Two Gαi1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity THOMAS ZIELINSKI,1 ADAM J. KIMPLE,2 STEPHANIE Q. HUTSELL,3 MARK D. KOEFF,1 DAVID P. SIDEROVSKI,2,4,5 and ROBERT G. LOWERY1 RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from Gα and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a Gαi1 mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. Gαi1(R178M/A326S) GTPase activity was stimulated 6- to 12-fold by RGS proteins known to act on Gαi subunits and not affected by those unable to act on Gαi, demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of Gαi1(R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on Gαi1(R178M/A326S), the authors developed a Transcreener fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gαi1(R178M/A326S) with a homogeneous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling. (Journal of Biomolecular Screening 2009:1195-1206) Key words: fluorescence polarization, GDP detection, regulators of G-protein signaling, surface plasmon resonance INTRODUCTION T HE STANDARD MODEL OF G-PROTEIN-COUPLED RECEPTOR (GPCR) SIGNAL TRANSDUCTION had long been restricted to a 3-component system: receptor, G-protein, and effector.1 The 7-transmembrane domain receptor is coupled to a membraneassociated heterotrimeric complex composed of a guanosine triphosphate (GTP)–hydrolyzing Gα subunit and a Gβγ dimeric partner. Agonist-induced conformational changes enhance the guanine nucleotide exchange activity of the receptor, leading to the release of guanosine diphosphate (GDP) (and subsequent binding of GTP) by the Gα subunit. On binding GTP, conformational changes within the 3 “switch” regions of Gα allow the release of Gβγ. Separated Gα⋅GTP and Gβγ subunits are then 1 BellBrook Labs, Madison, Wisconsin. Departments of 2Pharmacology, 3Biochemistry & Biophysics, 4Lineberger Comprehensive Cancer Center, and 5UNC Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill. Thomas Zielinski and Adam J. Kimple contributed equally to this work. Received Mar 28, 2009, and in revised form Aug 1, 2009. Accepted for publication Aug 4, 2009. Journal of Biomolecular Screening 14(10); 2009 DOI: 10.1177/1087057109347473 © 2009 Society for Biomolecular Sciences free to propagate intracellular signaling via diverse effectors.2 The intrinsic GTP hydrolysis (GTPase) activity of Gα resets the cycle by forming Gα⋅GDP, which has low affinity for effectors but high affinity for Gβγ. In this way, the inactive, GDP-bound heterotrimer (Gα⋅GDP/Gβγ) is reformed and capable once again to interact with activated receptor. Based on this cycle of receptor-catalyzed GTP exchange and intrinsic GTP hydrolysis by Gα, the duration of heterotrimeric G-protein signaling is thought to be controlled by the lifetime of the Gα subunit in its GTP-bound state. After the establishment of this basic model,1 RGS proteins (regulators of G-protein signaling) were subsequently discovered3-5 to bind Gα subunits (via a conserved ~120–amino acid RGS domain) and dramatically accelerate their intrinsic GTPase activity,6 thereby attenuating heterotrimer-linked signaling. Nearly 40 human proteins contain at least 1 RGS domain, with many of these proteins (e.g., RGS4, RGS16) serving as GTPase-accelerating proteins (GAPs) for Gαi/o subunits, yet others such as RGS2 and p115-RhoGEF are particularly attuned to Gαq/11 and Gα12/13 substrates, respectively.7 The discovery of this superfamily of Gα-directed GAPs resolved apparent timing paradoxes between observed rapid physiological responses mediated by GPCRs and the slow hydrolysis activity of the cognate G-proteins seen in vitro. Thus, in this capacity as negative regulators of GPCR signal transduction, the RGS www.sbsonline.org 1195 Zielinski et al. proteins present themselves as excellent potential drug discovery targets.7 For example, pharmacological inhibition of RGS domain GAP activity should lead to prolonged signaling from G-proteins activated by agonist-bound GPCRs. The most direct way to detect RGS protein function is by measuring the increased GTPase activity exhibited by its target Gα protein. However, accurate in vitro measurements of Gα-catalyzed GTP hydrolysis are difficult to obtain without laborious biochemical reconstitutions with purified Gβγ and an activated GPCR (e.g., Ingi et al.8). In the absence of GPCRmediated nucleotide exchange, it is GDP release (rather than GTP hydrolysis) that is the rate-limiting step in the Gα nucleotide cycle.9 Thus, to examine the effect of an RGS protein in accelerating GTP hydrolysis by an isolated Gα subunit in vitro, a single round of hydrolysis of radiolabeled GTP is usually performed (aka the “single-turnover GTPase assay”; Berman et al.6). This standard assay for measuring RGS domain-mediated GAP activity is low throughput and requires discrete steps of [γ-32P]GTP loading onto Gα, protein reactant admixture (with addition of the critical cofactor Mg2+ to initiate hydrolysis), isolation (in discrete time intervals) of released [32P]phosphate with activated charcoal precipitation and centrifugation, and finally scintillation counting. We have described an alternative single-turnover GTPase assay10 using a coumarin-labeled, phosphate-binding protein to facilitate fluorescence-based detection of inorganic phosphate production; however, this method demands stringent controls on multiple experimental steps to eliminate phosphate contaminants that interfere with the detection of GTPase activity. Such convoluted protocols of inorganic phosphate detection are difficult for the nonspecialist and especially not suited for high-throughput screening (HTS) of large compound libraries for RGS domain inhibitors. We and others have reported alternative, fluorescence-based strategies for detecting the binding between RGS protein and Gα substrate,11-13 but none has specifically facilitated a discrete endpoint measurement