscopy, drug discovery, cell culture, and molecular genetics, described below are those well known and commonly employed in the art. Standard techniques are typically used for preparation of signal detection, recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, and lipofection). The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Lakowicz, J. R. Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983) for fluorescence techniques, which are incorporated herein by reference) which are provided throughout this document. Standard techniques are used for chemical syntheses, chemical analyses, and biological assays. As employed throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

"Agent" as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering (i.e., eliciting or inhibiting) or mimicking a desired physiological function of a GPCR.

"Agonist" as used herein refers to a molecule or substance that binds to or otherwise interacts with a receptor or enzyme to increase activity of that receptor or enzyme.

"Antagonist" as used herein refers to a molecule that binds to or otherwise interacts with a receptor or enzyme to inhibit the activation of that receptor or enzyme. "Inverse agonist" as used herein refers to a molecule that binds to or otherwise interacts with a receptor to inhibit the basal activation of that receptor or enzyme.

"Biosensor" refers to an analytical device incorporating a biological material intimately associated with or integrated within a physicochemical transducer or transducing microsystem, which may be optical, electrochemical, thermometric, piezoelectric or magnetic. The usual aim of a biosensor is to produce either discrete or continuous digital electronic signals which are proportional to a single analyte or a related group of analytes.

A "cell free" complex" refers to a complex that is not located in the membrane of a functional cell. Preferably the complex is located in a portion of the membrane of the cell from which it is isolated.

A "complex" of the present invention may include one or more G-protein subunits and optionally may also include a GPCR.

"Comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

"Dye" refers to a molecule or part of a compound that absorbs specific frequencies of light, including but not limited to ultraviolet light. The terms "dye" and "chromophore" are synonymous.

As used herein, an "energy donor" is a molecule that, upon absorbing light, can transfer excitation energy to an energy acceptor or a quencher. This energy transfer can occur when the absorption spectrum of an energy acceptor overlaps the emissions spectrum of the energy donor. Other mechanisms also allow energy transfer when the acceptor is a quencher. In both cases, the light emitted by the donor is quenched. However, if the excitation energy is transferred to an acceptor, the acceptor will emit light at its own characteristic emission wavelength spectrum, whereas if the energy is transferred to a quencher, there is no secondary light emission. An "energy acceptor" is a molecule that can accept excitation energy transferred by an energy donor and use the transferred energy to emit light at its own characteristic emission wavelength spectrum.

As used herein, a "quencher" is a molecule that can accept energy from an energy donor, thereby reducing emitted signal of the donor.

"G-protein coupled receptors" and "GPCRs" as used interchangeably herein include but are not limited to all subtypes of the opioid, muscarinic, dopamine, adrenergic, adenosine, rhodopsin, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P and substance-R receptors, melanocortin, metabotropic glutamate, or any other GPCR known to couple via G-proteins. This term also includes orphan receptors which are known to couple to G-proteins, but for which no specific ligand is known. The term also encompasses biologically active or immunogenic fragments of GPCRs that are suitable for use in assays for detecting GPCR activity.

"G-protein subunit" as used herein can refer to any of the three subunits, alpha, beta or gamma that form the heterotrimeric G-proteins. The term also refers to a subunit of any class of G-protein, e.g., Gs, Gi /Go, Gq and Gz, and also includes recombinant G- protein constructs such as promiscuous and chimeric G-proteins. Representative G proteins include but are not limited to G_q.alpha., G_q-beta., G_q-gamma., Gπ.alpha., G_12/ι₃. alpha., G_12/13-beta., Gum-gamma., Gj-alpha., Gj.beta., Gj-gamma., G_s.alpha., G_s.beta., Gs-gamma., G-alpha₁₄ and G-alpha₁₆, various G-beta-gamma. dimers, and combinations thereof. In addition, recitation of a specific subunit (e.g., G alpha) is intended to encompass that subunit in each of the different classes, unless the class of G-protein is specifically otherwise specified.

"Ligand" as used herein refers to a naturally occurring or synthetic compound that binds to a protein receptor. Upon binding to a receptor, ligands generally lead to the modulation of activity of the receptor. The term is intended to encompass naturally occurring compounds, synthetic compounds and/or recombinanfly produced compounds. As used herein, this term can encompass agonists, antagonists, and inverse agonists.

The term "protein that normally interacts with a G-protein subunit" means a protein that is normally associated with the G-protein subunit, as the G-protein subunit exists in the cell. Such proteins can be those that permit the G-protein subunit to mediate its various biological activities, and can also be those having roles that have not been clearly implicated in GPCR activity, yet are found in close spatial proximity to a G-protein subunit at a given point in time. Examples of proteins that normally interact with G- protein subunits are RGS and AGS proteins.

The term "receptor" as used herein refers to a protein normally found on the surface of a cell which, when activated, leads to a signaling cascade in a cell.

The terms "tethered", "tethering" and the like as used herein refer to a physical or chemical modification of a biological component that leads to attachment of the component to a support.

Signal detection

In a preferred embodiment of the invention, the signal associated with a change in at least one G-protein is detected by a proximity based assay. A "proximity based" assay refers to an assay that employs an energy donor and an energy acceptor, wherein donors and acceptors are understood to mean members of a class of biological or chemical substances which can interact with one another within spatial vicinity, such as for example photosensitizers and chemiluminescers, photosensitizers and fluorophores, or fluorophores and fluorophores, or fluorophores and quenchers. The transfer of energy may be by means of light, electron radiation or non-radiative transfer and also by reactive chemical molecules.

A variety of proximity based assays have been developed based on the quenching, alteration or amplification of fluorescence to detect and measure the presence of an analyte of interest. With respect to fluorescence quenching as a measurement method, for example, it is known in the art that the fluorescence of a fluorochrome is quenched when a dye that quenches the light emitted by the fluorochrome is about 50 angstroms or closer to the fluorochrome (the spatial vicinity of influence is defined with reference to the Forster radius which differs between respective FRET pairs and is known to be influenced by the sixth power of the difference in distance of FRET pairs). These assays operate on the principle that when the dye is sufficiently close to the fluorochrome, energy emitted from the excited fluorochome is transferred to the nearby dye which results in a measurable alteration in the amount of the net magnitude of the light that can be detected. Examples of fluorescence signal detection methods such as FRET (Stryer, L. Ann Rev. Biochem. 47, 819-846, 1978), fluorescence cross- correlation spectroscopy ("FCCS") (Maiti et al, Proc. Nat'l Acad Sci USA 94, 11753- 11757, 1997) flow cytometry ((Hart and Greenwald, Molecular Immunology 16:265- 267, 1979; U.S. Pat. No. 4,658,649), luminiscence resonance energy transfer (LRET) (Mathis, G. Clin. Chem. 41, 1391-1397, 1995), direct quenching (Tyagi et al, Nature Biotechnology 16, 49-53, 1998), ground-state complex formation (Packard, B.Z., Toptygin, D. D., Komoriya, A., and Brand, L. Biophys. Chem. 67, 167-176, 1997), chemiluminescence energy transfer (CRET) (Campbell, A. K., and Patel, A. Biochem. J. 216, 185-194, 1983), bioluminiscence resonance energy transfer (BRET) (Xu, Y., Piston D. W., Johnson Proc. Natl. Acad. Sci., 96, 151-156, 1999), or excimer formation (Lakowicz, J. R. Principles of Fluoroscence Spectroscopy, Kluwer Academic/Plenum Press, New York, 1999) are compatible with the design of the assay.

FRET is the phenomenon whereby a fluorescent molecule - the donor - transfers energy by a nonradiative mechanism to a neighbouring chromophore - the acceptor. The absorption spectrum of the acceptor chromophore must overlap with the fluorescence emission spectrum of the donor. When the spectral overlap is more extensive, the efficiency of the FRET process will be higher and consequently, FRET can occur over longer distances. FRET is highly dependent on the proximity between the donor and acceptor, and, in general, only occurs when the molecules are separated by <100 A. It is characterized by the Fδrster radius (Ro). Ro values for common donor-acceptor pairs usually vary between 30 and 60 A and can be determined experimentally. They depend on the spectral overlap, relative orientation of the donor and acceptor chromophores, and the quantum yield of the donor fluorophore.

The degree of FRET can be determined by any spectral or fluorescence lifetime characteristic of the excited donor moiety. For example, intensity of the fluorescent signal from the donor, the intensity of fluorescent signal from the acceptor, the ratio of the fluorescence amplitudes near the acceptor's emission maxima to the fluorescence amplitudes near the donor's emission maximum, or the excited state lifetime of the donor can be monitored.

Preferably, changes in the degree of FRET are determined as a function of the change in the ratio of the amount of fluorescence from the donor and acceptor moieties, a process referred to as "ratioing". Changes in the absolute amount of indicator, excitation intensity, and turbidity or other background absorbances in the sample at the excitation wavelength affect the intensities of fluorescence from both the donor and acceptor approximately in parallel. Therefore the ratio of the two emission intensities is a more robust and preferred measure of disassociation than either intensity alone.

The donor moiety may be excited by light of appropriate intensity within the excitation spectrum of the donor moiety. The donor moiety preferably emits the absorbed energy as fluorescent light. When the acceptor fluorescent protein moiety is positioned to quench the donor moiety in the excited state, the fluorescence energy is transferred to the acceptor moiety which may emit fluorescent light. FRET can be manifested as a reduction in the intensity of the fluorescent signal from the donor moiety, reduction in the lifetime of the excited state of the donor moiety, or emission of fluorescent light at the longer wavelengths (lower energies) characteristic of the acceptor moiety. In the context of the present invention, when the conformation of one or more of the G- protein subunits changes upon activation via the GPCR, the fluorescent protein moieties come closer together (or physically separate), and FRET is increased (or decreased) accordingly.

Fluorescent molecules and quenchers are well known in the art, as are methods of binding fluorophores and quenchers to other molecules, for example, by coupling through active groups. Fluorophores and quenchers can also be indirectly bound to a target molecule, for example, through binding of a specific binding member that is coupled to a fluorophore.

In a preferred embodiment, the energy donor and acceptor moieties are fluorescent or luminescent donor and acceptor moieties. Preferred energy donor and acceptor are Terbium/Alexa546, Terbium/FlAsH-(EDT2), Terbium/fluorescein, Terbium/GFP, Terbium tetramethylrhodamine (TMR), Terbium/Cy3, Terbium/QSY-7, Terbium/R phycoerythrin, Europium/Cy5, Europium/Allophycocyanin (APC), Europium/Alexa 633, Alexa 488/Alexa 555, Alexa 568/Alexa 647, Alexa 594/Alexa 647, Alexa 647/Alexa 594, Cy3/Cy5, BODIPY FL/BODIPY FL, Fluorescein TMR, IEDANS/fluorescein, and fluorescein/fluorescein.

