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• W2048073308 abstract "The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein α subunits. To define the properties of this ∼20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Giα1,2 in protein interaction assays, inhibited guanosine 5′-O-(3-thiotriphosphate) binding to Giα, and stabilized the GDP-bound conformation of Giα. The GPR peptide had little effect on nucleotide binding to Goα and brain G-protein indicating selective regulation of Giα. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Giα. The GPR consensus peptide also blocked receptor coupling to Giαβγ indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Giα, this conformation of GiαGDPwas not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Giα- and Gβγ−regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems. The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein α subunits. To define the properties of this ∼20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Giα1,2 in protein interaction assays, inhibited guanosine 5′-O-(3-thiotriphosphate) binding to Giα, and stabilized the GDP-bound conformation of Giα. The GPR peptide had little effect on nucleotide binding to Goα and brain G-protein indicating selective regulation of Giα. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Giα. The GPR consensus peptide also blocked receptor coupling to Giαβγ indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Giα, this conformation of GiαGDPwas not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Giα- and Gβγ−regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems. G-protein regulatory tetratrico peptide repeats guanosine 5′-O-(3-thiotriphosphate) guanine nucleotide dissociation inhibitor hydroxy tryptamine glutathioneS-transferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid The G-protein regulatory (GPR)1 motif or GoLOCO repeat is a ∼20-amino acid domain found in several proteins that interact with and/or regulate G-proteins (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 2Siderovski D.P. Diverse-Pierlussi M.A. De Vries L. Trends Biochem. Sci. 1999; 24: 340-341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Such proteins include the activator of G-protein signaling AGS3, the AGS3-related protein PINS inDrosophila melanogaster, two members of the RGS family of proteins, and three proteins (LGN, Pcp2, and Rap1GAP) isolated in yeast two-hybrid screens using Giα or Goα as bait. Rat AGS3 was isolated in a yeast-based functional screen designed to identify receptor-independent activators of heterotrimeric G-protein signaling (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The AGS3-related protein PINS is required for asymmetric cell division of neuroblasts in D. melanogaster, where it is found complexed with Gi/Go (3Yu F.W. Morin X. Cai Y. Yang X.H. Chia W. Cell. 2000; 100: 399-409Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 4Schaefer M. Schevchenko A. Schevchenko A. Knoblich J.A. Curr. Biol. 2000; 10: 353-362Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), but neither the signal input nor output for this complex is known. Some insight as to how PINS may regulate Gi/Go is provided by studies with AGS3 (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). In the yeast-based system, AGS3 selectively activated Giα2 and Giα3. The action of AGS3 as a G-protein activator in the yeast-based system was independent of nucleotide exchange as it was not antagonized by overexpression of RGS4, and it was still observed following replacement of Giα2 with Giα2-G204A, a mutant that is deficient in making the transition to the GTP-bound state (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google Scholar). Both of these manipulations effectively prevent receptor-mediated activation of G-protein signaling in the yeast system and block the action of AGS1, which was isolated in the same screen and apparently behaves as a guanine nucleotide exchange factor for heterotrimeric G-proteins (5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google Scholar, 6Cismowski M. Ma C. Ribas C. Xie X. Spruyt M. Lizano J.S Lanier S.M. Duzic E. J. Biol. Chem. 2000; 275: 23421-23424Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). These data indicate that the interaction of AGS3 with G-protein influences a unique control mechanism within the activation/deactivation cycle of heterotrimeric G-proteins. AGS3 exists as a 650-amino acid protein enriched in brain and a 166-amino acid protein (AGS3-SHORT) enriched in heart (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). 