of RGS domain-mediated GAP activity per se. To develop a facile steady-state GTPase assay for RGS domain GAP activity, we first set out to increase the spontaneous GDP release rate of Gα (koff(GDP)) while also decreasing its intrinsic rate of GTP hydrolysis (kcat(GTPase)), thereby allowing detection of at least a 5-fold enhancement of steady-state GTP hydrolysis by RGS proteins to provide an adequate signal-to-noise ratio. Gαi1 and closely related Gα proteins have been the focus of extensive structure/function studies,14-17 and point mutations that affect both koff(GDP) and kcat(GTPase) without affecting functional interaction with the RGS domain have been identified previously15-18 (e.g., Fig. 1A). Two of the most striking Gα mutations have been made to the highly conserved active-site arginine (R178C; Coleman et al.15), which causes a ~100-fold reduction in GTPase activity, and to the alanine residue within the conserved TCAT loop that contacts the guanine ring (A326S; Posner et al.16), which results in a ~25-fold increase in koff(GDP) relative to wild-type yet an identical kcat(GTPase). 1196 www.sbsonline.org To detect RGS protein-accelerated GTPase activity, we adapted a monoclonal antibody and fluorescent tracer, previously developed for the Transcreener adenosine diphosphate (ADP) assay,19 for selective immunodetection of GDP with a fluorescence polarization readout. Measurement of GTPase activity using this Transcreener GDP assay overcomes the signal-to-noise limitations of phosphate detection methods and has been validated as a robust HTS method in the case of ADP detection for kinases and ATPases.20-22 Moreover, because it is a catalytic assay rather than a substrate-binding assay, it should enable detection of all types of modulators of RGS protein GAP activity, including those that bind at allosteric sites and affect RGS protein-catalytic activity without directly targeting the RGS domain Gα binding site.23 In this present study, we tested multiple-point mutant Gαi1 proteins with increased GDP dissociation and/or decreased GTP hydrolysis rates for their ability to enable detection of RGS domain GAP activity using a steady-state GTPase assay format (i.e., multiple rounds of turnover of GTP to GDP). Coupling one of these variants, Gαi1(R178M/A326S), to the Transcreener GDP detection system has not only allowed facile detection of RGS protein GAP activity but also was useful in helping establish (along with surface plasmon resonance spectroscopy) that the mutant Gαi1 interacted with RGS proteins with the same specificity and affinity as the wild-type Gαi1 protein. MATERIALS AND METHODS Chemicals and assay materials GDP and GTP were purchased from USB Corp. (Cleveland, OH). The monoclonal antibody and tracer used for GDP detection were developed at BellBrook Labs (Madison, WI) as described,19 with the tracer comprising ADP conjugated to Alexa Fluor 633 (Invitrogen/Molecular Probes, Carlsbad, CA). Unless otherwise specified, all additional reagents were of the highest quality obtainable from Sigma (St. Louis, MO) or Fisher Scientific (Hampton, NH). Protein expression and purification Wild-type, full-length human Gαi1 and various RGS proteins used in these studies were expressed in Escherichia coli and purified as previously described.24 Gαi1 point mutants were created using PCR-based site-directed mutagenesis (QuikChange II, Stratagene, La Jolla, CA) on the wild-type pProEXHTb-Gαi1 expression vector; mutagenesis primers were designed using Stratagene’s QuikChange primer design program and synthesized/PAGE purified by Sigma-Genosys. All mutant constructs were sequence verified at Functional Biosciences LLC (Madison, WI) before protein expression, purification, concentration, quantitation, and cryopreservation using established protocols.10,24 Journal of Biomolecular Screening 14(10); 2009 Fluorescent Steady-State RGS Protein GAP Assay FIG. 1. Increased guanosine diphosphate (GDP) release and decreased guanosine triphosphate (GTP) hydrolysis of the Gαi1(R178M/A326S) mutant compared to wild-type Gαi1 and single-point mutants, as measured by [35S]GTPγS binding and single-turnover [γ-32P]GTP hydrolysis, respectively. (A) Point mutations to Gαi-family subunits previously reported in the literature15-18 to alter intrinsic GTP hydrolysis and GDP dissociation rates. (B) Binding of [35S]GTPγS to wild-type or indicated mutant Gαi1 subunits. (C) Single-turnover GTP hydrolysis activities of wild-type or indicated mutant Gαi1 subunits. (D) Initial rates of GTP binding and hydrolysis for the Gαi1(R178M/A326S) mutant, as well as other Gαi1 point mutants derived from data similar to that of panels B and C. Radiolabeled nucleotide binding and single-turnover GTPase assays Assessments of spontaneous GDP release and single-turnover GTP hydrolysis rates by wild-type and mutant Gαi1 subunits, using measurements of [35S]GTPγS binding and [γ-32P]GTP hydrolysis, respectively, were conducted exactly as previously described.24,25 Briefly, for [35S]GTPγS binding by 100 nM of Gαi1 subunits at 20 °C, timed aliquots were removed, filtered through nitrocellulose, and washed 4 times with 10 mL of wash buffer before scintillation counting. Assays were conducted in duplicate, counts were subtracted from analogous reactions in “nonspecific binding” buffer,24 and normalized data were plotted as mean ± SEM. For single-turnover [γ-32P]GTP hydrolysis assays, Gαi1 subunits (100 nM) were prebound to [γ-32P]GTP in the absence of Mg2+ for 10 min at 30 °C. Reactions were then initiated by the addition of 10 mM MgCl2 (final concentration), and the production of 32Pi was measured by activated charcoal filtration and liquid scintillation counting.9,25 Initial rates were obtained by data analysis using GraphPad Prism (La Jolla, CA). Radiolabeled nucleotide steady-state GTPase assays Assessments of steady-state [γ-32P]GTP hydrolysis rates by wild-type and mutant Gαi1 subunits were conducted essentially as previously described.26 Briefly, Gαi1 protein was diluted to Journal of Biomolecular Screening 14(10); 2009 50 nM in a buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 0.05% C12E10, 1 mM dithiothreitol (DTT), 5 mM EDTA, 10 mM MgCl2, and 5 µg/ml bovine serum albumin (BSA). Assays were initiated with the addition of [γ-32P]GTP (and RGS4 if used), aliquots stopped at indicated time intervals, and free [γ-32P]Pi quantified as previously described.26 Transcreener GDP assays Standard curves and GTPase reactions were both run at 30 °C in kinetic mode on a Tecan Safire2 multiwell reader in Corning 384-well black round-bottom, low-volume polystyrene nonbinding surface microplates (part 3676). Fluorescence polarization was read using 635-nm excitation (20 flashes per well) and 670-nm emission. A free tracer reference was set to 20 mP by adjusting the photomultiplier tubes, and buffer containing GDP antibody alone was used as a blank for sample and reference wells. EC50 and EC85 values, Hill slopes, and curves were generated by GraphPad Prism. Unless otherwise indicated, reactions were run in 20 mM Tris (pH 7.5), 1 mM EDTA, 10 mM MgCl2, 10 µM GTP, 8 µg/mL GDP antibody, and 2 nM tracer in a final 20-µL volume. GDP antibody was used at a concentration 85% of the amount required for saturated binding to tracer (i.e., the EC85). Where shown, polarization data were converted to the amount of GDP produced using standard curves. Reaction rates were then determined in GraphPad www.sbsonline.org 1197 Zielinski et al. Prism using linear regression to estimate slope. For GTPase and GAP assays, reactions were started with the addition of GTP with or without RGS protein. Compound interference test To assess the robustness of the Transcreener GDP assay for practical screening applications, we performed a control screen using the GenPlus library of 960 bioactive molecules from Microsource Discovery Systems, many of which are approved drugs. GDP assay reagents (as denoted above) were added to duplicate wells containing 10 µM compound and either 10 µM GTP to mimic no-enzyme control reactions or 9 µM GTP plus 1 µM GDP to mimic completed enzyme reactions in 1% DMSO. Pilot screen and counterscreen of GenPlus Library Screens of the GenPlus library with the Transcreener GDP assay (10 µM final compound concentration) for modulators of RGS4 GAP activity on Gαi1(R178M/A326S), as well as for nonspecific modulators of intrinsic GTPase activity of Gαi1(R178M/ A326S) alone, were conducted as mentioned above with the following changes. GTPase reactions containing 50 nM Gαi1(R178M/ A326S) with or without 250 nM RGS4 were run in Corning 384-well microplates at 30 °C in 20 mM Tris (pH 7.5), 1 mM EDTA, 10 mM MgCl2, 10 µM GTP, 12 µg/µL GDP antibody, 2 nM tracer, and 0.5% DMSO (v/v) in a final volume of 20 µL. Fluorescence polarization was read at 60, 90, 120, and 180 min of elapsed reaction time on a Tecan Safire2 multiwell reader as described above. Surface plasmon resonance spectroscopy Optical detection of surface plasmon resonance (SPR) was performed using a BIAcore 3000 (GE Healthcare, Piscataway, NJ). Wild-type and mutant Gαi1 proteins were immobilized onto nickel-nitrilotriacetic acid SPR sensor chips (GE Healthcare) by hexahistidine tag-mediated capture coupling as previously described.27 Affinities of RGS proteins for immobilized Gαi1 proteins were obtained from dose-response sensorgrams using equilibrium saturation binding analyses as previously described.24 RESULTS AND DISCUSSION Profiling multiple Gαi1 point mutations for nucleotide cycling rate alterations Using PCR-based site-directed mutagenesis, we created several amino acid substitutions at various positions within Gαi1 known to affect koff(GDP) and/or kcat(GTPase) (e.g., Fig. 1). These mutants included aspartate, serine, or threonine replacing Ala-326; cysteine, lysine, or methionine replacing Arg-178; alanine, serine, or valine replacing Thr-181; single mutants 1198 www.sbsonline.org K192A and F336A; and double mutants K192A/F336A, R178C/A326S, R178C/A326T, R178M/A326S, R178C/A326T, and T181A/A326S. Note that multiple different substitutions were made at several sites, including amino acids intended to be more or less disruptive than the original reported mutation. For instance, R178K and R178M were tested as more conservative substitutions at the catalytic arginine position relative to the original R178C variant; it was thought that either of these alternative substitutions might result in a smaller decrease in kcat(GTPase) than the cysteine replacement, which reduces kcat(GTPase) by 2 orders of magnitude.15 Although the R178C mutation leads to a substantial decrease in kcat(GTPase), Berman et al.6 have shown that the single-turnover GTPase rate of this Gα mutant can still be increased by RGS domain-mediated GAP activity, whereas the more conventional GTPase-crippling mutation of Q204L renders Gαi1 truly dead in terms of responsiveness to RGS proteins. Thus, the Gαi1(Q204L) mutant was not pursued in this study. Gαi1 mutants were initially profiled for enhanced GDP release and/or reduced GTPase rate sufficient to see a change in steady-state GTP hydrolysis upon RGS protein addition. This initial profiling led us to focus on 2 positions: Arg-178 and Ala326. Binding of the nonhydrolyzable GTP analog, [35S]GTPγS, to Gαi1⋅GDP was used to measure the rate of GDP dissociation (e.g., Fig. 1B); the prevailing assumption for Gα subunits is that kon for [35S]GTPγS binding is much more rapid than koff(GDP).28 Single-turnover GTP hydrolysis measuring 32Pi released from Gα-bound [γ-32P]GTP—an assay format that is not rate limited by GDP dissociation9—was used to assess intrinsic kcat rates for the Gαi1 mutants (e.g., Fig. 1C). As expected, Gαi1 variants with mutation