Other lanthanides which can be used as energy donors include Dysprosium and Samarium. These energy donors can be used in combination with acceptors such as Alexa dyes. Fluorescent polypeptides can also be used in the invention, including proteins that fluoresce due to either intramolecular re-arrangements or the addition of cofactors that promote fluorescence. For example, green fluorescent proteins of cnidarians, which act as their energy-transfer acceptors in bioluminescence, are suitable fluorescent proteins for use in the fluorescent indicators. A green fluorescent protein ("GFP") is a protein that emits green light, a blue fluorescent protein ("BFP") is a protein that emits blue light, and a yellow fluorescent protein ("YFP") is a protein that emits yellow light. GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renilla reniformis, and Phialidium gregarium. See, Ward et al., (1982) Photochem. Photobiol., 35, 803; and Levine et al, (1982) Comp. Biochem. Physiol., 72B, 77). Other fluorescent proteins can be used, such as, for example, yellow fluorescent protein from Vibrio flscheri strain Y-l, Peridinin-chlorophyll a binding protein from the dinoflagellate Symbiodinium sp.phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin, or oat phytochromes from oat reconstructed with phycoerythrobilin. These fluorescent proteins have been described in Baldwin et al., (1990) Biochemistry 29, 5509, Morris et al, (1994) Plant Molecular Biology, 24, 673, and Wilbanks et al, (1993) J. Biol. Chem. 268, 1226, and Li et al., (1995) Biochemistry 34, 7923.

Examples of additional fluorescent donor and acceptor moieties that may be used in the present invention are described in Wu and Brand (1994) Anal. Biochem. 218, 1.

As mentioned above, it is possible to use a fluorophore bound to a first component of the GPCR/G-protein complex that can be quenched by a fluorescence quencher bound to a second component of the GPCR/G-protein complex, or the first component of the GPCR/G-protein complex can comprise or bind a quencher and the second component used in the assay is bound to a donor fluorophore. Non-limiting examples of fluorescence quenchers that can be used in the methods of the present invention include DABCYL, DABSYL, QSY 7, QSY 9, QSY 21, and QSY 35.

One preferred FRET pair for use in the present invention is a Terbium-DPTA-csl24- EDA-fluor (available commercially from Panvera.com), coupled with an Alexa 546 fluor (available commercially from Molecular Probes.com) to report on molecular associations and/or rearrangements occurring at the level of the G-protein subunits consequent upon receptor stimulation. Fluors would have functionalised groups (e.g. isothiocyanate, succinimidyl ester, maleimide and the like) for conjugation to proteins. Terbium can be excited by light at 340nm. The emitted light, Stokes shifted to 550nm, may either be detected at that wavelength or may interact with adjacent Alexa 546 fluor attached to the other G-protein subset if, in the latter case, the molecular arrangement was conductive to such an interaction occurring via FRET and LRET. If such an interaction occurred, light would then be emitted from the Alexa fluor at a wavelength of 570nm.

Fluorescence in a sample is measured using a fluorometer or any other instrumentation that incorporates within a means to detect fluorescent radiation over a range of wavelengths. In general, excitation radiation, from an excitation source having a first wavelength, passes through excitation optics. The excitation optics cause the excitation radiation to excite the sample. In response, fluorescent moieties on the protein sample emit radiation which have a wavelength that is different from the excitation wavelength. Collection optics then collect the emission from the sample. The device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned. According to one embodiment, a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed. The multi-axis translation stage, temperature controller, auto- focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer. The computer also can transform the data collected during the assay into another format for presentation.

Methods of performing assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y. -L., San Diego: Academic Press (1989), pp. 219- 243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjarnin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

In a preferred embodiment, the fluorescent probes are separately attached via amine or thiol linkages to either the G-alpha subunit or the G-beta/gamma subunit complex prior to reconstitution of the complete signalling complex. Alternatively, one member of the LRET/FRET pair may be attached to the GPCR under investigation and the other member of the pair attached to one of the G-protein subunits. In a further preferred embodiment, two or more different FRET pairs are attached to two or more different sets of the GPCR/G-protein complex components, thereby enabling analysis of the interaction between different sets of components simultaneously. For example, probes of a first FRET pair may be attached to the G- alpha subunit and the G-beta/gamma subunit; and probes of a second different FRET pair may be attached to the GPCR and the G-alpha subunit. This allows simultaneous analysis of the interaction between the (i) the G-alpha subunit and the G-beta/gamma subunit; and (ii) the GPCR and the G-alpha subunit or the G-beta/gamma subunit.

In a further preferred embodiment, a FRET pair of the type mentioned above could be attached to one of the G-proteins (particularly the G-alpha subunit) and to an RGS or AGS protein to determine the interaction between that RGS or AGS and the G-alpha subunit by LRET/FRET and to identify those compounds, drugs, products of combinatorial chemistry etc., which could influence or modulate this interaction.

In a further preferred embodiment, one FRET pair is attached to a G-alpha and to a G- beta/gamma dimer, and another FRET pair is attached to a G-alpha and a RGS or AGS protein to determine simultaneously the interaction between that RGS or AGS and the heterotrimeric G-proteins subunits by LRET/FRET and to identify those compounds, drugs, products of combinatorial chemistry etc., which could influence or modulate this interaction.

Luminescent lanthanide chelates have highly unusual spectral characteristics that make them useful non-isotopic alternatives to organic fluorophores, particularly where there are problems of background autofluorescence. Due to their relatively long luminescent emission lifetime resolved signals can be employed. They are also useful donors in fluorescence (luminescence) resonance energy transfer to measure nanometer conformational changes and binding events. Emission primarily arises from electric dipole transitions. Energy transfer is therefore termed lanthanide based or luminescent resonance energy transfer (LRET). LRET also provides the opportunity to undertake homogenous (no separation step) assays as opposed to heterogeneous assays requiring a separation step such as filtering.

Examples of suitable lanthanide/luminescence (LRET) pairs that can be used are described in Selvin (2002) Annu Rev Biophys Biomol Struct. 31:275-302. Donor:Acceptor pairs and RO (A): Terbium to fluorescein 45, Terbium to GFP (green fluorescent protein) 43.1, Terbium to tetramethylrhodamine (TMR) 57, Terbium to Cy3 61.2, Terbium to phycoerythrin 97.5, Europium to Cy5 55.2, Europium to allophycocyanin 71.

Preferably, LRET-based and FRET-based emission measurements are time-resolved (TR). Measurement of time-resolved resonance energy transfer can reduce the interference from background fluorescence or luminescence, for example, from the wells that contain the samples. When long enough delay times (time between pulsed excitation and starting of emission recording) can be used, all background interferences can be eliminated (for a review see. e.g. Hemmil (1991); Gudgin Dickinson et al, (1995) J PhotochemPhotobiol 27, 3).

In other embodiments of the present invention, fluorescence polarization may be detected. Fluorophores that can be directly or indirectly bound to a GPCR or G-protein subunit for fluorescence polarization detection include any fluorophores known in the art or later developed, for example, fluorescein, rhodamine, Alexa dyes, Cy dyes, TMR, JOE, FAM, TAMRA, BODIPY, pyrene, Europium or other lanthanide compounds, and fluorescent proteins such as phycoerythrin, phycocyanin, allophycocyanin, GFP and its derivatives.

Fluorescence polarization measurements provide information on molecular orientation and mobility and processes that modulate them, including receptor-ligand interactions, proteolysis, protein-DNA interactions, membrane fluidity and muscle contraction. Because polarization is a general property of fluorescent molecules (with certain exceptions such as lanthanide chelates), polarization-based readouts are somewhat less dye-dependent and less susceptible to environmental interferences such as pH changes than assays based on fluorescence intensity measurements. Experimentally, the degree of polarization is determined from measurements of fluorescence intensities parallel and perpendicular with respect to the plane of linearly polarized excitation light, and is expressed in terms of fluorescence polarization or anisotropy.

Methods for conducting fluorescence polarization measurements will be known to those skilled in the art and can be readily adapted for use in the present invention. The principle of the fluorescence polarization immunoassay was first described by W. B. Dandliker and G. A. Feigen, "Quantification of the Antigen- Antibody Reaction by the Polarization of Fluorescence", Biochem. Biophys. Res. Comm. 5:299 (1961).

Assays of the present invention

Methods for detecting or identifying G-protein activation through GPCRs are important for numerous applications in medicine and biology. The present invention provides methods that can be used for a number of different purposes including: (1) determining the function or activity of any given GPCR when stimulated by a cognate ligand (as depicted in Figure 2); (2) screening for new drugs affecting selected GPCRs which may include novel agonists, antagonists or inverse agonists (as depicted in Figure 3); (3) detecting GPCRs in a sample (as depicted in Figure 4); and (4) screening for new drugs affecting G-protein interactions which may include novel agonists, antagonists or inverse agonists (as depicted in Figure 5); . The basic assays described herein and variations thereof can also be used in other applications, as will be apparent to those skilled in the art upon reading the present application.

A significant advantage of the assays of the invention is that they can directly detect G- protein activation through GPCRs in a cell free environment either qualitatively or quantitatively, and thus are particularly amenable to high throughput screening of or using large numbers of GPCRs. This can be useful, for example, in determining the receptors activated by a particular drug or receptors that are activated upon exposure to a particular stimulus, such as an odor or taste (e.g., activation of olfactory GPCRs).

Another important advantage is that the assays can be performed in a homogeneous format. Homogeneous assays are single step procedures that require only the mixing of sample with reagents prior to incubation and the reading of results. Separation and washing steps are not required.

GPCR polypeptides, each of which encompasses biologically active or immunogenic fragments or oligopeptides thereof, can be used for screening compounds that affect GPCR receptor activity by, for example, specifically binding the GPCR and affecting its function, thereby affecting GPCR activity and G-protein activation.

Identification of such compounds can be accomplished using any of a variety of drug screening techniques. Of particular interest is the identification of agents that have activity in affecting GPCR function. Such agents are candidates for development of treatments for, conditions associated at least in part with GPCR activity. The screening assays of the invention are generally based upon the ability of the agent to bind to the GPCR polypeptide component of a cell free complex comprising a GPCR and at least one G-protein subunit, and thereby effect a detectable change in one of the G-protein subunits of the complex.

Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 40 and less than about 7,800 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably with at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, lipids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts (including extracts from human tissue to identify endogenous factors affecting GPCRs) are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. The invention also contemplates the use of competitive drug screening assays in which GPCR receptor-specific neutralizing antibodies compete with a test compound for binding of the GPCR polypeptide. In this manner, the antibodies can be used to detect the presence of any polypeptide that shares one or more antigenic determinants with a GPCR polypeptide.

A variety of other reagents may be included in the screening assays described herein. Where the assay is a binding assay, these include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding, protein-DNA binding, and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. Components that modify G-protein function, such as RGS and AGS proteins may also be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high- throughput screening. Typically between 0.1 and 1 hours will be sufficient.

Identification and Design of Therapeutic Compounds

A major asset of the invention is its ability to vastly increase, over current methods, the rate at which compounds can be evaluated for their ability to act as agonists, antagonists, and/or inverse agonists for GPCRs. As additional GPCR genes are identified and characterized, the activity of these receptors in response to various compounds, as well as to methods such as site directed mutagenesis, can be used to gain detailed knowledge about the basic mechanisms at work in these receptors. A fundamental knowledge of the basic mechanisms at work in these receptors will be of great use in understanding how to develop promising new drugs and/or to identify the fundamental mechanisms behind specific tastes, smells and the like.

Assays using the cell free complexes of the present invention can identify promising agonists, antagonists and/or inverse agonists for specific GPCRs. In an exemplary assay, the cell free complexes of the invention can be contacted with a candidate therapeutic, and the activity of the G-protein activation through the receptor measured, e.g., by measuring a signal associated with a change in a G-protein subunit of the complex. Agonists can be identified by their ability to increase GPCR activity over basal levels in the system. Antagonists can be identified by contacting the cell free complexes of the assay system with a native ligand or agonist and a candidate antagonist; compounds that inhibit the ability of the ligand or agonist to increase activity of the GPCR are identified as antagonists.

