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J. Biol. Chem., Vol. 277, Issue 9, 6767-6770, March 1, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/9/6767    most recent C100699200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peterson, Y. K. Articles by Lanier, S. M. Search for Related Content PubMed PubMed Citation Articles by Peterson, Y. K. Articles by Lanier, S. M.

ACCELERATED PUBLICATION
Identification of Structural Features in the G-protein Regulatory Motif Required for Regulation of Heterotrimeric G-proteins^*

Yuri K. Peterson, Starr Hazard III, Stephen G. Graber^§, and Stephen M. Lanier^¶

From the Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, New Orleans, Louisiana 70118, the  Department of Library and Informatics, Medical University of South Carolina, Charleston, South Carolina 29425, and the ^§ Department of Biochemistry and Molecular Pharmacology, West Virginia University School of Medicine, Morgantown, West Virginia 26506

Received for publication, December 3, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The G-protein regulatory (GPR) motif, a conserved 25-30 amino acid domain found in multiple mammalian proteins, stabilizes the GDP-bound conformation of G_i, inhibits guanosine 5'-O-(3-thiotriphosphate) (GTPS) binding to G_i and competes for G binding to G. To define the core GPR motif and key amino acid residues within a GPR peptide (TMGEEDFFDLLAKSQSKRMDDQRVDLAG), we determined the effect of truncation, insertion, and alanine substitutions on peptide-mediated inhibition of GTPS binding to purified G_i1. The bioactive core GPR peptide consists of 17 amino acids (⁷F-R²³). Within this core motif, two hydrophobic sectors (⁷FF⁸ and ¹⁰LL¹¹) and Q²² are required for bioactivity, whereas M19A and R23A increased IC₅₀ values by 70-fold. Disruption of spatial relationships between the required sectors in the amino and carboxyl regions of the peptide also resulted in a loss of biological activity. Mutation of three charged sectors (⁴EED⁶, R¹⁸, ²⁰DD²¹) within the 28-amino acid GPR decreased peptide affinity by ~10-fold. Alanine substitutions of selected residues within the core GPR peptide differently influenced peptide inhibition of GTPS binding to G_i versus G_o. These data provide a platform for the development of novel, G-protein-selective therapeutics that inhibit G_i-mediated signaling, selectively activate G-sensitive effectors, and/or disrupt specific regulatory input to G-proteins mediated by GPR-containing proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The activation/deactivation cycle of heterotrimeric G-proteins, key players in cell signaling events, involves guanine nucleotide exchange, GTP hydrolysis, and a number of dynamic, conformationally sensitive protein interactions. In addition to the extensively studied activation of G-proteins by the superfamily of G-protein-coupled receptors, the G-protein activation/deactivation cycle is regulated by nonreceptor proteins that influence subunit interactions, GTPase activity, and guanine nucleotide binding properties of G. One signature motif for such regulatory proteins is the regulator of G-protein signaling (RGS)¹ domain, a ~120-amino acid motif found in all members of the RGS family (1). Another signature motif (~25-30 amino acids) is defined by the G-protein regulatory (GPR) domain in activator of G-protein signaling (AGS) 3 (2-6), which was discovered in a functional screen for receptor-independent activators of G-protein signaling. The GPR motif was also recognized in RGS12 and RGS14 by general sequence analysis/alignment and termed the GoLOCO motif (7, 8).

Surprisingly, interaction of the GPR motif with G_i stabilizes the GDP bound conformation of G_i, competes with G for G binding, and inhibits guanine nucleotide exchange (3-6, 9-12). Thus, the GPR motif acts as a guanine nucleotide dissociation inhibitor of G_i. The GPR motif is evolutionarily conserved within individual orthologs and among proteins with apparently diverse functions (see S.M.A.R.T. data base at dylan.embl-heidelberg.de/). Four spatially conserved GPR motifs are found in AGS3 (3) and LGN (13), which were isolated as G_i-regulatory/binding proteins. Recombinant AGS3 constructs with more than one GPR motif actually bind more than one G_i at the same time (5), suggesting a scaffolding role for such proteins. The AGS3/LGN-related protein PINS, which plays key roles in cell polarity (14-17), possesses a similar domain structure. The interaction of the PINS protein GPR domains with G is involved in the function of PINS in cell polarity and asymmetric cell division (17). AGS3 is also involved in synaptic adaptation in rat models of addiction (22). Single GPR motifs are found in Rap1GAP, Pcp2, RGS12, and RGS14, which are all implicated as G-protein regulators. Protein interaction studies and/or functional screens in yeast indicate that the AGS3 GPR motif interacts with G_i1-3, but not G_s, G_q, G_z, G₁₂, or G₁₆ (3, 5, 11). Specific GPR motifs are capable of interacting with G_o, albeit with apparently lower affinity than observed for G_i (10). Rap1GAP was actually isolated in yeast two-hybrid screens using G_o (18) and G_z (19), whereas Pcp2 was isolated in similar screens using G_o (20). Thus the GPR motif appears to serve as a discrete motif to anchor a variety of proteins that influence the guanine nucleotide binding/hydrolysis properties of G-proteins.