to the active-site catalytic residue Arg-178 had very low or undetectable levels of GTP hydrolysis, whereas Gαi1(A326S), the single mutation reported to only affect GDP dissociation, had a GTPase rate similar to wild-type Gαi1 (Fig. 1C,D). [35S]GTPγS binding assays showed that 2 variants with mutations only at the catalytic site, R178M and R178C, had GDP dissociation rates similar to wild-type Gαi1, whereas introduction of the A326S mutation, either alone or in combination with R178C, caused a 3-fold acceleration in GDP dissociation (Fig. 1B,D). When the A326S mutation was combined with methionine at Arg-178 (instead of cysteine), the GDP dissociation rate increased more than 10-fold over wild-type: from 0.008 min–1 to 0.130 min–1 (Fig. 1D). We currently do not have a precise structural explanation for why the particular combination of R178M and A326S mutations results in more rapid GDP release than the single A326S mutation alone; it is not an additive effect because the singly mutated Gαi1(R178M) variant exhibits wild-type GDP dissociation (Fig. 1B). It is interesting to note that Posner and colleagues,16 when reporting the crystal structure of the Gαi1(A326S) mutant, suggested the presence of an indirect interaction between the Arg-178 and Ser-326 residues (via contacts Journal of Biomolecular Screening 14(10); 2009 Fluorescent Steady-State RGS Protein GAP Assay FIG. 2. RGS4 GTPase-accelerating protein (GAP) activity is observed as an increase in steady-state guanosine triphosphate (GTP) hydrolysis only for the rate-altered Gαi1(R178M/A326S) variant. Time courses of steady-state [γ-32P]GTP hydrolysis by (A) 50 nM Gαi1(R178M/A326S) mutant or (B) 50 nM wild-type Gαi1 in the presence or absence of 250 nM RGS4 at 20 °C. Results are the mean (± SEM) of duplicate samples. with nucleotide and Gly-45), thereby providing a possible mechanism for the functional interaction we have observed here between the R178M and A326S mutations. Combined action of 2 Gαi1 mutations allows steady-state measurement of GAP activity With the R178M/A326S mutant of Gαi1 demonstrating the largest change in GDP release rate of all mutants tested, we next examined whether this particular Gαi1 variant would be affected by RGS domain-mediated GAP activity in steadystate [γ-32P]GTP hydrolysis assays. Addition of purified RGS4 protein to the Gαi1(R178M/A326S) variant (in the presence of free [γ-32P]GTP and Mg2+) resulted in a dramatic increase in [32P]Pi detected over time. In contrast, there was no effect of RGS4 on wild-type Gαi1 in this steady-state assay (Fig. 2A vs. 2B), as expected given the original report by Berman et al.6 Development of a Transcreener GDP assay The Transcreener platform relies on highly selective antibodies for detection of nucleotides produced in enzyme reactions.29 To allow measurement of RGS protein-mediated acceleration of steady-state GTP hydrolysis in a homogeneous format without radioactivity, we developed a Transcreener assay for GDP (Fig. 3A) using a competitive fluorescence polarization immunoassay format. For this method, a recently developed monoclonal antibody that recognizes GDP with >100-fold higher affinity than GTP19 is added to the reaction, along with a fluorescent tracer that binds to the antibody with high affinity. When no free, unlabeled GDP is present in the reaction, the fluorescent tracer remains antibody bound and exhibits a high polarization given its high apparent molecular weight. GDP produced in the reaction displaces the tracer from Journal of Biomolecular Screening 14(10); 2009 the antibody, thereby reducing its apparent molecular weight, increasing its rotational motion, and thus reducing the degree of polarization of emitted light. A similar Transcreener assay has been widely used for detection of ADP produced by kinases and other ATP-hydrolyzing proteins (e.g., Huss et al.,20 Klink et al.,21 Liu et al.22; reviewed in Lowery and Kleman-Leyer29). Figure 3B shows typical fluorescence polarization standard curves mimicking the conversion of GTP to GDP by a GTPase. An important aspect of flexibility for a GTPase assay is the ability to accommodate a range of initial GTP concentrations, so that diverse enzymes and screening strategies can be employed; therefore, these studies were performed using different GTP concentrations of 1, 10, and 100 µM. Because the antibody cross-reacts to some degree with GTP, its concentration must be increased as higher GTP concentrations are used, to buffer for the total guanine nucleotide pool. Thus, for this analysis, the EC85 concentrations of monoclonal antibody were first determined in the presence of the indicated initial GTP concentrations (2.2, 12, and 64 µg/mL Ab for 1, 10, and 100 µM GTP, respectively), and the standard curves for GTP to GDP conversion were performed with 16 replicates at those antibody concentrations. At a GDP concentration equivalent to 10% conversion of GTP, which is generally considered to be well within the initial velocity region, polarization shifts of 108, 134, and 148 mP were observed for the 1-, 10-, and 100-µM GTP concentration curves, respectively (Fig. 3B). Acceptable Z′ factor values30 of greater than 0.5 were observed down to 2% conversion for the 2 higher initial GTP concentrations and to 5% for the 1-µM initial GTP curve (Fig. 3C), suggesting that the Transcreener GDP assay should be capable of very robust detection of GTPase enzyme initial velocity over at least a 100-fold range of initial GTP concentration. To assess the potential for compound interference with the Transcreener GDP assay readout, we performed a control screen www.sbsonline.org 1199 Zielinski et al. FIG. 3. Fluorescence polarization immunoassay for the detection of guanosine diphosphate (GDP). (A) Schematic representation of methodology underlying the Transcreener GDP assay as applied to steady-state guanosine