A number of G-protein coupled receptors have been shown to exhibit activity in the absence of agonist (so-called spontaneous, constitutive or basal activity). This agonist- independent basal activity can be inhibited by compounds previously considered to be antagonists (e.g., the antipsychotic drugs). Therefore, these compounds are inverse agonists rather than antagonists.

In an exemplary embodiment, cell free complexes are prepared in which two different G-protein subunits are labeled with Fluorescence Resonance Energy Transfer (FRET) or Luminescence Resonance Energy Transfer (LRET) probes. These complexes are then exposed to a candidate GPCR binding compound and the effect of exposure to the compound monitored by detecting a change in the fluorescence or luminescence generated by the probes. A change in the fluorescence or luminescence generated by the probes is caused by a change in the positioning of the G-protein subunits within the complex. Candidate compounds that have agonist activity are those that, when contacted with the cell free complex, elicit a GPCR-mediated G-protein subunit rearrangement. Similarly, functional assays can be used to identify candidate compounds that block activity of a known GPCR agonist (e.g., block the activity of or compete with a known agonist), block activity of a known GPCR antagonist (e.g., block the activity of or compete with a known antagonist) and/or block the activity of a known GPCR inverse agonist.

In another embodiment, cell free complexes which do not contain GPCRs are prepared for the purpose of screening for agonists, antagonists and/or inverse agonists. In this embodiment, the cell free complex comprises at least one G-protein subunit that is labeled with an energy donor or energy acceptor.

In one particular example the complex comprises at least two G-protein subunits, more preferably the complex comprises a heterotrimeric G-protein complex comprising a G- alpha subunit and a G-beta-gamma dimer. In this example, the G-alpha subunit may be labeled with an energy donor and the G-beta-gamma dimer may be labeled with an energy acceptor. The candidate agent may then be exposed to this complex and any rearrangement or change in conformation in the G-protein complex caused by the candidate agent can be detected by a change in fluorescence or luminescence generated by the energy donor/energy acceptor pair. It will be appreciated that a rearrangement or change in conformation in the G-protein complex caused by the candidate agent indicates that the candidate agent is a potential modulator of GPCR signalling.

In another example, the cell free complex comprises only a G-protein subunit (for example, a G-alpha subunit) which is labeled with an energy donor. A protein such as an RGS or AGS, labeled with an energy acceptor, is then exposed to the complex under conditions that allow interaction between the G-protein subunit and the RGS or AGS. The candidate agent may then be exposed to this complex and any change in the interaction between the G-protein subunit and the RGS or AGS caused by the candidate agent can be detected by a change in fluorescence or luminescence generated by the energy donor/energy acceptor pair. It will be appreciated that a change in the interaction between the G-protein subunit and the RGS or AGS caused by the candidate agent indicates that the candidate agent is a potential modulator of GPCR signalling.

As many more GPCRs with constitutive activity are found (including native as well as mutated receptors), a newly recognized class of drugs termed inverse agonists are becoming increasingly important therapeutic agents. Inverse agonism is demonstrated when a drug binds to a receptor that exhibits constitutive activity and reduces this constitutive activity.

The assays of the present invention using cell free complexes are particularly well suited to identify promising inverse agonists for specific receptors, as they provide a direct method for detecting a decrease in GPCR activity in response to a compound. In an exemplary assay, cell free complexes can be contacted with a candidate inverse agonist, and the activity of the G-protein activation measured, e.g., by measuring binding of GTP analogs, or rearrangement of G-protein subunits within the complex. Compounds that decrease the basal activity of the receptor in the assay are identified as inverse agonists, and may then be subject to further testing for therapeutic use.

As will be readily understood by those skilled in this field the methods of the present invention provide a rational method for designing and selecting compounds including antibodies which interact with and modulate the activity of GPCRs. In the majority of cases these compounds will require further development in order to increase activity. It is intended that in particular embodiments the methods of the present invention includes such further developmental steps. For example, it is intended that embodiments of the present invention further include manufacturing steps such as incorporating the compound into a pharmaceutical composition in the manufacture of a medicament.

Accordingly, in a preferred embodiment , the method further comprises formulating the identified compound for administration to a human or a non-human animal as described herein.

The present invention clearly encompasses the use of any analytical method and/or industrial process for carrying the screening methods described herein into a pilot scale production or industrial scale production of a compound identified in such screens. This invention also provides for the provision of information for any such production. Accordingly, the present invention also provides a process for identifying or determining a modulator of GPCR signalling, said method comprising: (i) performing a method as described herein to thereby identify or determine a compound or modulator; (ii) optionally, determining the structure of the compound or modulator; and (iii) providing the compound or modulator or the name or structure of the compound or modulator such as, for example, in a paper form, machine- readable form, or computer-readable form.

Naturally, for compounds that are known albeit not previously tested for their function using a screen provided by the present invention, determination of the structure of the compound is implicit in step (i). This is because the skilled artisan will be aware of the name and/or structure of the compound at the time of perfoπning the screen.

As used herein, the term "providing the compound or modulator" shall be taken to include any chemical or recombinant synthetic means for producing said compound or modulator or alternatively, the provision or a compound or modulator that has been previously synthesized by any person or means. In a preferred embodiment, the compound or modulator or the name or structure of the compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.

The present invention also provides a process for producing a modulator of GPCR signalling, said method comprising: (i) performing a method as described herein to thereby identify or determine a compound or modulator; (ii) optionally, determining the structure of the compound or modulator; (iii) optionally, providing the name or structure of the compound or modulator such as, for example, in a paper form, machine-readable form, or computer- readable form; and (iv) producing or synthesizing the compound or modulator.

In a preferred embodiment, the synthesized compound or modulator or the name or structure of the compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.

Identification of Ligands for Orphan GPCRs

An assay system according to the invention can also be used to classify compounds for their effects on G-protein coupled receptors, such as on orphan receptors, to identify candidate ligands that are the native ligands for these orphan receptors. Cell free complexes comprising orphan GPCRs can be exposed to a series of candidate ligands, and the ligands with the ability to activate signaling through the receptors can be identified through changes in the G-protein subunits of the complex, eg. by measuring rearrangement of G-protein subunits within the complex.

Assays to Determine the Presence of a GPCR-activating agents in a Sample

The present invention also provides a rapid and reliable method for determining G- protein activation through receptors known to be involved in drug responses. The present invention thus can also be used to test for drugs, e.g., narcotics, e.g., cocaine, heroin, morphine or designer opiates in foods or bodily fluids, e.g., blood or urine. An exemplary assay for identifying a drug in a substance (e.g., an opioid) would comprise the steps of contacting a cell free complex of the invention (e.g., where the GPCR is an opioid receptor) with a sample suspected of containing the drug. The presence of the drug can be verified by detecting, either qualitatively or quantitatively, changes in the G-protein subunits of the complex, eg. by detecting rearrangement of G-protein subunits within the complex.

The present invention can also be used for detection of aromas or odorants that stimulate GPCRs. The assays of the present invention can therefore be applied, for example, to detection of aromas in samples of food and wine or to the detection of odors that may be released by patients suffering from diseases.

Assays to Determine the Presence of a GPCR in a Sample

In another aspect the present invention provides a method for identifying or determining the levels of a GPCR in a sample. In this aspect, combinations of G- protein subunits are used to capture GPCRs in a sample. Detection of the captured GPCR is then achieved by interrogation with a known ligand.

Until recently, 17 alpha, 5 beta and 13 gamma subunits had been identified. This allows the production of at leastl755 distinct permutations of G-protein heterotrimeric complexes to use in methods of the present invention. These G-protein heterotrimeric complexes can be tethered to a support to effectively provide "hooks" or "baits" for capturing different types of GPCRs. Once caught by a degree of specific binding, the captured GPCR can then be interrogated with a range of ligands for identification. For example, a known ligand would identify its cognate, captured receptor via signalling through the G-proteins as per the invention. This aspect of the invention therefore allows detection of changes in GPCR levels and functionality as diagnost c biomarkers for human health and disease.

In one embodiment, the GPCRs expressed on a variety of cells are obtained from a biological fluid (or even biopsy material) by isolating cells (for example, by differential centrifugation) and performing a detergent extraction in order to solubilise membrane associated GPCRs. A urea step is then performed to remove intrinsic G-proteins leaving the "naked" GPCR available to bind to a multi-addressable, heterotrimeric G- protein array. Identification of GPCRs involved in Various Biological Processes

The GPCRs that are involved in biological responses, both normal responses (e.g., taste, smell, etc.) and pathological responses (e.g., the biological response to a GPCR involved in a disease or disorder) can be determined using assays of the invention. An assay using an array of cell free complexes of the invention, each sample of the array comprising a cell free complex with a particular GPCR, can be exposed to a particular stimulus (e.g., the odor, flavor compound, disease related complex, and the like), and the activity of each sample of the array can be determined. This can identify multiple receptors in a high throughput manner that are involved in the transduction of signals in response to that particular stimulus.

For example, the high throughput assays of the invention can be especially useful in determining the spectrum of GPCRs, e.g., olfactory receptors, that are activated or inverse agonized by a specific substance or mixture of substances. For example, a liquid can be contacted with an array of cell free complexes comprising particular GPCRs, and the GPCRs activated or suppressed can be identified.

Microarrays

The basics and theoretical considerations of protein microarrays were described in the 1980's by Roger Ekins and colleagues. (Ekins R.P., J Pharm Biomed Anal 1989. 7: 155; Ekins R.P. and Chu F.W., Clin Chem 1991. 37: 1955; Ekins R.P. and Chu F.W., Trends in Biotechnology, 1999, 17, 217-218), incorporated herein by reference.

More recently protein microarrays- protein spots printed on a glass have been described, G. MacBeath and S.L. Schreiber, Printing Proteins as Microarrays for High- Throughput Function Determination, Science 2000 September 8; 289(5485): p. 1760- 1763. Proteomics 2002 2:48-57 Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology. Weng S, Gu K, Hammond PW, Lohse P, Rise C, Wagner RW, Wright MC, Kuimelis RG. Phylos, Lexington, MA 02421, USA. An mRNA-protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion process), link phenotype to genotype and enable powerful directed evolution schemes. Microarray platform

The complexes of the invention may be immobilised on any of a number of commercially available array platform Hydrogel® based array system on glass slides as a commercially available array platform for this project; however the arraying of the complexes is not limited to the hydrogel covered chip. Proteins are printed onto the HydroGel substrate are captured within the porous structure. Proteins added or captured are kept in their native form and are freely accessible for assay binding reaction. No modifications to the proteins or oligonucleotides are required prior to immobilization, insuring protein integrity and assay dependability.

Uses

GPCR ligands (which typically involve hormones, neurotransmitters, chemokines and the like) are involved in many physiological and pathophysiological processes associated with but not limited to the cardiovascular system, the central nervous system, the endocrine system, the gastrointestinal system and in inflammation, immunity and cancer, (see Table 1). For example, seventeen or more distinct GPCR ligands profoundly modulate cardiovascular activity by interacting with their respective cognate receptors. The cell free complexes of the present invention enable rapid and efficient measurement of these compounds (ligands) for diagnostic purposes.