The existence of such a fairly discrete and highly conserved binding motif that inhibits GDP dissociation is of particular interest. As a first step toward developing a small organic molecule that would mimic the action of a GPR peptide, we defined the key structural features of the GPR motif required for biological activity. These data provide a platform for the development of novel, G-protein-selective therapeutics that target this critically important signaling protein within the cell. Such agents might inhibit G-mediated signaling by G-protein-coupled receptors, selectively activate G-sensitive effectors, and/or disrupt specific regulatory input to G-proteins mediated by GPR-containing proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Peptides were synthesized and purified by Bio-Synthesis, Inc. (Lewisville, TX) and peptide identity verified by matrix-assisted laser desorption ionization mass spectrometry. Peptides were synthesized with an acetylated amino terminus and an amidated carboxyl terminus. All other materials were obtained as described elsewhere (3-5).

GTPS Binding and Protein Interaction Assays-- GTPS binding assays and protein interaction assays were conducted as described previously (4, 5). The GPR domain of AGS3 (⁴⁶³P-S⁶⁵⁰) was generated as a gutathione S-transferase fusion protein in pGEX4T1. GST-AGS3 was expressed in and purified from BL21 bacteria using glutathione-Sepharose 4B. G_i1 and G_o were purified in the GDP bound state from Sf9 insect cells infected with recombinant virus as described previously (21). Concentration response curves with GPR peptides were analyzed by PRISM (Graphpad Software, Inc, San Diego, CA) to calculate IC₅₀ values.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Stabilization of the GDP-bound conformation of G_i by a 28-amino acid peptide encompassing the GPR motif presents an unexpected aspect of regulation within the G-protein activation/deactivation cycle. This activity of the peptide essentially prevents nucleotide exchange on G and binding of GTP to G-protein. The GPR-G_iGDP complex is quite stable and is observed in the absence and presence of added magnesium, which is generally required for high affinity binding of GTPS (Fig. 1). As part of a broad approach to define the structural properties of this regulation and develop a small molecule ligand that would target this novel regulatory site on G subunits, we defined the core GPR motif and the key amino acid residues within this core motif. A panel of peptides derived from the GPR motif were characterized in GTPS binding assays and protein interaction assays using purified mammalian G-protein  subunits.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of magnesium on inhibition of GTPS binding to Gi. GTP35S (500 nM) binding (using 0 and 25 mM MgCl2) to Gi (100 nM) was measured in absence and presence of increasing amounts of GPR peptide as described under "Experimental Procedures." Data are expressed as the percent of specific binding (~2 pmol) observed in the absence of added peptide and are expressed as the mean ± S.E. of two experiments with duplicate determinations.