triphosphate (GTP) hydrolysis (and resultant GDP production) by a ratealtered Gα protein. Fluorescent tracer is illustrated with a jagged oval; when bound to the GDP-selective monoclonal antibody, emitted light remains polarized, whereas there is low polarization of emitted light when tracer is displaced by free GDP. (B) The Transcreener GDP assay was used to generate standard curves for conversion of GTP into GDP at initial GTP concentrations of 1, 10, and 100 µM using appropriate EC85 concentrations of GDP antibody established for these initial GTP concentrations (2.2, 12, and 64 µg/mL, respectively). (C) Z′ factor values, reflecting both assay signal window and signal variability,30 were determined in 16 replicates for each of the points in the GDP detection standard curves presented in panel B. Although the assay window was reduced at lower percent conversions (e.g., 57 mP for 3% conversion to GDP of 10 µM initial GTP; panel B), acceptable Z′ factors30 of >0.5 were observed down to 2% conversion for the 2 higher initial GTP concentrations and to 5% for the 1-µM initial GTP curve, given the very low signal variability. (D) Control screen using the 960-compound GenPlus library. Assay components were added to duplicate wells containing 10 µM compound and either 10 µM GTP (to mimic no-enzyme control reactions) or 9 µM GTP plus 1 µM GTP (to mimic completed GTPase reactions). The range of signal observed (3 standard deviations about the mean; “µ ± 3σ”) in each condition is demarked with a dotted gray line. (Fig. 3D) with the GenPlus library of 960 bioactive molecules, many of which are approved drugs. This control screen was done under conditions mimicking 10% conversion to GDP for a GTPase reaction run at 10 µM initial GTP concentration. All wells were run in duplicate. The vast majority of the compounds clustered very tightly around the means for the 10-µM GTP and the 9-µM GTP/1-µM GDP conditions; the Z′ factor for the nocompound controls in this screen was 0.93. Only 3 compounds in the control screen caused the signal to vary more than 3 standard deviations from the mean: dirithromycin, metazolamide, and lonidamin (Fig. 3D). There is no obvious structural similarity 1200 www.sbsonline.org between them, nor are any of them similar in structure to a guanine nucleotide. These data suggest that compound interference with the Transcreener GDP assay readout will be minimal. Fluorescence polarization–based detection of RGS protein GAP activity is dependent on 2 rate-altering mutations Having validated the utility of the Transcreener assay in detecting GDP in the presence of GTP, we next tested its use in measuring RGS protein GAP activity on several rate-altered Gαi1 variants (Fig. 4). In these experiments, the Gαi1 proteins Journal of Biomolecular Screening 14(10); 2009 Fluorescent Steady-State RGS Protein GAP Assay FIG. 4. RGS4 increases the steady-state GTPase activity of Gαi1(R178M/A326S) but not wild-type Gαi1, as measured using the Transcreener guanosine diphosphate (GDP) assay and reported in absolute change in polarization (left panels) and GDP produced per Gα protein in reaction (right panels). (A,B) R178M/A326S double-mutant and (C,D) wild-type Gαi1 proteins were present at a 50-nM final concentration. Dashed lines represent reactions conducted in the presence and solid lines (“mock”) in the absence of 250 nM RGS4 protein. (A,C) Change in polarization (∆mP) at each time point for indicated Gαi1 protein was calculated as ∆mP = |mP(Gαi1) – mP(no Gαi1)|. (B,D) Data from panels A and C were converted to GDP produced per mol of input Gαi1 using previously established standard curves for GDP detection in the presence of guanosine triphosphate (GTP) (e.g., Fig. 3B). (E) Summary of initial rates obtained by the Transcreener GDP assay for each Gαi1 mutant tested. “GAP factor” is defined as the ratio between steady-state GTPase rate in the presence of RGS protein and steady-state GTPase rate in the absence of RGS protein. were incubated with and without the well-characterized, Gαi-directed RGS protein RGS431 in the presence of the Transcreener GDP assay reagents, and plates were read at intervals starting at 15 min. The change in the absolute value of polarization at each time point (∆mPt = |mPt(Gαi1) – mPt(no G αi1)|) was plotted over a time course of 6 h; in addition, the plotted change in polarization that occurred in the linear region (over the first hour) was converted to GDP formation using standard curves (akin to those of Fig. 3B) and normalized to the amount of Gαi1 protein present in the reaction, with Journal of Biomolecular Screening 14(10); 2009 the resultant initial rates of GTP hydrolysis calculated from the data shown in Figure 4E. The 2 Gαi1 variants with single mutations at the catalytic arginine only, R178C or R178M, each had lower steady-state GTPase activity than wild-type Gαi1 and, like wild-type, were unaffected by RGS4 (GAP factors of 0.9 and 1.2, respectively; Fig. 4E). These results are expected because their steady-state GTPase rate is limited by slow GDP dissociation. Conversely, the A326S variant exhibited a much higher steady-state GTPase rate than wild-type, as expected from its higher koff(GDP) (Fig. 1); www.sbsonline.org 1201 Zielinski et al. FIG. 5. Structural features of the RGS16/Gαi1⋅GDP⋅AlF4– complex highlighting the locations of Arg-178 and Ala-326 residue positions mutated in the Gαi1(R178M/A326S) variant. The RGS16/Gαi1 complex (PDB id 2IK8; Soundararajan et al.24) was rendered using PyMOL with the RGS16 RGS domain in orange and Gαi1 protein in blue, respectively. Gαi1 switch regions are depicted in gray; switches 1 and 2 (SI, SII) are visible in the foreground, whereas switch 3 is in the background and thus unlabeled. GDP is shown in magenta, the AlF4– ion is red, and the Mg2+ ion is depicted as a yellow sphere. Residues arginine-178 and alanine-326 