The nearly 200 functionally known GPCRs directly or indirectly account for nearly 60% of all currently-prescribed prescription drugs generating over $60 billion in sales annually. Members of this therapeutically prolific and valuable superfamily of proteins (receptors) are ubiquitous in human physiology and play vital roles in the most fundamental processes of cells including biochemical communication, growth, differentiation, and survival. The present invention can be used in the discovery of human biomarkers of disease as well as determining therapeutic products targeting known GPCRs and orphan GPCRs.

In addition, GPCR ligand assays of the present invention have potential applications in areas such as:

Foods, beverages and cosmetics (e.g., human sensory GPCRs; biosensors) Diagnostic applications (e.g., detection of GPCR-based ligands) Environmental biosensors (e.g., detection of GPCR-based toxins) Agrichemical products (e.g., plant & insect GPCRs) Veterinary medicines (e.g., animal GPCRs).

Since many of the natural ligands of GPCRs are hormones, their concentrations in body fluids (e.g., serum samples) are often measured as a diagnostic aid. Concentrations outside of "normal limits" may suggest hypo- or hyper-secretive conditions (e.g., hyperparathyroidism). In addition, serum concentrations of many GPCR-based drugs (e.g., theophylline) are routinely monitored to individualize drug dosage adjustments and maintain therapeutic levels while avoiding toxic levels. Serum concentrations are also measured to confirm cases of suspected toxicity or poisoning. In addition, many GPCR ligands differ significantly in their chemical composition (eg biogenic amine, peptide, lipid etc.) necessitating a wide range of techniques for their individual analysis and detection. Currently, the assays used by clinical laboratories to measure such ligands are time-consuming and expensive (e.g., RIA, ELISA, etc.), and their specificity is often limited (i.e., cross-reactive with other drugs or hormones). The compositions and methods of the invention provide a more rapid and cost-effective approach.

The present invention may be adapted for use in a critical care, point of care instrument. Critical care refers to the treatment of patients with acute, life-threatening illness or injury. Care may be provided at the scene of an accident, in an ambulance, in a hospital trauma center or emergency room. Critical care is provided by multidisciplinary teams of healthcare professionals - physicians, nurses, respiratory care technicians and other health personnel. For example, Ambri has developed the SensiDxTM System to ensure that these professionals have a method of critical care testing that will provide fast, accurate and reliable results within minutes. The present invention may be adapted to a similar format.

Animal GPCRs, depending upon their conservation and function, are clearly targets for new and improved veterinary products. Agrichemical applications in crop protection could derive from targeting the GPCRs of insects, fungi, and algae (i.e., new pesticides), whereas the G-protein linked processes that regulate plant growth could yield improvements in food production.

The present invention is further described by the following non-limiting Examples. EXAMPLES

Materials and Methods

Cell culture and protein expression

Sf9 (Spodoptera frugiperdά) insect cells (Invitrogen, Mount Waverley, Vic, Australia) were grown in Sf-900 II SFM serum-free media (Invitrogen) and maintained at 28°C with agitation at 140 oscillations/min in an orbital shaker. Recombinant baculo virus samples encoded either the recombinant porcine α2A-adrenergic receptor (α2A-AR) or the G-protein subunits Gail or Gail (his) or βl or β4 or γ2 or γ2(his) or the recombinant muscarinic M₂ receptor (M₂R). For expression of G-proteins or M₂R or α2A-AR, Sf9 cells (at 2x10⁶ cells/ml) were infected with the appropriate combinations of Gail (± His6-tagged) and βlγ2 (± His6-tagged) or M₂R or α2A-AR baculo virus(es) at a multiplicity of infection ratio of 1-2 and harvested at 72 h. Cell viability following 72 h infection was approximately 40-70% as determined by trypan blue staining.

Membrane preparation

Infected Sf9 cells were collected and centrifuged at lOOOxg for 10 min and suspended 125 mL ice-cold "lysis buffer" (50 mM HEPES pH 8.0, 0.1 mM EDTA, pH 8, 3 mM MgCl₂, 10 mM β-mercaptoethanol) with protease inhibitors; 0.02 mg/ml phenylmethyl sulfonyl fluoride (PMSF), 0.03 mg/ml benzamidine, 0.025 mg/ml bacitracin, 0.03 mg/ml Lima bean trypsin inhibitor. Cells were subjected to N2 cavitation at 500 psi (3400 kPa) for 15 min, followed by sedimentation of nuclei and unbroken cells (750xg, 10 min). Membranes were isolated by centrifugation of the supernatant at 100,000xg for 30 min at 4°C.

Preparation of urea-treated membranes

A modification of the following method was used to remove endogenous G-proteins from Sf9 membranes expressing M₂R or α2A-AR (Lim & Neubig, 2001, Biochem. J. 354:337-344). The 100,000xg membrane pellet was resuspended (50 ml per 1 L of original infected Sf9 cell culture) in "incubation buffer" (250 mM Sucrose, 10 mM Tris pH 8.0, 3 mM MgCl₂) containing 7 M urea and protease inhibitors. After 30 min incubation on ice, membranes were diluted to 4 M urea with "incubation buffer" and protease inhibitors and centrifuged at 100,000xg at 4°C for 30 min. The urea-treated membrane pellet was washed twice in "incubation buffer". The final urea-treated membrane pellet was resuspended to approximately 1-3 mg/ml protein and rapidly frozen in liquid N2 and stored at -80°C until use. Confirmation and quantification of α2A-AR expression was done by [³H] MK912 (Perkin Elmer Life Sciences Inc., Boston, MA, USA) binding. Confirmation and quantification of M₂R expression was done by [³H]-Scopolamine (Perkin Elmer Life Sciences Inc., Boston, MA, USA) binding.

Fluorescent Probes

The following fluorescent probes were purchased from Invitrogen (Mount Waverley, Vic, Australia, as agent for Molecular Probes), Alexa Fluor 546 carboxylic acid, succinimidyl ester; Alexa Fluor 546 C₅ maleimide; and TMR (tetramethylrhodamine) maleimide. The carbostyril-124-diethyletriamine-pentaacetic acid - based Terbium (Tb) chelates as the amine-reactive form, CS124-DTPA-Phe-NCS (Tb-NCS), and the thiol reactive form, CS124-DTPA-EMCH (Tb-mal) were from Invitrogen (Mount Waverley, Vic, Australia as agent for PanVera Corp).

Yohimbine was from Sigma Australia (Castle hill, NSW, Australia). All other compounds were as described elsewhere herein.

Baculovirus Clones/Vectors

G-protein subunits including Gαπ_hi_s, β₄ and γ₂ and α_2A-AR were from baculovirus cDNA clones expressed in the Sf9 insect cell system. RGS4hi_S was expressed from pQE60 (Qiagen, Australia) in M15[ρREP4] E.coli induced with ImM IPTG for 3 hours.

Purification ofG-proteins/Conjugation to fluorescent probes

For conjugation to G-proteins, the following was performed at 4°C unless otherwise indicated. A modification of the following method was used (Kozasa & Gilman, 1995, J Biol. Chem. 270:1734-1741). Frozen membranes (at > 5 mg/ml protein) containing combinations of G-protein α and βγ subunits were thawed and diluted to 5 mg/ml protein with "wash buffer" (50 mM HEPES pH 8.0, 3 mM MgCl₂, 10 mM β- mercaptoethanol, 50 mM NaCl and 10 μM GDP) containing fresh protease inhibitors and 1% (w/v) cholate (final concentration). Membranes were stirred on ice for 1 hr to extract. The sample was then centrifuged at 100,000xg for 40 min and the supernatant collected (termed membrane extract) and diluted 5-fold with buffer A (20 mM HEPES, 100 mM NaCl, 1 mM MgCl₂, 10 mM β-mercaptoethanol, 0.5% (w/v) polyoxyethylene- 10-lauryl ether and 10 μM GDP.

The sample was then loaded onto a 1 ml nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen Pty Ltd, Clifton Hill, Vic, Australia) and allowed to pass through (under gravity) and then the sample was collected and re-applied to the column. The column containing His6-tagged proteins was washed with buffer A (at a volume equating to 5% of the original cell volume) containing 5 mM imidazole and 300 mM NaCl. The column was washed with 50 ml of buffer A (minus the β-mercaptoethanol for G- proteins labelled with fluorescent labels of the maleimide form) to remove imidazole (and remove β-mercaptoethanol for the malemide label). The column was warmed to 22°C (room temperature) for 15 min and a five-fold molar excess of the fluorescent label in buffer A (minus β-mercaptoethanol for the fluorescent labels of the maleimide form) was added. The column containing the labelled G-proteins was incubated at room temperature for 2 hr with occasional mixing. The non His6-tagged G-protein subunits eluted with 1 ml fractions of buffer E (20 mM HEPES pH 8.0, 50 mM NaCl, 10 mM β- mercaptoethanol, 10 μM GDP, 1 % (w/v) cholate, 50 mM MgCl₂, 5 mM imidazole, 10 mM NaF and 30 μM A1C1₃. The remaining His6-tagged G-protein subunits were eluted from the nickel column with buffer E containing 150 mM imidazole. Protein expression was confirmed by SDS-polyacrylamide gel electrophoresis and compared with known molecular weight protein standards. Elution fractions containing the appropriate G- protein subunit were pooled (to < 3ml) and dialysed against 5 x 200 ml buffer F (20 mM HEPES pH 8.0, 3 mM MgCl₂, 10 mM NaCl, 10 mM β-mercaptoethanol, 1 μM GDP and 0.1% (w/v) cholate) using a Slide-a-lyzer (Pierce Chemical Company, Rockford, IL, USA). Following dialysis, the G-protein subunits were stored at -80°C in buffer F or in 50% glycerol in buffer F. Final protein concentration was determined by SDS-polyacrylamide gel electrophoresis of G-protein subunits followed by laser scanning densitometry.

For RGS4_his labelling using Alexa Fluor 546 C₅ maleimide, a procedure similar to that described above was carried out on E.coli lysates, except no detergents were required. Reconstitution and capture of M₂R or a2A-AR/G-protein signalling complex (transductosome)

Membranes expressing α2A-AR were diluted approximately 2-3 fold (to give 0.8 mg protein/ml) with ice-cold reconstitution buffer (50 mM Tris pH 7.6, 100 mM NaCl, 10 mM MgCl₂, 1 mM DTT and 0.5 μM GDP). After addition of the indicated amounts of G-protein subunits in buffer F, the reaction mix was incubated on ice for 60 min. For capture of the α2A-AR/G-protein complex (termed transductosome), α2A-AR membranes at 0.8 mg/ml with reconstituted G-proteins (+His6 tag) were added to the indicated amounts of Ni-NTA beads (45 - 165 μm diameter in a 50% solution of reconstitution buffer) for a further 60 min on ice. To initiate ³⁵S-GTP binding, samples were transferred to 26°C and after 5 min various agonists and antagonists (clonidine, UK14303 ("UK") and rauwolscine (from Sigma, Castle hill, NSW, Australia) were added (final protein was 0.4 mg/ml). In contrast, there was no preincubation of the M₂R/G-protein complex and Ni²⁺-NTA beads. To initiate ³⁵S-GTP binding, samples were transferred to 28°C and after 5 min, various agonists (carbachol or acetylcholine) and antagonists (atropine or pirenzepine) were added. Samples were filtered over GF/C's or Whatman No.l filters following 90 min reaction. Binding of GTPγS35 (³⁵S- GTP, Perkin Elmer Life Sciences Inc., Boston, MA, USA) at 0.2 nM (final) was determined following filtration through GF/C filters (transductosome without Ni-NTA beads) or Whatman No.l filters (transductosome with Ni-NTA beads) and two 3 ml washes with ice-cold 20 mM Tris pH 7.4, 25 mM MgCl₂ and 100 mM NaCl. Filters were air-dried and then counted by liquid scintillation counting.