Several conserved features constitute the GPR motif. The invariant Q¹⁵ essentially divides the peptide into two general regions of conserved residues (Fig. 2A). Both helical wheel and hydrophobic moment analysis indicate that the area upstream of Q¹⁵ likely exists as an alpha helix. We first asked if either of the two general regions of conserved residues (i.e. upstream and downstream of Q¹⁵) were active by themselves. Neither GPR1-15 nor GPR15-28 inhibited GTPS binding to G_i. The region upstream of Q¹⁵ is distinguished by the negatively charged ⁴EED⁶ followed by two hydrophobic sectors (⁷FF⁸, ¹⁰LL¹¹) (Fig. 2). The region downstream of the Q¹⁵ peptide is characterized by the conserved sequence ¹⁸RMDDQR²³. Deletion of the first eight residues at the amino terminus, which removed ⁷FF⁸ or mutation of R²³ to phenylalanine (3-5), resulted in an inactive peptide indicating that the minimal sequence for bioactivity is ⁷F-R²³ (Fig. 2A). The ²⁰DDQR²³ sequence is one of the most highly conserved regions of the peptide among different species and proteins. To test the importance of the spatial relationship between this region and the remainder of the conserved residues found elsewhere in the peptide, we inserted two and four alanines just upstream of the ²⁰DDQR²³ region. Insertion of the alanines completely abolished bioactivity in GTPS binding assays (Fig. 2A). Thus, these data indicated that there is a core GPR motif (residues 7-23) in which a defined spatial relationship among the conserved residues is required for bioactivity.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Identification of the core GPR motif. A: left panel, amino acid sequence of GPR peptide and truncated GPR peptides. Consensus amino acids are depicted in red. The right panel portrays GTPS binding (500 nM with 2 mM MgCl2) to Gi (100 nM) measured in the absence and presence of GPR peptides (100 µM) as described under "Experimental Procedures." Data are expressed as the percent of specific binding (~1 pmol) observed in the absence of added peptide and are expressed as the mean ± S.E. of two experiments with duplicate determinations. Similar results were obtained in two to five separate experiments. B: left panel, sequence alignment and IC50 values of mutated GPR peptides. Conserved amino acids are depicted in red, and mutated amino acids are colored green. Right panel, effect of increasing concentrations of competing peptides on GTPS (500 nM with 2 mM MgCl2) binding to Gi (100 nM). Data are expressed as the percent of specific binding (~1 pmol) observed in the absence of added peptide and are expressed as the mean ± S.E. of two experiments with duplicate determinations. Similar results were obtained in two to five separate experiments. Inactive, inhibition of GTPS binding was less than 30% at peptide concentrations of 100 µM.

We then addressed the relative importance of conserved amino acids within the core GPR motif by alanine substitutions and subsequent peptide evaluation in GTPS binding assays (Fig. 2B). Peptides with alanine substitutions at ⁷FF⁸, ¹⁰LL¹¹, and Q²² were inactive and thus identify the key residues of the peptide. Both ⁷FF⁸ and ¹⁰LL¹¹ likely constitute the core of the predicted alpha helix in this region, indicating the importance of this structural feature for bioactivity (Fig. 1). The importance of this region is further emphasized by loss of biological activity observed with an F⁸R mutation in the context of a GST-AGS3 fusion protein (3, 5, 17). The IC₅₀ values for D9A, K13A, Q15A, and S16A peptides were similar to the consensus GPR peptide (Fig. 2B). Alanine substitutions at the other key residues within the GPR motif indicated that the residues could be grouped as those causing 7-10-fold (⁴EED⁶, R¹⁸, and ²⁰DD²¹) or 50-70-fold (M¹⁹ and R²³) shifts in IC₅₀ values (Fig. 2B). The loss of affinity observed with the alanine substitutions at ⁴EED⁶ are consistent with the retention of bioactivity by the truncation mutant GPR3-24, whereas the loss of activity with the ⁷FF⁸ alanine substitutions was also observed by elimination of these residues in the GPR10-24 truncated peptide. The larger reduction in affinity observed with the M¹⁹ and R²³ alanine substitutions indicate the importance of these residues within the core GPR peptide. Indeed, reversal of charge or hydrophobicity at these residues (M19D and R23F) essentially rendered these peptides inactive (3-5).²

The inhibition of GTPS binding and apparent stabilization of the GDP-bound conformation of G_i by the GPR peptide likely involves two events, the initial binding of the peptide to G-protein, followed by a conformational change in G_i itself. To determine whether the two events can be functionally dissociated, we performed two series of experiments. We first asked if mutant peptides that exhibited lower affinity in GTPS binding assays were also deficient in inhibiting binding of a GPR-containing GST-AGS3 fusion protein to G_i. In the second series of experiments, we determined whether truncated GPR peptides could act in a complementary fashion and whether they could antagonize the action of the GPR consensus peptide. Data generated from this series of experiments indicated that the loss of function in binding studies was also reflected in the protein interaction assays (Fig. 3, A and B). The partial inhibition of GST-AGS3 binding to G-protein in the presence of GPR6-24 indicates that this peptide is not as potent as GPR3-24 or wild type peptide, likely due to the loss of the ⁴EED⁶ acidic cluster (Fig. 2, A and B, and Fig. 3B). These data indicated that G-protein interaction and inhibition of GTPS binding could not be dissociated from each other. This point is further emphasized by data generated in another series of experiments (Fig. 3C). A combination of the inactive GPR truncation peptides (GPR 1-15 and GPR 15-28) did not inhibit binding of GST-AGS3 to G_i. Neither of the peptides antagonized the action of the consensus GPR peptide nor were they able to rescue the activity of nonfunctional, alanine-substituted GPR peptides (Fig. 3). These data confirm the importance of the spatial relationship within the core GPR motif. These data also indicate that the GPR domain must function as an intact unit and that there are multiple points of contact between the GPR domain and G.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Effect of GPR peptides on binding of Gi to AGS3. Protein interaction assays were performed as described under "Experimental Procedures" using 75 nM Gi1 and 300 nM GST-AGS3 in the presence of 10 µM GDP. Peptide concentration is 100 µM in A and B. The peptide concentration was 10 µM in C except for the four lanes at the right of the panel where the truncated GPR peptides were present at 100 µM. Similar results were obtained in two to three separate experiments.