are rendered as “sticks” in green with CPK atomic coloring (nitrogen = blue, oxygen = red). however, its steady-state GTPase rate was unaffected by RGS4 (GAP ratio of 1.1; Fig. 4E), presumably because a further rate increase in GTPase is limited by koff(GDP). Most important, the 2 double mutants, R178C/A326S and R178M/A326S, had very low basal steady-state GTPase activities that became demonstrably higher in the presence of RGS4 (e.g., Fig. 4A,B); the GAP effect on Gαi1(R178M/A326S) was greater than with the Gαi1(R178C/A326S) variant (GAP factors of 6.5 and 3.6, respectively; Fig. 4E). Given its high GAP factor response in both the steady-state [γ-32P]GTP hydrolysis assay (Fig. 2) and the Transcreener GDP assay (Fig. 4), the Gαi1(R178M/A326S) variant was used in subsequent analyses. Gαi1(R178M/A326S) interacts with RGS proteins with the same affinity and specificity as wild-type A possible concern about the use of a mutated Gα protein for RGS protein GAP assays is that the mutation(s) could disrupt the global fold of Gα or, at the very least, affect the disposition 1202 www.sbsonline.org of the switch regions and other surface contact points to which RGS proteins interact,24,31 thereby altering the normal affinity and specificity that RGS proteins show for their various Gα substrates. The 2 point mutations of R178M and A326S are interior to the guanine nucleotide binding pocket (Fig. 5) but could nevertheless affect the RGS domain interaction surface. To test for this possibility, we used SPR to compare the binding interactions of RGS2 and RGS16 with wild-type Gαi1 versus the Gαi1(R178M/A326S) variant. Multiple previous studies8,24,32 have established that RGS2, a potent GAP for Gαq, does not interact with wild-type Gαi1 in vitro; this same lack of interaction was observed with the Gαi1(R178M/A326S) mutant (data not shown). Conversely, RGS16 is known to be a Gαiinteracting RGS protein24 and was found by SPR to bind equivalently to immobilized wild-type Gαi1 and Gαi1(R178M/ A326S) proteins (Fig. 6). This equivalence included RGS16 only interacting with high affinity to the Gα subunits in their transition state-mimetic form (namely, Gα in complex with GDP and aluminum tetrafluoride31). These binding results Journal of Biomolecular Screening 14(10); 2009 Fluorescent Steady-State RGS Protein GAP Assay FIG. 6. RGS16 binds equivalently to wild-type Gαi1 and the rate-altered Gαi1(R178M/A326S) mutant. (A,B) Sensorgrams derived from 600-s injections of various concentrations (3 nM to 10 µM) of RGS16 over surface plasmon resonance (SPR) biosensors of immobilized (A) wild-type Gαi1⋅GDP⋅AlF4– or (B) Gαi1(R178M/A326S)⋅GDP⋅AlF4–. SPR experiments were also conducted with both Gαi1 subunits in their inactive, guanosine diphosphate (GDP)–bound state (data not shown). (C,D) Resultant sensorgrams were used in equilibrium saturation binding analyses (as previously described24) to derive dissociation constants (KD values). RGS16 bound to wild-type Gαi1⋅GDP⋅AlF4– with a dissociation constant of 124 nM (95% confidence interval of 76-174 nM; panel C), whereas RGS16 bound to Gαi1(R178M/A326S)⋅GDP⋅AlF4– with a dissociation constant of 115 nM (64-166 nM; panel D). Note that interactions were not observed (for either Gα subunit) when the Gα was GDP bound (as expected; Soundararajan et al.24 and Tesmer et al.31) or when RGS2 was injected (data not shown). suggest that no long-range perturbations have been made to the RGS domain interaction sites on Gαi1 by the 2 rate-altering mutations of R178M and A326S. Using the Transcreener GDP assay, we performed an additional test of the Gαi1(R178M/A326S) variant to control for any unintended changes the 2 point mutations could have engendered within Gα to alter its interaction specificity with various RGS proteins. With the same RGS protein spectrum used in the SPR binding experiments, we found that RGS2 (highly selective for Gαq over Gαi substrates) had no effect on increasing steady-state GTPase activity of Gαi1(R178M/ A326S), whereas RGS16 increased steady-state GTPase activity 12-fold over the basal rate (Fig. 7). Pilot screen for inhibitors of RGS4 GAP activity on Gαi1(R178M/A326S) Given the robust performance of the Transcreener GDP assay in the control screen for potential compound interference Journal of Biomolecular Screening 14(10); 2009 (Fig. 3D) and evidence that the 2 mutations to Gαi1 affected neither the affinity nor specificity of the Gα/RGS domain interaction (Figs. 6, 7), we proceeded to a pilot screen with Gαi1(R178M/A326S) and RGS4 using the GenPlus library of 960 bioactive molecules (Fig. 8). The screening window was first optimized by varying the concentrations of Gα and RGS protein inputs; at 50 nM Gαi1(R178M/A326S) and 250 nM RGS4, a maximal signal-to-background difference of 112 mP units was obtained after 120 min of elapsed reaction time before fluorescence polarization (FP) measurement. The thiol-reactive compound CCG-4986 was used in the screen as a positive control for RGS4 inhibition.13,33 The GenPlus library screen was conducted with Gαi1(R178M/A326S) and RGS4 (Fig. 8A); a separate counterscreen of the library was performed with Gαi1(R178M/A326S) and no RGS4 (Fig. 8B) to identify compounds having either nonspecific effects or modifying Gαi1 GTPase activity (rather than RGS4 GAP activity per se). Z′ factors of 0.60, 0.83, 0.83, and 0.82 were obtained at 60, 90, 120, and 180 min elapsed reaction time, as calculated based www.sbsonline.org 1203 Zielinski et al. FIG. 7. The steady-state GTPase activity of Gαi1(R178M/A326S) is increased by RGS4 and RGS16 but not by the Gαq-selective RGS2. Transcreener guanosine diphosphate (GDP) assays were performed as in Figure 4, using 250 nM of the indicated RGS protein. Moles of GDP produced per mol input Gαi1(R178M/A326S) protein were first plotted over time using GraphPad Prism and linear