2M. Bernard, Y. K. Peterson, P. Chung, and S. M. Lanier, submitted for publication.2M. Bernard, Y. K. Peterson, P. Chung, and S. M. Lanier, submitted for publication.,3 The 650-amino acid protein consists of two functional domains defined by a series of seven amino-terminal tetratrico peptide repeats (TPR) and four carboxyl-terminal GPR motifs. Site-directed mutagenesis, protein interaction studies, and subcellular localization experiments indicated that the GPR motifs of AGS3 were likely responsible for binding G-protein, whereas the TPR domain is a site for binding of regulatory proteins (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 3Yu F.W. Morin X. Cai Y. Yang X.H. Chia W. Cell. 2000; 100: 399-409Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 4Schaefer M. Schevchenko A. Schevchenko A. Knoblich J.A. Curr. Biol. 2000; 10: 353-362Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). 2M. Bernard, Y. K. Peterson, P. Chung, and S. M. Lanier, submitted for publication., 3N. Pizzinat, A. Takesono, and S. M. Lanier, submitted for publication. AGS3 preferentially binds to Gα in the presence of GDP (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). AGS3-GPR effectively competed with Gβγ subunits for binding to Gtα and inhibited guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding to Giα1.2 Such an activity likely has significance in a number of aspects of G-protein-mediated signaling events and presents a novel opportunity to control the basal activity of G-protein signaling, as well as influence receptor-mediated activation of G-protein. These observations also raise many interesting questions relative to basic aspects of G-protein structure/function and alternative modes of regulation and functional roles for G-protein signaling systems in the cell. To address these issues, we generated a series of peptides based upon the consensus GPR motif in AGS3 and evaluated their effects on the nucleotide binding properties of Giα. A 28-amino acid GPR peptide effectively blocked the interaction of AGS3 with Giα and inhibited GTPγS binding to Giα by a mechanism that involved stabilization of the GDP-bound conformation of Giα. The GPR consensus peptide also blocked receptor coupling to Giαβγ indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Giα, this conformation of GiαGDP was not recognized by a G-protein-coupled receptor. 35S-GTPγS (1250 Ci/mmol),3H-GDP (29.6 Ci/mmol), and 3H-5-hydroxy tryptamine (HT) (21.8 Ci/mmol) were purchased from PerkinElmer Life Sciences. Peptides were synthesized and purified by Bio-Synthesis, Inc. (Lewisville, TX), and peptide mass was verified by matrix-assisted laser desorption ionization mass spectrometry. GDP, GTPγS, and 5-HT were obtained from Sigma. Acrylamide, bisacrylamide, protein assay kits, and sodium dodecyl sulfate were purchased from Bio-Rad. Ecoscint A was purchased from National Diagnostics (Manville, NJ). CytoScint was purchased from ICN Biomedicals (Costa Mesa, CA). Thesit (polyoxyethylene-9-lauryl ether) was obtained from Roche Molecular Biochemicals. Polyvinylidene difluoride membranes were obtained from Pall Gelman Sciences (Ann Arbor, MI). Nitrocellulose BA85 filters were purchased from Schleicher & Schuell (Keene, NH). Whatman GF/C FP200 filters were purchased from Brandel Inc.(Gaithersburg, MD). Purified bovine brain G-protein was kindly provided by Dr. John Hildebrandt (Department of Pharmacology, Medical University of South Carolina) (7Dingus J. Wilcox M.D. Kohnken R. Hildebrandt J.D. Methods Enzymol. 1994; 237: 457-471Crossref PubMed Scopus (29) Google Scholar). All other materials were obtained as described elsewhere (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 8Clawges H.M. Depree K.M. Parker E.M. Graber S.G. Biochemistry. 1997; 36: 12930-12938Crossref PubMed Scopus (69) Google Scholar). The GPR domain of AGS3 (Pro463-Ser650) containing the four GPR motifs was generated as a glutathione S-transferase fusion protein by polymerase chain reaction using the full-length cDNA of AGS3 as a template. The AGS3-Pro463-Ser650segment was also cloned into the pQE-30 vector (Qiagen, Valencia, CA) to generate an amino-terminal His-tagged protein. His-tagged AGS3 was expressed in and purified from bacteria using a nickel affinity matrix (ProBond™ resin; Invitrogen, Carlsbad, CA). The His-tagged AGS3 was eluted from the matrix with imidazole and desalted by centrifugation as with the GST fusion protein (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The interaction of GST-AGS3-GPR and HIS-tagged AGS3-GPR with G-proteins was assessed by protein interaction experiments using purified G-protein as described previously (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Giα1–3 and Goα were purified from Sf9 insect cells infected with recombinant virus as described (8Clawges H.M. Depree K.M. Parker E.M. Graber S.G. Biochemistry. 