Fluorescence Measurements

Time-resolved, luminescent resonance energy transfer (TR-LRET) and other fluorescent measurements were performed using a Victor 3 (Perkin Elmer, Melbourne Australia) multilabel plate counter fitted with a 1500V Xenon Flash light source for time-resolved fluorometric absorbance measurements. For TR-LRET measurements involving Terbium containing compounds conjugated to various G-protein subunits, excitation was at 340nm with emission being measured at 550 nm and 572 nm with a 500μs delay and 1400μs counting duration. The time-resolved excitation/emission was gated as indicated in the results. Fluorescence measurements of Alexa 546 and TMR G- protein conjugates by themselves were made at an excitation of 485nm and an emission of 570nm with no time gating in place. Signalling of reconstituted a2A-AR receptor/ fluorescent labelled G-protein subunit complex using the S-GTP binding assay

Membranes expressing α2A-AR were reconstituted with his-tagged or non his-tagged Gail and βlγ2 (or β4γ2 where indicated) in a buffer containing 50 mM Tris pH 7.6, 100 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 5 μM GDP and lOμM AMP-PNP. Either one or both of the G-protein subunits were fluorescent labelled as detailed in the figure legends. The final concentration in the reconstitution mix was 0.1 mg protein/ml for the α2A-AR membranes and 40nM for the G-proteins. Following mixing and incubation on ice for 10 min., the reconstituted membranes were transferred to v-shaped 96 well plates and further incubated in the presence or absence of the α2A-AR agonist, UK (10 μM) or the α2A-AR antagonist, Yohimbine (lOOuM) for 10 minutes. ³⁵S-GTP binding was initiated with the addition of [³⁵S]GTPγS (PerkinElmer Life Sciences, Boston, MA, USA) at a final concentration of 0.2nM. After 90 minutes at 27°C samples were transferred to 96 well filtration plates containing glass fibre filters (Millipore Corporation, Bedford, MA, USA), filtered using a MultiScreen Vacuum Manifold (Millipore Corporation, Bedford, MA, USA) and washed with 4 x 200μL ice-cold 20 mM Tris pH 7.4, 25 mM MgCl₂ and 100 mM NaCl. Filters were air-dried and then counted by liquid scintillation counting.

For assays using capture of the α2A-AR/G-protein complex (termed transductosome), onto nickel beads, lOuL Ni-NTA agarose beads (45-165 μm diameter in a 50% solution of reconstitution buffer) (Qiagen, Clifton Hill, Victoria, Australia) were added to 96 well filtration plates containing 1.2μM Durapore® membranes (Millipore Corporation, Bedford, MA, USA). The reconstituted membranes were incubated for 10 minutes with the Ni-NTA beads. Agonists or antagonists were added to the transductosome/bead complex and binding initiated by the addition of [^3sS]GTPγS as described above. After 90 minutes at 27°C samples were filtered, washed and counted as described above.

Time-resolved luminescence resonance energy transfer (TR-LRET): Association and dissociation of Gau_his-Tb(mal) andβ₄γ₂-Alexa(mal)

Gαπ_hi_s-Tb mal) and β₄γ₂-Alexa(mal) were preincubated on ice for 15 minutes to enhance association of the subunits prior to the addition of the G-protein complex to

96-well plates in a final volume of lOOμl at the final concentrations indicated in figures. All dilutions of proteins were done in TMND buffer consisting of 50mM TRIS pH 7.6, lOmM MgCl₂, lOOmM NaCl, 10 mM dithiothreitol and lμM GDP. To enhance dissociation of the protein complex (i.e., dissociation of Gαπ_hi_s-Tb(mal) and β γ₂- Alexa(mal)), A1F₄ ^" was used (lOmM NaF and 30μM A1C1₃). Time-resolved luminescent energy transfer was carried out using a Victor³V (Perkin Elmer) fluorescence plate reader with a time delay of 500μs, counting duration of 1400μs and using an excitation wavelength of 340nm and monitoring emission at both 550nm and 570nm.

Example 1: Reconstitution and testing of α2A-AR/G-protein signalling complex (transductosome).

α2A-AR membranes were treated with 7 M urea to inactivate endogenous Sf9 cell G- protein α and βγ subunits as described previously (Lim & Neubig, 2001, Biochem. J. 354:337-344). Urea treatment left antagonist [³H]MK912 binding intact. α2A-AR membrane expression was approximately 1000 fmol/mg with a Kd of approximately 0.2 nM as determined by [3H]MK912 binding (data not shown).

To determine the time-course of ³⁵S-GTP binding to α2A-AR membranes (0.4 mg/ml) reconstituted with G-proteins, clonidine (a partial α2A-AR agonist) was used at 100 μM. Figure 6 shows that ³⁵S-GTP binding increases over 60 min in the presence or absence of clonidine. However, the addition of 50 nM Gail to the a2A-AR membranes significantly increases the rate of ³⁵S-GTP binding in the presence of clonidine. Additionally, reconstitution of α2A-AR membranes with the G-protein subunits 50 nM Gail and 50 nM βlγ2 further accelerates the rate of ³⁵S-GTP binding over 60 min in the presence of clonidine. These results demonstrate the specificity and requirements for the G-protein subunits for activation of α2A-AR mediated ³⁵S-GTP binding.

The concentration of GDP in the ³⁵S-GTP binding assay is important for obtaining an optimal signal in the receptor activation assay. GDP is required to aid receptor coupling to the heterotrimer in the receptor:GαilGDP;βlγ2 basal state, whilst GTP is required as the substrate for the receptor activated α-subunit. The amount of ³⁵S-GTP bound determines the amount of signal observed. To determine whether high concentrations of the guanine nucleotide, GDP, could reduce non-specific binding, increasing concentrations of GDP were incubated with clonidine-stimulated α2A-AR membranes (Figure 7). Figure 7 shows that GDP at concentrations between 0.5 - 10 μM dose- dependently decreased the background binding of ³⁵S-GTP to Gail as well as the α2A- AR activated signal using clonidine.

To determine the sensitivity of the α2A-AR-induced binding of ³⁵S-GTP to Gαilβlγ2, the α2A-AR agonist UK14303 was used. Figure 8 shows that increasing the concentration of UK concomitantly increased the binding of ³⁵S-GTP to the reconstituted transductosomes with a significant signal being detected with UK at concentrations as low as 10 nM. Additionally, the α2A-AR antagonist, rauwolscine (500 μM) completely abolished the UK-induced ³⁵S-GTP binding, indicating specificity of the receptor-mediated signal. Maximal UK-induced signalling occurred between 0.1 and 1 μM resulting in an approximate 7-fold increase of ³⁵S-GTP binding.

To determine the requirement of βlγ2 for α2A-AR transductosome signalling, increasing concentrations of βlγ2 were used with a constant concentration of Gail (52 nM). Figure 9 shows that increasing concentrations of βlγ2 did not alter the basal (non-receptor-stimulated) level of ³⁵S-GTP binding, however, βlγ2 dose-dependently increased receptor-activated ³⁵S-GTP signalling (using UK at 10 μM as the agonist) in the presence of 52 nM Gail and 0.4 mg/ml α2A-AR. Under the conditions tested, maximal ³⁵S-GTP binding occurred at 1.5 nM β lγ2 in combination with 52 nM Gail .

To determine the requirement of Gail for a2A-AR transductosome signalling, increasing concentrations of Gail were used with a constant concentration of βlγ2 (3.1 nM). Figure 10 shows that increasing concentrations of Gail did not alter the basal (non-receptor-stimulated) level of ³⁵S-GTP binding, however, Gail dose-dependently increased receptor-activated ³⁵S-GTP signalling (using UK at 10 μM as the agonist) in the presence of 3.1 nM βlγ2 and 0.4 mg/ml α2A-AR.

In order to capture the Gαilβlγ2/α2A-AR transductosome on Ni²⁺-coated beads (Ni- NTA beads), it was necessary to determine whether positively charged His6-tagged G- protein subunit modification (on the amino terminus) displayed functional properties similar to native (non His6-tagged) G-protein subunits.

To determine whether His6-tagged Gail were functional in the α2A-AR transductosome signalling assay, increasing concentrations of Gail (his) were used in combination with a constant concentration of βlγ2 (20 nM). Figure 11 shows that increasing concentrations of G il (his) increased the basal (non receptor-stimulated) level of ³ S-GTP binding under the conditions tested. However, stimulation of α2A- AR with lOOμM UK further increased ³⁵S-GTP signalling with 20 nM βlγ2 and 0.4 mg/ml α2A-AR. Only 1 nM Gail (his) was required to obtain an adequate signal-to- noise ratio, with a 4.3-fold increase in ³⁵S-GTP binding at this concentration. These results suggest that the recombinant His6-tagged form of Gail (Gail (his)) could be used in the reconstituted a2A-AR/Gailβlγ2 transductosome without significant loss of signalling activity.

In separate experiments, His6-tagged βlγ2 (βlγ2(his)) was tested in the reconstituted functional ³⁵S-GTP binding assay with α2A-AR. As shown in Figure 12, substitution of βlγ2(his) in exchange for βlγ2 or Gail (his) for Gail (as shown in Figure 11) resulted in similar UK-mediated ³⁵S-GTP binding compared with control (i.e., non His6-tagged G-proteins) with 6.3 - 6.6-fold increases in the presence of these G- proteins. This indicates that His-tagging of βlγ2 (or Gail) did not significantly alter the functional properties of the Gαilβlγ2/α2A-AR transductosome. It is significant that in the absence of G-proteins, UK-induced signalling (³⁵S-GTP binding) was negligible (approximately 4% of UK-stimulated binding in the presence of G-proteins), further demonstrating the absolute requirement of the transductosome signalling complex for appropriate G-protein subunits.

These results suggest that the recombinant His6-tagged (positively charged) form of Gail (Gail (his)) and βlγ2 (βlγ2(his)) could be utilised in the reconstituted α2A- AR/Gαilβlγ2 transductosome without significant loss of activity. Furthermore, there is potential to utilise the His6 high-binding affinity to a Ni²⁺-coated surface for capture of transductosome to an appropriate substratum.

To test whether Ni²⁺-coated agarose beads were amenable to "capture" of the Gαilβlγ2/α2A-AR functionally active transductosome complex, Gαil(his) was used in place of Gail. As shown in Figure 13, reconstitution of the transductosome complex followed by incubation with 50 μl of Ni-NTA beads, resulted in significant UK-induced ³⁵S-GTP binding. Similar amounts of ³⁵S-GTP binding were achieved in the presence of 50 μl Ni-NTA beads with captured transductosome compared with reconstituted membranes only (i.e., not captured on beads) indicating that 50 μl of beads could efficiently capture the transductosome complex. Furthermore, prior removal of Ni²⁺ (by several washes of beads in 150 mM EDTA) from the surface of Ni- NTA beads significantly reduced the amount of ³⁵S-GTP binding. This suggests that the Gαil(his)βlγ2/α2A-AR transductosome complex requires the Ni²⁺ binding sites for the high affinity Gail (his) :Ni²⁺ interactions. Additionally, when the assay was carried out in the presence of 150 mM imidazole, this also resulted in significant loss of ³⁵S- GTP binding captured on the surface of the Ni-NTA beads, since imidazole competitively competes with Gail (his) for Ni²⁺ binding sites.