regression performed to determine steady-state GTPase rates. Presented bar graph denotes GTPase-accelerating protein (GAP) factors derived from these steady-state GTPase rates. on data from control wells containing either Gαi1(R178M/ A326S) only or Gαi1(R178M/A326S) plus RGS4. Note that these Z′ factor values reflect only the RGS4-dependent increase in GTPase activity and not the total observed GTPase activity relative to no-enzyme controls. The Z factor for the GenPlus library screen at the 120-min time point (shown in Fig. 8A) was 0.73, which was calculated by excluding values from wells containing hit compounds. Of the 960 compounds in the GenPlus library, 17 were initially considered hits in the RGS4/ Gα screen (i.e., those data points that fell outside the µ ± 3σ range). However, 10 of these 17 hits also resulted in a greater than ±3σ change in the mean signal within the Gα-only counterscreen and thus were excluded because these compounds are likely either to affect the Gα subunit or to otherwise interfere with the assay. Thus, the RGS4-specific hit rate was 0.7%: 7 compounds from the 960-compound library exhibited an modulatory effect on GDP production that was specific to RGS4stimulated GTPase activity (Fig. 8A vs. 8B). Six compounds (id # 62, 63, 244, 413, 524, 812) exhibited an RGS4-specific inhibitory effect (i.e., a change in polarization greater than the [mean + 3 SD] signal threshold), and 1 compound (#596) exhibited an RGS4-specific activating effect on GDP production (i.e., a change in polarization less than the [mean – 3 SD] signal threshold). This hit rate may be artificially high in this pilot screen given that the collection of compounds surveyed (GenPlus library) is not a diverse sampling of chemical space but a collection of US-, European-, and Japanese-approved drugs and other bioactive compounds. As expected,13,33 the thiol-reactive RGS4 inhibitor CCG-4986 consistently exhibited inhibition of RGS4-stimulated GDP production (Fig. 8A). 1204 www.sbsonline.org FIG. 8. Pilot screen and counterscreen using the 960-compound GenPlus library. Transcreener guanosine diphosphate (GDP) assay components were added to wells containing 50 nM Gαi1 (R178M/ A326S) with (A) or without (B) 250 nM RGS4 protein and 10 µM compound (in 0.5% [v/v] final concentration of DMSO), 150 µM of reactive RGS4 inhibitor CCG-4986, or 0.5% DMSO only, as indicated in the legends. The range of signal observed (3 standard deviations [σ] about the mean [µ]) is denoted by the dashed lines for the 960-compound library screen using RGS4 and Gαi1 (R178M/A326S) (black; panel A, coefficient of variation [CV%] = 8.8%) and library counterscreen using Gαi1 (R178M/A326S) alone (gray; panel B, CV% = 3.0%). Data in panel A were obtained at 120 min of elapsed reaction time; data in panel B were obtained after 210 min of elapsed reaction time, given the slower GTPase (and GDP production) rate of Gαi1 (R178M/A326S) in the absence of RGS4 GTPase-accelerating protein (GAP) activity. To our knowledge, the combined use of a GDP detection assay with a rate-altered Gα subunit represents a unique strategy to detect RGS protein GAP activity. Even though the 2 primary components of Gα catalysis, GTP hydrolysis rate and product release, were altered significantly by mutation, the resultant Gα subunits still served as functional substrates for the GTPaseaccelerating activity of RGS proteins. Using this doublemutation strategy to develop a steady-state RGS protein GAP Journal of Biomolecular Screening 14(10); 2009 Fluorescent Steady-State RGS Protein GAP Assay assay that is easy for the nonspecialist to perform and is well suited for HTS removes a major technical barrier preventing the exploration of RGS proteins as therapeutic targets. Moreover, Gαi1 is a substrate for the GAP activity of several RGS protein family members24 in addition to those we have tested here; thus, the reagents and methods that we have described should have broad applicability across the protein family. Employing the rate-altered Gαi1(R178M/A326S) mutant in a homogeneous, endpoint-based, enzymatic HTS assay will be useful in screening for RGS protein inhibitors, and unlike existing assays based on the RGS domain/Gα binding interaction,11-13 this enzymatic assay should also facilitate identification of small-molecule activators of RGS domain-mediated GAP activity. The lipid moiety phosphatidylinositol-3,4,5-trisphophate (PIP3) has been shown to bind to and thereby inhibit, in an allosteric fashion, the GAP activity of select RGS domains such as that of RGS423; Ca2+/calmodulin reverses this PIP3mediated inhibition by competing for the PIP3 binding site.23 A small molecule targeting this site of allosteric modulation over RGS domain GAP activity could potentially be quite valuable therapeutically in pathophysiological situations that may arise from a loss of RGS protein activity, such as RGS2 in hypertension34 and RGS4 in schizophrenia.35 ACKNOWLEDGMENTS Thanks to Drs. Christopher Johnston and Francis Willard (UNC) for initial discussions regarding rate-altering Gα mutations and Dr. Steve Hayes (BellBrook Labs) for discussion and critical appraisal of the manuscript. Work at BellBrook Labs was supported by NIH SBIR grant R43 NS059082, and work in the Siderovski lab was funded by National Institutes of Health (NIH) grant R01 GM082892. A.J.K. acknowledges early support from NIH training grant T32 GM008719 and current support from NIH fellowship F30 MH074266. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. REFERENCES 1. 2. 3. 4. 5. 