1997; 36: 12930-12938Crossref PubMed Scopus (69) Google Scholar). All purified G-proteins used in these studies were isolated in the GDP-bound form, and G-protein interaction assays contained 10 μm GDP. A separate series of protein interaction experiments were designed to determine whether the Giα complexed with AGS3 contained bound GDP. Giα1 (100 nm) was loaded with 3H-GDP (0.5 μm; 2.0 × 104 dpm/pmol) by incubation for 20 min at 24 °C in binding buffer (50 mm Hepes-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, 50 μm adenosine triphosphate, and 10 μg/ml bovine serum albumin). The 3H-GDP-loaded Giα1 was incubated with 300 nm GST or GST-AGS3-GPR in the presence and absence of 10 μm GPR peptide and processed as described (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The washed resin containing bound proteins was transferred to vials for measurement of 3H-GDP by liquid scintillation spectroscopy. GTPγS binding assays were generally conducted as described (9Ross M. Higashijima T. Methods Enzymol. 1994; 237: 26-37Crossref PubMed Scopus (96) Google Scholar). G-proteins (100 nm) were preincubated for 20 min at 24 °C in the presence and absence of GPR peptides. Binding assays (duplicate determinations) were initiated by addition of 0.5 μm GTPγS (4.0 × 104 dpm/pmol), and incubations (total volume = 50 μl) were continued for 30 min at 24 °C. Both preincubations and GTPγS binding assays were conducted in binding buffer containing 2 mm MgCl2. Reactions were terminated by rapid filtration through nitrocellulose filters with 4 × 4-ml washes of stop buffer (50 mm Tris-HCl, 5 mmMgCl2, 1 mm EDTA, pH 7.4, at 4 °C). Radioactivity bound to the filters was determined by liquid scintillation counting. Nonspecific binding was defined by 100 μm GTPγS. Giα1 (100 nm) was loaded with 3H-GDP (0.5 μm; 2 × 104 dpm/pmol) by incubation for 20 min at 24 °C in binding buffer without MgCl2. 45-μl aliquots of the preincubation mixture (∼500,000 dpm) were then added to incubation tubes containing 5 μl of vehicle or peptide, and samples were incubated for 30 min at 24 °C. Two sets of tubes were set up for each time point to be analyzed. Each set contained duplicate samples for determination of total binding, nonspecific binding, or binding in the presence of peptide. For each time point, one set of tubes served as an internal time control, whereas the other set received added GTPγS or GDP to initiate dissociation. Data are expressed as % of control, where control represents the level of 3H-GDP binding at each time point in the set of tubes that did not receive added nucleotide to initiate dissociation. The amount of3H-GDP bound following the 20-min preincubation (∼30,000 dpm) was identical to that observed at the 30-min incubation time point following addition of vehicle or peptide. 3H-GDP dissociation was initiated by addition of GTPγS or GDP in a volume of 5 μl (final concentration, 100 μm). Reactions were terminated at specified time points by rapid filtration through nitrocellulose filters (BA85; Schleicher & Shuell) with 4 × 4-ml washes of stop buffer. Radioactivity bound to the filters was determined by liquid scintillation counting. Nonspecific binding was defined by 100 μm GDP. Sf9 cell membranes expressing 5-HT1A receptors were reconstituted with Gαβγ, and high affinity agonist binding was measured with3H-5-HT as described previously (8Clawges H.M. Depree K.M. Parker E.M. Graber S.G. Biochemistry. 1997; 36: 12930-12938Crossref PubMed Scopus (69) Google Scholar, 10Bae H. Anderson K. Flood L.A. Skiba N.P. Hamm H.E. Graber S.G. J. Biol. Chem. 1997; 272: 32071-32077Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Membrane aliquots (100 μg of membrane protein, 85 nm receptor) were preincubated for 15 min at 25 °C with G-proteins (2125 nm Gαβγ) with or without GPR peptides in a total volume of 17 μl (reconstitution buffer, 5 mm NaHEPES, 100 mm NaCl, 5 mm MgCl2, 1 mm EDTA, 500 nm GDP, 0.04% CHAPS, pH 7.5). The reconstitution mixtures were then diluted 10-fold with binding buffer (50 mm Tris-HCl, 5 mm MgCl2, 0.5 mm EDTA, pH 7.5), and 50 μl were added to binding tubes (total volume = 150 μl) containing 2 nm3H-5-HT. The final concentrations of receptor, G-protein, and peptide in the binding tubes were 2.8 nm, 70.8 nm, and 114 μm, respectively. Nonspecific binding was determined in the presence of 100 μm 5-HT. Binding reactions were incubated at 25 °C for 1.5 h and terminated by filtration over Whatman GF/C FP200 filters using a Brandel cell harvester. The filters were rinsed thrice with 4 ml of ice-cold washing buffer (50 mm Tris-Cl, 5 mmMgCl2, 0.5 mm EDTA, 0.01% sodium azide, pH 7.5, at 4 °C), placed in 4.5 ml of CytoScint, and counted to constant error in a scintillation counter. The ∼20-amino acid GPR motif is repeated four times in AGS3-related proteins, with the exception of the three repeats found in the Drosophila protein PINS (Fig.1). Alignment of the four GPR repeats from five species revealed a GPR consensus sequence (Fig. 1). The GPR consensus sequence is characterized by the upstream negative