To further confirm whether Ni²⁺-coated beads capture the transductosome complex, several volumes of beads were used. As little as 5 μl Ni-NTA beads captured the transductosome complex as shown in Figure 14, with saturation of bead binding at > 20 μl beads as determined by ³⁵S-GTP binding. Additionally, in separate experiments we demonstrated that coating the Ni-NTA beads firstly with Gail (his) followed by addition of βlγ2 then α2A-AR (with wash steps in between each addition of protein), also resulted in the sequential building up of the transductosome complex (data not shown) .

Example 2: Reconstitution and testing of MϊR G-protein signalling complex (transductosome) .

M₂R membranes were treated with 7 M urea to inactivate endogenous Sf9 cell G- protein α and βγ subunits. Urea treatment left antagonist [3H]-Scopolamine binding intact. Figure 15 demonstrates that M₂R membrane expression was approximately 25.8 pmol/mg with a Kd of approximately 2.1 nM as determined by [3 HJ-Scopolamine binding. Figure 16 shows the EC50 for inhibition of specific binding of the nonspecific muscarinic receptor antagonist [3H]-Scopolamine (at InM) was inhibited by atropine at 5.2 nM (non-specific muscarinic receptor antagonist) compared with the Ml-muscarinic receptor antagonist pirenzepine (1100 nM). Furthermore, the EC50 for carbachol was 39.3μM.

To determine the time-course of ³⁵S-GTP binding, M₂R membranes (0.4 mg/ml) were reconstituted with G-proteins (5 nM Gαπ(_hi_s) and 5 nM βιγ₂) and carbachol (a M₂R agonist) was used at 25mM. Figure 17 shows that ³⁵S-GTP binding increased above basal binding following 90 min of incubation. These results demonstrate that the agonist carbachol activated M₂R mediated ³⁵S-GTP binding. To determine the sensitivity of the M₂R-induced binding of ³⁵S-GTP to G ii his):βιγ₂, the muscarinic receptor agonist carbachol was used. Figure 18 shows that increasing the concentration of carbachol concomitantly increased the binding of ³⁵S-GTP to the reconstituted transductosomes. Additionally, the muscarinic receptor antagonist, atropine (500 μM) completely abolished the carbachol-induced ³⁵S-GTP binding, indicating specificity of the receptor-mediated signal. The ECs₀ for carbachol-induced ³⁵S-GTP binding was 10.2 μM resulting in an approximate 8-fold increase of ³⁵S-GTP binding at maximal stimulation.

Since His₆-tagged G-protein subunit modification (on the amino terminus) displayed functional properties with the M₂R, we determined whether the G u^β^/T^bR transductosome could be captured on Ni²⁺-coated beads (Ni-NTA beads). To determine whether captured His₆-tagged Gαϋ(_hi_S) signalling was functional in the M2R transductosome signalling assay, increasing concentrations of carbachol were used in the presence of Ni-NTA beads. Figure 19 shows that increasing concentrations of carbachol increased the basal (non receptor-stimulated) level of S-GTP with an EC₅₀ of 5.3 μM with an approximate 7-fold stimulation at maximum stimulation. Additionally, the muscarinic receptor antagonist, atropine (500 μM) abolished the carbachol-induced ³⁵S-GTP binding, indicating specificity of the receptor-mediated signal.

In the above experiments, the Gαi₁(_hi_S)βιγ₂/M₂R transductosome was tested in the reconstituted functional ³⁵S-GTP binding assay with a relatively high concentration of GDP (25 μM). As shown for the α_2A-AR (Example 1), it was expected that lowering the GDP concentration would increase the amount of ³⁵S-GTP (fmol) bound by decreasing the competition of GDP:GTP exchange. Figure 20 shows that increasing the concentration of carbachol increased the binding of ³⁵S-GTP to the reconstituted transductosomes. Note that approximately 0.9 fmol ³⁵S-GTP was bound in the presence of carbachol at concentrations above ImM. However under conditions when the GDP concentration was 25 μM (as in Figure 18) maximal carbachol stimulation resulted in only 0.1 fmol ³⁵S-GTP bound. However, the EC₅₀ for carbachol-stimulated ³⁵S-GTP binding (with 1 μM GDP) was similar to that at 25 μM GDP (18.4 μM and 10.2 μM, respectively). Therefore, the variable GDP concentration in the assay (with constant ³⁵S-GTP set to 0.2 nM) does not alter the affinity for receptor-stimulated ³⁵S- GTP binding, however, the maximal binding is reduced at higher concentrations of GDP. This may indicate that more Gαα(his) subunits are occupied by GDP at higher concentrations of GDP (with subsequently less opportunity for GTP: GDP exchange due to GDP saturation) i.e., this may limit the maximal exchange rate but not the affinity for receptor-modulated Gauφis) activation.

To determine whether captured His₆-tagged Gαπ(_hi_S) signalling was functional in the M2R transductosome signalling assay using lμM GDP, increasing concentrations of carbachol were used in the presence of Ni-NTA beads. Figure 21 shows that increasing concentrations of carbachol increased the basal (non receptor-stimulated) level of ³⁵S- GTP with an EC₅₀ of 16.9 μM (compared with EC₅₀ = 5.3 μM with 25μM GDP as in Figure 19). At maximal stimulation with carbachol, this resulted in an approximate 9.5-fold stimulation of ³⁵S-GTP binding. Additionally, the muscarinic receptor antagonist, atropine (500 μM) abolished the carbachol-induced S-GTP binding, indicating specificity of the receptor-mediated signal.

Example 3: Determination of signalling activity for GPCR-G-protein complex associated with a surface

To determine whether signalling activity was altered when the complete signalling complex was attached to the surface of beads, dose-response measurements were carried out. Figure 22 shows the dose-response of the α2A-AR - G-protein complex (termed transductosome) to the specific agonist UK 14304 (measured by the sensitivity of the receptor to a specific agonist to be detected by heterotrimeric G-proteins using the 35S-GTP binding assay) when the transductosome was either associated with Ni(NTA) agarose beads or was not. The EC50 values for stimulation of the α2A-AR by its agonist, UK 14304, when the transductosome complex was either associated with Ni(NTA) agarose beads or was not, were not significantly different. As a further indication that attachment of the transductosome signalling complex was unaltered in terms of signalling activity when associated with a surface, the dose efficacy of antagonists at the α2A-AR when the receptor-G-protein signalling complex (transductosome) was either associated with Ni(NTA) agarose beads or was not, was determined. As shown in Figure 23 the rank order potency of a number of adrenergic receptor subtype-specific antagonists determined from their respective IC50 values, is indicative of the specificity of the α2A-AR, further indicating that the receptor signalling complex is unaltered with regard to sensitivity (Figure 22) or specificity (Figure 23) when associated with a surface. Example 4: Determination of functionality of fluorescently labeled G-protein subunits in GPCR reconstitution assay

To determine whether the labelling procedure inhibited G-protein signalling the ³⁵S- GTP binding experiments were carried out using either unlabelled Gαπ, β₁γ2, βrY₂ labelled with TMR-NCS or TMR-maleimide or Gα_;ι labelled with TMR-maleimide. Figure 24 demonstrates signalling in unlabelled G-proteins was greater than the labelled proteins, however the concentration of labelled proteins in the ³⁵S-GTP binding assay was considerably lower and may explain this difference. Nevertheless, labelling of the G-protein subunits yielded significant α_2A-AR-induced G-protein dependent signalling under the conditions tested. In each experiment presented in Figure 24 the subunit being tested (e.g., a labelled Gαilhis-TMR(mal)) was paired with a corresponding subunit (eg β^) that was known to have signalling activity. This demonstrates that at least when one of the subunits (i.e, Gαu or β^) is fluorescently labelled, then α_2A-AR-induced signalling activity is maintained.

Similarly, in Figure 25, labelling of Gα protein subunits with Terbium chelate (either NCS or EMCH, ie succinimidyl ester or maleimide, respectively) indicated that Terbium labelled proteins are functional. Preliminary experiments indicated that signalling using β4γ2 in place of βlγ2 did not alter α_2A-AR signalling as determined by ³⁵S-GTP biding. Additionally, higher levels of expression of β4γ2 compared with βlγ2 were achieved, therefore we chose to label β4γ2 subunits to improve yield of labelled samples. Furthermore, storage of the G-protein subunits in glycerol did not decrease the signalling activity as shown in Figure 23 considering half the concentration of subunits in glycerol were used for these experiments.

Figure 26 demonstrates that labelling of βγ protein subunits with Terbium chelate (either NCS or EMCH ie succinimidyl ester or maleimide, respectively) indicated that Terbium labelled proteins are functional. Furthermore, storage of the G-protein subunits in glycerol did not decrease the signalling activity as shown in Figure 24.

To determine whether α_2A-AR-induced signalling activity was present following reconstitution of dual labelled G-proteins, Gαilhis was labelled with Terbium-EMCH and βlγ2 labelled with TMR (maleimide form). Figure 27 (A) shows that under these conditions signalling activity was maintained. Furthermore, switching the labelling of fluorophores ie., labelling of Gαilhis with TMR (maleimide form) and βlγ2 with Terbium-EMCH resulted in significant G-protein signalling.

Association ofGauM_s-Tb(mal) and β₄γ₂-Alexa(mal)

lOnM Gαπhis labelled with Tb-chelate (Gαπ_hi_sTb) had a background fluorescence at 570 nm of approximately 1500 arbitrary units (a.u.) which declined to approximately 1000 a.u. over a 20 minute time course (Figure 28). Figure 28 also demonstrates the rapid association of Gα^is (labelled with Tb-chelate i.e., Gαu_hi_s b)- and β γ₂ labelled with (Alexa 546 i.e., β₄γ₂- Alexa) at lOnM using time resolved-fluorescence resonance energy transfer techniques. The TR-FRET at equilibrium resulted in a 5 -fold increase in signal under the conditions tested. The time constant (ty₂) for association was less than 1 minute at lOnM G-proteins. Addition of a (20x) molar excess of unlabelled Gam after 3 minutes (near equilibrium) of

in a rapid decline of TR-FRET indicating dissociation of the G-protein subunits as shown in Figure 28. In other experiments (data not shown) the Kd for Gαπω_sTb and β₄γ₂-Alexa binding was calculated as approximately InM, consistent with data from the literature.

Figure 29 demonstrates that at lower concentrations of G-proteins i.e. at 3nM Gαu_hisTb and 8nM β₄γ₂-Alexa, the total TR-FRET signal was lower at equilibrium in comparison to that observed following the addition of lOnM G-proteins (Figure 28)although there was still a 3 -fold increase in the TR-FRET signal. Figure 29 also demonstrates that adding a molar (25x) excess of unlabelled β₄γ₂ resulted in a rapid decline of TR-FRET signal indicating dissociation of G-protein subunits.

With respect to the signal to background ratio, it was found that in addition to a 50μs delay in the TR-FRET counting cycle, a lOOμs counting duration window gave the optimum result as shown in Figure 30. The maximum signal to noise ratio observed was approximately 13 -fold above background at approximately 12 minutes.