6. Gilman AG: G proteins: transducers of receptor-generated signals. Annu Rev Biochem 1987;56:615-649. McCudden CR, Hains MD, Kimple RJ, Siderovski DP, Willard FS: G-protein signaling: back to the future. Cell Mol Life Sci 2005;62:551-577. Siderovski DP, Hessel A, Chung S, Mak TW, Tyers M: A new family of regulators of G-protein-coupled receptors? Curr Biol 1996;6:211-212. Koelle MR, Horvitz HR: EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins. Cell 1996;84:115-125. Druey KM, Blumer KJ, Kang VH, Kehrl JH: Inhibition of G-proteinmediated MAP kinase activation by a new mammalian gene family. Nature 1996;379:742-746. Berman DM, Wilkie TM, Gilman AG: GAIP and RGS4 are GTPaseactivating proteins for the Gi subfamily of G protein alpha subunits. Cell 1996;86:445-452. Journal of Biomolecular Screening 14(10); 2009 20. 21. 22. 23. Neubig RR, Siderovski DP: Regulators of G-protein signalling as new central nervous system drug targets. Nat Rev Drug Discov 2002;1: 187-197. Ingi T, Krumins AM, Chidiac P, Brothers GM, Chung S, Snow BE, et al: Dynamic regulation of RGS2 suggests a novel mechanism in G-protein signaling and neuronal plasticity. J Neurosci 1998;18:7178-7188. Ross EM: Quantitative assays for GTPase-activating proteins. Methods Enzymol 2002;344:601-617. Willard FS, Siderovski DP: Purification and in vitro functional analysis of the Arabidopsis thaliana regulator of G-protein signaling-1. Methods Enzymol 2004;389:320-338. Willard FS, Kimple RJ, Kimple AJ, Johnston CA, Siderovski DP: Fluorescence-based assays for RGS box function. Methods Enzymol 2004;389:56-71. Willard FS, Kimple AJ, Johnston CA, Siderovski DP: A direct fluorescence-based assay for RGS domain GTPase accelerating activity. Anal Biochem 2005;340:341-351. Roman DL, Talbot JN, Roof RA, Sunahara RK, Traynor JR, Neubig RR: Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay. Mol Pharmacol 2007;71: 169-175. Sondek J, Lambright DG, Noel JP, Hamm HE, Sigler PB: GTPase mechanism of Gproteins from the 1.7-A crystal structure of transducin alpha-GDP-AIF-4. Nature 1994;372:276-279. Coleman DE, Berghuis AM, Lee E, Linder ME, Gilman AG, Sprang SR: Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. Science 1994;265:1405-1412. Posner BA, Mixon MB, Wall MA, Sprang SR, Gilman AG: The A326S mutant of Gialpha1 as an approximation of the receptor-bound state. J Biol Chem 1998;273:21752-21758. Nishina H, Nimota K, Kukimoto I, Maehama T, Takahashi K, Hoshino S, et al: Significance of Thr182 in the nucleotide-exchange and GTPhydrolysis reactions of the alpha subunit of GTP-binding protein Gi2. J Biochem (Tokyo) 1995;118:1083-1089. Marin EP, Krishna AG, Sakmar TP: Rapid activation of transducin by mutations distant from the nucleotide-binding site: evidence for a mechanistic model of receptor-catalyzed nucleotide exchange by G proteins. J Biol Chem 2001;276:27400-27405. Kleman-Leyer KM, Klink TA, Kopp AL, Westermeyer TA, Koeff MD, Larson BR, et al: Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay. Assay Drug Dev Technol 2009;7:56-67. Huss KL, Blonigen PE, Campbell RM: Development of a Transcreener kinase assay for protein kinase A and demonstration of concordance of data with a filter-binding assay format. J Biomol Screen 2007;12:578-584. Klink TA, Kleman-Leyer KM, Kopp A, Westermeyer TA, Lowery RG: Evaluating PI3 kinase isoforms using Transcreener ADP assays. J Biomol Screen 2008;13:476-485. Liu Y, Zalameda L, Kim KW, Wang M, McCarter JD: Discovery of acetyl-coenzyme A carboxylase 2 inhibitors: comparison of a fluorescence intensity-based phosphate assay and a fluorescence polarizationbased ADP assay for high-throughput screening. Assay Drug Dev Technol 2007;5:225-235. Popov SG, Krishna UM, Falck JR, Wilkie TM: Ca2+/calmodulin reverses phosphatidylinositol 3,4, 5-trisphosphate-dependent inhibition of regulators of G protein-signaling GTPase-activating protein activity. J Biol Chem 2000;275:18962-18968. www.sbsonline.org 1205 Zielinski et al. 24. 25. 26. 27. 28. 29. 30. 31. 1206 Soundararajan M, Willard FS, Kimple AJ, Turnbull AP, Ball LJ, Schoch GA, et al: Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits. Proc Natl Acad Sci USA 2008; 105:6457-6462. Johnston CA, Lobanova ES, Shavkunov AS, Low J, Ramer JK, Blaesius R, et al: Minimal determinants for binding activated G alpha from the structure of a G alpha(i1)-peptide dimer. Biochemistry 2006;45:11390-11400. Afshar K, Willard FS, Colombo K, Johnston CA, McCudden CR, Siderovski DP, et al: RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos. Cell 2004;119:219-230. Willard FS, Siderovski DP: Covalent immobilization of histidine-tagged proteins for surface plasmon resonance. Anal Biochem 2006;353:147-149. Ferguson KM, Higashijima T, Smigel MD, Gilman AG: The influence of bound GDP on the kinetics of guanine nucleotide binding to G proteins. J Biol Chem 1986;261:7393-7399. Lowery RG, Kleman-Leyer K: Transcreener: screening enzymes involved in covalent regulation. Expert Opin Ther Targets 2006;10:179-190. Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73. Tesmer JJ, Berman DM, Gilman AG, Sprang SR: Structure of RGS4 bound to AlF4-activated G(i alpha1): stabilization of the transition state for GTP hydrolysis. Cell 1997;89:251-261. www.sbsonline.org 32. Heximer SP, Watson N, Linder ME, Blumer KJ, Hepler JR: RGS2/G0S8 is a selective inhibitor of Gqalpha function. Proc Natl Acad Sci USA 1997;94:14389-14393. 33. Kimple AJ, Willard FS, Giguere PM, Johnston CA, Mocanu V, Siderovski DP: The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face. Biochim Biophys Acta 2007;1774: 1213-1220. 34. Heximer SP, Knutsen RH, Sun X, Kaltenbronn KM, Rhee MH, Peng N, et al: Hypertension and prolonged vasoconstrictor signaling in RGS2deficient mice. J Clin Invest 2003;111:445-452. 35. Mirnics K, Middleton FA, Stanwood GD, Lewis DA, Levitt P: Diseasespecific changes in regulator of G-protein signaling 4 (RGS4) expression in schizophrenia. Mol Psychiatry 2001;6:293-301. Address correspondence to: David P. Siderovski, Ph.D. UNC–Chapel Hill 4073 Genetics Medicine Bldg, 120 Mason Farm Road, CB#7365 Chapel Hill, NC 27599-7365 E-mail: dsiderov@med.unc.edu Journal of Biomolecular Screening 14(10); 2009