Although the GPR peptide clearly prefers G_i over G_o, subtle mutations within the core motif differentially effect peptide interaction with the two G-proteins (Fig. 4A). A screen of the mutant GPR peptides for selectivity indicated that inhibition of GTPS binding to G_o is completely eliminated by alanine substitutions within the core GPR motif that only minimally altered GTPS binding to G_i (Fig. 4A). The difference in the interaction of the GPR peptide with G_i versus G_o is also observed by comparing the relative inhibition of GTPS binding in the presence and absence of magnesium. The inhibition of GTPS binding to G_o is much more sensitive to magnesium as compared with the effects of the peptide on G_i (Fig. 4B). These data indicate that the GPR peptides can differentially target G-protein  subunits. Amino acids outside the GPR motif may also influence G-protein selectivity or bioactivity (see Discussion in Ref. 4).³

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of GPR peptides on GTPS binding to Gi versus Go. A, GTP35S binding (500 nM) to purified G-protein (100 nM) was determined as described under "Experimental Procedures" as detailed in the legend to Fig. 1 in the absence and presence of 10 µM peptide. Assays were conducted in parallel using the same stocks of GTPS and peptides. Data are expressed as the percent of specific binding (Gi ~ 2 pmol, Go ~ 1 pmol). B, GTP35S (500 nM) binding (using 0 and 25 mM MgCl2) to Gi or Go (100 nM) was measured in absence and presence of 100 µM GPR peptide as described under "Experimental Procedures." The increased GTPS binding to Go in the presence of selected peptides was also observed with similar amounts of bovine serum albumin, suggesting that it does not likely represent an actual increase in nucleotide exchange, per se, but rather enhanced protein recovery. Data are expressed as the percent of specific binding (~1 pmol for Gi and Go) observed in the absence of added peptide and are expressed as the mean ± S.E. of two experiments with duplicate determinations.

The core GPR motif identified in the present study is indicated in Sequence 1. 

Within this core, ⁷FF⁸, ¹⁰LL¹¹, and Q²² are absolutely required for bioactivity. The carboxyl terminus R²³ and the M¹⁹ residue also play key roles in bioactivity as their mutation results in large shifts in affinity. Within the larger GPR peptide (Sequence 2),

⁴EE⁵, ²⁰DD²¹, and the internal R¹⁸ all exert effects on affinity (~7-10-fold). Minimizing the core GPR motif to its essential pharmacophores identified the chemical moieties within the GPR motif that regulate G_i/G_o nucleotide exchange. By virtue of its novel mode of regulation of G-proteins, the core GPR motif provides an unexpected platform for the development of G-protein selective therapeutics.

	ACKNOWLEDGEMENTS

We thank Joe Blumer (Louisiana State University Health Sciences Center) for suggestions and review of this work. We also appreciate discussions with Michael Bernard (Medical University of South Carolina) and Stephen Sprang (University of Texas Southwestern); the footnoted communication from Randall Kimple, David Siderovski, and John Sondek (University of North Carolina School of Medicine); and the technical assistance of Jane Jourdan and Maureen Fallon.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants NS24821 and MH5993 (to S. M. L.) and National Science Foundation Grant MCB9870839 (to S. G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, MEB Suite 7103, LSUHSC, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-4744; Fax: 504-568-2361; E-mail: slanie@lsuhsc.edu.

Published, JBC Papers in Press, December 27, 2001, DOI 10.1074/jbc.C100699200

² Y. K. Peterson and S. M. Lanier, unpublished observations.

³ R. Kimple, J. Sondek, and D. P. Siderovski, personal communication.

	ABBREVIATIONS

The abbreviations used are: RGS, regulator of G-protein signaling; GPR, G-protein regulatory; AGS, activator of G-protein signaling; GTPS, guanosine 5'-O-(3-thiotriphosphate); GST, glutathione S-transferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/9/6767    most recent C100699200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peterson, Y. K. Articles by Lanier, S. M. Search for Related Content PubMed PubMed Citation Articles by Peterson, Y. K. Articles by Lanier, S. M.