Dissociation ofGau_hi_s-Tb(mal) and β₄Y₂-Alexa(mal)

Dissociation of heterotrimeric G-protein subunits can be effected by addition of A1F ^" as is shown in Figure 30. The addition of A1F₄ ^" rapidly (less than 1 minute) dissociated the Gαπ i_sTb and β₄γ₂-Alexa bound subunits at equilibrium, resulting in an approximate 50% decrease in TR-FRET signal. This new steady-state binding remained stable for at least 25 minutes.

Association and Dissociation ofGa _hts-Alexa546 and β₄Y₂-Tb

In all above data showing TR-FRET, Gαπ_hi_s-Tb and β₄γ₂-Alexa were used. To determine whether TR-FRET would occur on reverse labelled G-proteins, Gauhis was labelled with Alexa-546 (Gαπ_hi_S-Alexa546) and β₄γ₂ was labelled with Terbium chelate (β₄γ₂-Tb) as shown in Figure 31. In this set of experiments, the β₄γ₂-Tb had lower background fluorescence at 570nm compared with Gαπ_hi_S-Tb (approximately 50 a.u.). Importantly, the t for G-protein association as determined by "reverse" TR-FRET was similar to that in Figures 28-30 indicating that labelling in this reverse fashion did not alter the ability of G-protein subunit association. The total TR-FRET signal was somewhat lower than that shown in Figures 28-30 indicating that the labelling efficiency of β₄γ with Terbium chelate may well be lower. However, the signal to noise ratio was equivalent to that shown in Figure 30 (approximately 13 -fold). As is also shown in Figure 31, the addition of A1F₄ ^" rapidly dissociated the Gα^his Alexa and β₄γTb bound subunits at equilibrium resulting in an approximate 50%) decrease in TR- FRET signal. This new steady-state binding equilibrium remained stable for at least 15 minutes.

To determine the dissociation constant for the Gαilhis-Tb and β4γ2-Alexa interaction, TR-FRET measurements were done using four concentrations of Gαilhis-Tb (3nm to 15nm) tested over a range of β4γ2-Alexa concentrations (Onm -lOOnm). The mean dissociation constant for the interaction was 2.7nm (see Figure 32). This is indicative of a very tight association between these binding partners as has been estimated by other techniques and is also indicative that the TR-FRET technology used here does not perturb the normal high binding affinity of these heterotrimeric G-proteins.

Interaction of a regulator of G-protein signalling (RGS4) with Ga or β₄γ2 as determined by TR-FRET

In a separate series of experiments we aimed to determine whether a regulator of G- protein signalling (RGS4) interacted with either Gα or β γ₂ as determined by TR- FRET. Figure 33 demonstrates that there is a rapid TR-FRET between 150nM

RGS4his labelled with Alexa-546 (RGS4_hi_SAlexa) in the presence of 50nM Gαπhis b, indicating a rapid "on" binding between the Gα^his subunit and RGS4M_S. The resulting increase in signal was approximately 5 -fold at steady state. Addition of a (20x) molar excess of unlabelled Gαπhi_s after 6 minutes of GαπhisTb:RGS4 i_SAlexa association resulted in a rapid decline of the TR-FRET signal indicating dissociation of Gαπ_hi_sTb and RGS4hj_S Alexa as shown in Figure 33. However, there was no significant association evident between RGS4_hj_SAlexa and β₄γ₂Tb, consistent with the reported interaction of RGS with heterotrimeric G-proteins favouring association with Galpha in vivo, even taking into account the relatively lower background TR-FRET signal from the β₄γ₂Tb dimer compared with Gαπ_hi_sTb labelled subunit.

A dose curve of RGS4_hi_SAlexa against 50nM Gαπ_hi_sTb demonstrated that increasing concentrations of RGS4_hi_SAlexa increased the TR-FRET signal until a stable maximum was reached as shown in Figure 34. From this, a disassociation constant (K_d) of 15nM was calculated indicating a high affinity association between RGS_hi_SAlexa and Gα_ϋhi_sTb.

To determine parameters associated with the interaction of RGS4 with the G πωsTb labelled subunit, TR-FRET measurements were carried out using RGS4hi_S Alexa and Gαπ_hi_sTb in various states of activation, inactive in the presence of GDP, activated in the presence of GTPγS and transitional in the presence of aluminium fluoride (aluminium fluoride has been reported to act as a transition state analogue of GTP; the latter compound being known to influence the properties of the Gα subunit). These results showed that in the presence of aluminium fluoride, steady state TR-FRET was significantly increased compared to when GDP or GTP was present as shown in Figure 35. RGS4 has previously been reported to have greater affinity for the transitional state Gα subunit and this increase in signal could be a result of greater amounts of binding due to increased affinity. Alternatively, this increase could be a result of conformational changes in the Gαπ_hi_sTb subunit that brings labelled residues into closer proximity with labelled residues of RGS4_hi_sAlexa resulting in more efficient energy transfer.

From Figure 36 it could also be observed that steady state TR-FRET was reached at differing intervals depending on the activation state of Gαπ i_sTb. Figure 36 shows that the rate constant (K) of association was slowest when Gαπ_hi_sTb was activated in the presence of GTPγS, this rate was increased significantly when GαπhisTb was inactive bound to GDP and further increased in the presence of aluminium fluoride. This data shows that the transitional state of Gαπ_hi_sTb mimicked in the presence of aluminium fluoride, is likely to be the preferred conformation for interaction with RGS4.

Example 5: Optimisation of fluorescent signal

Optimisation of the fluorescent signal may be accomplished by rational location of the FRET-interacting fluorescent probes in accord with the likely molecular changes occurring in the spatial arrangements and re-arrangements (association and diss- association) of the heterotrimeric G-proteins consequent upon their activation by a liganded GPCR. For example, donor and acceptor TR-FRET pairs may be placed either covalently, or by high affinity binding, to sites on the G-proteins which are known to rearrange during signal transduction. These sites of attachment may be based on the likely changes in the distance between FRET pairs taking into account the Forster radius of the interacting FRET pairs which in part dictates the generation of the FRET signal. Fluorescent probes may be attached by varying means which could include the production of fusion proteins or selected amino acid motifs within the sequence of one or more G-proteins (G-alpha, G-beta, G-gamma and any variants of these which may increase coupling to GPCRs such as chimeric or promiscuous variants, and including other interacting proteins such as RGS, AGS and the like). Fusion proteins or selected amino acid motifs may include, for example, a lanthanide binding tag motif (LBT) as a fusion protein (as described in Nitz et al. (2003) Chem.BioChem 4:272-276) on one or other of the G-proteins so as to allow the binding of a lanthanide (preferably Terbium) to specific locations, with excitation of the Terbium being induced by a Tryptophan or other aromatic amino acid within the LBT. Furthermore, spectrally-matched acceptor fluors such as a biarsenical Fluorescein or like molecules may be covalently bound to a tetracysteine motif of the type described in Adams (2002) J Am Chem Soc 124:6063-6067 ', engineered into a known site on a G- protein or other interacting molecule of the type listed above. This would allow significant choice and great precision in localising the FRET pairs to enable optimisation of the signal as well as design of the signal to be one which could selectively increase or decrease upon GPCR activation depending on fluorescent probe localisation.

All references cited above are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Table 1 GPCR ligands and site of action

Claims

Claims:

1. A method for determining the activity or function of a GPCR, the method comprising providing a cell free complex comprising a GPCR coupled to at least one G- protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor; and detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex.

2. A method according to claim 1, wherein the at least one G-protein subunit is a G-alpha subunit.

3. A method according to claim 1 or claim 2, wherein the complex comprises at least two G-protein subunits.

4. A method according to claim 3 wherein the tertiary event is a change in proximity of the at least one G-protein subunit with respect to another G-protein subunit.

5. A method according to claim 1 or claim 2, wherein the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta-gamma dimer.

6. A method according to claim 5 wherein the tertiary event is dissociation of the G-alpha subunit from the G-beta-gamma heterodimer.

7. A method according to claim 1 wherein the tertiary event is an interaction that occurs between the at least one G-protein subunit and a protein that normally interacts with a G-protein subunit.

8. A method according to claim 7 wherein the protein that normally interacts with a G-protein subunit is an RGS or AGS.

9. A method according to claim 5 or claim 6, wherein the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa.

10. A method according to claim 7, wherein the G-protein subunit is labeled with an energy donor and the protein that normally interacts with a G-protein subunit is labeled with an energy acceptor or vice versa.

11. A method according to claim 9 or claim 10, wherein the energy donor and energy acceptor are fluorescent or luminescent moieties.

12. A method according to claim 11, wherein the energy donor/acceptor moieties are selected from the group consisting of Terbium/ Alexa546, Terbium/FlAsH-(EDT2),

Terbium/fluorescein, Terbium/GFP, Terbium/tetramethylrhodamine(TMR),

Terbium/Cy3, Terbium/QSY-7, Terbium/R phycoerythrin, Europium/Cy5, Europium/ Allophycocyanin (APC), Europium/ Alexa 633, Alexa 488/Alexa 555, Alexa 568/Alexa 647, Alexa 594/Alexa 647, Alexa 647/Alexa 594, Cy3/Cy5, BODIPY FL/BODIPY FL, Fluorescein/TMR, IEDANS/fluorescein, and fluorescein/fluorescein.

13. A method according to claim 9 or claim 10, wherein the energy acceptor is a quencher.

14. A method according to claim 13, wherein the quencher is selected from the group consisting of DABCYL, DABSYL, QSY 7, QSY 9, QSY 21, and QSY 35.

15. A method according to any one of claims 12 to 14 wherein the energy donor and/or acceptor moiety is attached to a G-protein subunit or a protein that normally interacts with a G-protein by way of a fusion protein or amino acid motif present within the G-protein subunit or the protein that normally interacts with a G-protein.

16. A method according to claim 15 wherein the amino acid motif is a lanthanide binding tag motif.

17. A method according to any one of claims 1 to 16, wherein complex is tethered to a support.

18. A method according to claim 16, wherein the complex is tethered to a support via a G-alpha subunit.

19. A method according to claim 17 or claim 18, wherein the support is selected from the group consisting of a dish, tray, plate, multi-well plate, microtiter plate, bead, dendrimer, nanoparticle, nanobead, nano-tube, micro-chip, micro-well, microarray, silica gel, hydrogel, cellulosic bead, glass, filter paper, nitrocellulose and nylon membrane.

20. A method according to claim 19 wherein the solid support is part of a biosensor device.

21. A method according to any one of claims 1 to 20, wherein the GPCR is a known receptor.

22. A method according to any one of claims 1 to 20, wherein the GPCR is an "orphan" GPCR.

23. A method according to claim 21 or claim 22, wherein the cell free complex is stimulated prior to detection of the signal.

24. A method according to claim 23, wherein the cell free complex is stimulated by exposing the complex to a sample comprising a cognate ligand of the GPCR or by exposing the GPCR to an odor or taste.

25. A method according to claim 24, wherein the ligand is an agonist, antagonist or inverse agonist of the GPCR.

26. A method according to claim 24 or claim 25, wherein the level of signal detected is used to determine the amount of ligand in the sample.

27. A method of screening for an agent that modulates G-protein interactions, the method comprising exposing a candidate agent to a cell free complex comprising at least one G- protein subunit, wherein the at least one G-protein is labeled with an energy donor or an energy acceptor; and detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex.

28. A method according to claim 27, wherein the candidate agent is an agonist, inverse agonist or antagonist of a ligand of a GPCR.

29. A method according to claim 27, wherein the candidate agent is a G-protein regulator.

30. A method according to any one of claims 27 to 29, wherein the at least one G- protein subunit is a G-alpha subunit.

31. A method according to any one of claims 27 to 30, wherein the complex comprises at least two G-protein subunits.

32. A method according to claim 31 wherein the tertiary event is a change in proximity of the at least one G-protein subunit with respect to another G-protein subunit.

33. A method according to any one of claims 27 to 32, wherein the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta- gamma dimer.

34. A method according to claim 33 wherein the tertiary event is dissociation of the G-alpha subunit from the G-beta-gamma heterodimer.

35. A method according to claim 27 wherein the tertiary event is an interaction that occurs between the at least one G-protein subunit and a protein that normally interacts with a G-protein subunit.

36. A method according to claim 35 wherein the protein that normally interacts with a G-protein subunit is an RGS or AGS.

37. A method according to claim 33 or claim 34, wherein the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa.

38. A method according to claim 35, wherein the G-protein subunit is labeled with an energy donor and the protein that normally interacts with a G-protein subunit is labeled with an energy acceptor or vice versa.

39. A method according to claim 37 or claim 38, wherein the energy donor and energy acceptor are fluorescent or luminescent moieties.

40. A method according to claim 39, wherein energy donor/acceptor moieties are selected from the group consisting of Terbium/Alexa546, Terbium/FlAsH-(EDT2), Terbium/fluorescein, Terbium/GFP, Terbium/tetramethylrhodamine(TMR),

Terbium/Cy3, Terbium/QSY-7, Terbium/R phycoerythrin, Europium/Cy5, Europium/AUophycocyanin (APC), Europium/Alexa 633, Alexa 488/Alexa 555, Alexa 568/Alexa 647, Alexa 594/Alexa 647, Alexa 647/Alexa 594, Cy3/Cy5, BODIPY FL/BODIPY FL, Fluorescein/TMR, IEDANS/fluorescein, and fluorescein/fluorescein.

41. A method according to claim 37 or claim 38, wherein the energy acceptor is a quencher.

42. A method according to claim 41, wherein the quencher is selected from the group consisting of DABCYL, DABSYL, QSY 7, QSY 9, QSY 21, and QSY 35.

43. A method according to any one of claims 40 to 42 wherein the energy donor and/or acceptor moiety is attached to a G-protein subunit or a protein that normally interacts with a G-protein by way of a fusion protein or amino acid motif present within the G-protein subunit or the protein that normally interacts with a G-protein.

44. A method according to claim 43 wherein the amino acid motif is a lanthanide binding tag motif.

45. A method according to any one of claims 27 to 44 wherein the cell free complex further comprises a GPCR coupled to the at least one G-protein subunit.

46. A method according to claim 45, wherein the GPCR is a known receptor.

47. A method according to claim 45, wherein the GPCR is an "orphan" GPCR.

48. A method according to any one of claims 45 to 47 wherein the candidate agent is a ligand of the GPCR.

49. A method according to any one of claims 27 to 48, wherein the complex is tethered to a support.

50. A method according to claim 49, wherein the complex is tethered to a support via a G-alpha subunit.

51. A method according to claim 49 or claim 50, wherein the support is selected from the group consisting of a dish, tray, plate, multi-well plate, microtiter plate, bead, dendrimer, nanoparticle, nanobead, nano-tube, micro-chip, micro-well, microarray, silica gel, hydrogel, cellulosic bead, glass, filter paper, nitrocellulose and nylon membrane.

52. A method according to claim 51 wherein the solid support is part of a biosensor device.

53. A method for detecting a GPCR in a sample, the method comprising exposing a sample to a cell free G-protein complex comprising at least one

G-protein subunit which is labeled with an energy donor or an energy acceptor, wherein an associated GPCR present in the sample is capable of coupling to the G- protein complex such that stimulation of the GPCR leads to generation of a detectable signal associated with a tertiary event involving at least one G-protein subunit in the complex, and detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex.

54. A method according to claim 53, wherein the at least one G-protein subunit is a G-alpha subunit.

55. A method according to claim 53 or claim 54, wherein the complex comprises at least two G-protein subunits.

56. A method according to claim 55 wherein the tertiary event is a change in proximity of the at least one G-protein subunit with respect to another G-protein subunit.

57. A method according to claim 55 or claim 56, wherein the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta-gamma dimer.

58. A method according to claim 57 wherein the tertiary event is dissociation of the G-alpha subunit from the G-beta-gamma heterodimer.

59. A method according to claim 53 wherein the tertiary event is an interaction that occurs between the at least one G-protein subunit and a protein that normally interacts with a G-protein subunit.

60. A method according to claim 59 wherein the protein that normally interacts with a G-protein subunit is an RGS or AGS.

61. A method according to claim 57 or claim 58, wherein the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa.

62. A method according to claim 59, wherein the G-protein subunit is labeled with an energy donor and the protein that normally interacts with a G-protein subunit is labeled with an. energy acceptor or vice versa.

63. A method according to claim 61 or claim 62, wherein the energy donor and energy acceptor are fluorescent or luminescent moieties.

64. A method according to claim 63, wherein energy donor/acceptor moieties are selected from the group consisting of Terbium/Alexa546, Terbium/FlAsH-(EDT2),

Terbium/fluorescein, Terbium/GFP, Terbium/tetramethylrhodamine(TMR),

Terbium/Cy3, Terbium/QSY-7, Terbiurn/R phycoerythrin, Europium/Cy5, Europium/AUophycocyanin (APC), Europium/Alexa 633, Alexa 488/Alexa 555, Alexa 568/Alexa 647, Alexa 594/Alexa 647, Alexa 647/Alexa 594, Cy3/Cy5, BODIPY FL/BODIPY FL, Fluorescein/TMR, IEDANS/fluorescein, and fluorescein/fluorescein.

65. A method according to claim 61 or claim 62, wherein the energy acceptor is a quencher.

66. A method according to claim 65, wherein the quencher is selected from the group consisting of DABCYL, DABSYL, QSY 7, QSY 9, QSY 21, and QSY 35.

67. A method according to any one of claims 64 to 66 wherein the energy donor and/or acceptor moiety is attached to a G-protein subunit or a protein that normally interacts with a G-protein by way of a fusion protein or amino acid motif present within the G-protein subunit or the protein that normally interacts with a G-protein.

68. A method according to claim 67 wherein the amino acid motif is a lanthanide binding tag motif.

69. A method according to any one of claims 53 to 68, wherein complex is tethered to a support.

70. A method according to claim 69, wherein the complex is tethered to a support via a G-alpha subunit.

71. A method according to claim 69 or claim 70, wherein the support is selected from the group consisting of a dish, tray, plate, multi-well plate, microtiter plate, bead, dendrimer, nanoparticle, nanobead, nano-tube, micro-chip, micro-well, microarray, silica gel, hydrogel, cellulosic bead, glass, filter paper, nitrocellulose and nylon membrane.

72. A method according to claim 71 wherein the solid support is part of a biosensor device.

73. A method according to any one of claims 53 to 72, wherein the GPCR is a known receptor.

74. A method according to any one of claims 53 to 72, wherein the GPCR is an "orphan" GPCR.

75. A cell free complex comprising a GPCR coupled to at least one G-protein subunit, wherein the at least one G-protein is labeled with an energy donor or an energy acceptor; and wherein stimulation of the GPCR leads to a detectable tertiary event involving the at least one G-protein subunit in the complex.

76. A cell free complex according to claim 75, wherein the at least one G-protein subunit is a G-alpha subunit.

77. A cell free complex according to claim 75 or claim 76, wherein the complex comprises at least two G-protein subunits.

78. A cell free complex according to claim 77 wherein the tertiary event is a change in proximity of the at least one G-protein subunit with respect to another G-protein subunit.

79. A cell free complex according to claim 76 or claim 77, wherein the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta- gamma dimer.

80. A cell free complex according to claim 79 wherein the tertiary event is dissociation of the G-alpha subunit from the G-beta-gamma heterodimer.

81. A cell free complex according to claim 75 wherein the tertiary event is an interaction that occurs between the at least one G-protein subunit and a protein that normally interacts with a G-protein subunit.

82. A method according to claim 81 wherein the protein that normally interacts with a G-protein subunit is an RGS or AGS.

83. A cell free complex according to claim 79 or claim 80, wherein the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa.

84. A cell free complex according to claim 81, wherein the G-protein subunit is labeled with an energy donor and the protein that normally interacts with a G-protein subunit is labeled with an energy acceptor or vice versa.

85. A cell free complex according to claim 83 or claim 84, wherein the energy donor and energy acceptor are fluorescent or luminescent moieties.

86. A cell free complex according to claim 85, wherein energy donor/acceptor moieties are selected from the group consisting of Terbium/ Alexa546, Terbium/FlAsH- (EDT2), Terbium/fluorescein, Terbium/GFP, Terbium/tetramethylrhodamine(TMR), Terbium/Cy3, Terbium/QSY-7, Terbium/R phycoerythrin, Europium/Cy5₅ Europium/AUophycocyanin (APC), Europium/Alexa 633, Alexa 488/Alexa 555, Alexa 568/Alexa 647, Alexa 594/Alexa 647, Alexa 647/Alexa 594, Cy3/Cy5, BODIPY FL/BODIPY FL, Fluorescein/TMR, IEDANS/fluorescein, and fluorescein/fluorescein.

87. A cell free complex according to claim 83 or claim 84, wherein the energy acceptor is a quencher.

88. A cell free complex according to claim 87, wherein the quencher is selected from the group consisting of DABCYL, DABSYL, QSY 7, QSY 9, QSY 21, and QSY 35.

89. A cell free complex according to any one of claims 75 to 88, wherein complex is tethered to a support.

90. A cell free complex according to claim 89, wherein the complex is tethered to a support via a G-alpha subunit.

91. A cell free complex according to claim 89 or claim 90, wherein the support is selected from the group consisting of a dish, tray, plate, multi-well plate, microtiter plate, bead, dendrimer, nanoparticle, nanobead, nano-tube, micro-chip, micro-well, microarray, silica gel, hydrogel, cellulosic bead, glass, filter paper, nitrocellulose and nylon membrane.

92. A cell free complex according to claim 91 wherein the solid support is part of a biosensor device.

93. A cell free complex according to any one of claims 75 to 92, wherein the GPCR is a known receptor.

94. A cell free complex according to any one of claims 75 to 92, wherein the GPCR is an "orphan" GPCR.

95. A composition comprising a plurality of cell free G-protein coupled receptor protein subunit complexes according to any one of claims 75 to 94.

96. A microarray comprising a plurality of cell free G-protein coupled receptor protein subunit complexes according to any one of claims 75 to 94.

97. A biosensor comprising at least one cell free G-protein coupled receptor protein subunit complex according to any one of claims 75 to 94 and a means for detecting a signal associated with a change in at least one of the G-protein subunits in the complex.

98. A kit for screening for an agent that modulates the activity of a GPCR, the kit comprising a cell free complex comprising at least one G-protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor.

99. A kit according to claim 98, wherein the cell free complex comprises a heterotrimeric G-protein complex comprising a G-alpha subunit and a G-beta-gamma dimer.

100. A kit according to claim 98 or claim 99, wherein the cell free complex further comprises a GPCR coupled to the at least on G-protein subunit.

101. A kit according to claim 98 or claim 99, wherein the cell free complex is tethered to a support.