charge (Glu-Glu) and hydrophobic cluster (Phe-Phe), Leu/Met10, Leu/Ile11, Gln15, Ser/Ala16, Arg18, Met/Leu19, and the Asp-Asp-Gln-Arg sequence at the carboxyl end of the motif. Helical wheel and Chou-Fasman analysis indicated that this region is capable of existing as an amphipathic helix. Each of the GPR motifs illustrated in Fig. 1 possess a varying number of Proline residues just after and in some cases before the core consensus sequence, which may exert an important influence within the overall organization of the four GPR motifs. As part of an effort to define the structural basis of the interaction of AGS3 with Giα and the functional consequences of this interaction, we asked whether a consensus sequence peptide effectively interacted with Giα. The core GPR consensus sequence was bracketed by additional residues (three amino terminus, five carboxyl terminus) derived from AGS3-GPR-IV, and the carboxyl terminus was amidated (Fig. 1). The 28-amino acid GPR consensus peptide completely blocked the binding of Giα1 or Giα2 to GST-AGS3-GPR with an IC50 of ∼200 nm (Fig. 2 A andB). The GPR consensus peptide also inhibited GTPγS binding to Giα1 and Giα2 (IC50 ∼200 nm) (Fig. 2,C and D) consistent with the preferential binding of AGS3 to Giα in the presence of GDP (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The inhibitory effect of the GPR consensus peptide on GTPγS binding was selective for Giα as it only minimally affected nucleotide binding to Goα or brain G-protein (Fig. 2 D). The activity of the GPR consensus peptide in both the protein interaction assays and GTPγS binding assays was lost upon substitution of Phe for the highly conserved Arg23 (Fig. 2, B, C, andD). However, substitution of Ala for the invariant Gln15 did not alter the activity of the GPR peptide (Fig.2 B). 4Y. K. Peterson and S. M. Lanier, unpublished observations. Similar results were obtained when these amino acid substitutions were made in the context of GST-AGS3 fusion protein, which contained the terminal 74 amino acids of AGS3 including part of GPR-III and all of GPR-IV (Fig. 1) (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar).2 We then addressed the mechanism by which the GPR consensus peptide inhibited GTPγS binding to Giα2 and determined the effect of the GPR motif on receptor coupling to G-protein. The inhibition of GTPγS binding to Giα by the GPR consensus peptide may reflect a reduction in the rate of nucleotide exchange. Indeed, the rate of GDP dissociation was markedly diminished in the presence of the GPR consensus peptide (Fig.3 A). 5M. Natochin, B. Lester, Y. K. Peterson, M. L. Bernard, S. M. Lanier, and N. O. Artemyev, submitted for publication. The R23F mutation, which eliminated the effectiveness of the peptide to block interaction of AGS3 with Giα and GTPγS binding to Giα, also did not alter GDP dissociation (Fig. 3 A). The inhibition of GDP dissociation by the GPR consensus peptide suggests that the GPR motif is stabilizing the GDP-bound conformation of Giα. To address this issue we evaluated the interaction of GST-AGS3-GPR with Giα2, which had been preloaded with 3H-GDP. Subsequent analysis of the G-protein complexed with AGS3-GPR on the glutathione affinity matrix indicated that the nucleotide binding site of G-protein bound to AGS3 indeed contained GDP (Fig. 3 B). GiαGDP binding to AGS3-GPR was blocked by the GPR consensus peptide (Fig.3 B) consistent with the ability of this peptide to inhibit interaction of GST-AGS3-GPR with Giα1/2 (Fig. 2, A andB). The stabilization of the GDP-bound conformation of Giα by the GPR consensus peptide indicates that the AGS3-GPR motif can influence subunit interactions by interfering with Gβγ binding to Giα.2 This apparent effect may account for the results obtained in protein interaction assays using GST-AGS3-GPR and brain lysates, where Gβγ is absent from the AGS3-Giα complex (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The influence of the GPR motif on subunit interactions would have significant implications for signal processing. First, interaction of the AGS3-GPR motif with Gαβγ would release Gβγ for regulation of downstream signaling events, while stabilizing GαGDP(1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Such a mode of signal input may be of utility where there is a need for selective regulation of Gβγ-sensitive effectors. The time frame for termination of such a signaling event (i.e.reassociation of Gβγ with GiαGDP) likely differs from that of a more typical signaling event in which there has been an exchange of nucleotide bound to Giα, and signal termination involves GTP hydrolysis along with subunit reassociation. A second implication of stabilization of GαGDP by a GPR domain is related to receptor G-protein coupling. We addressed this issue experimentally using a membrane assay system where receptor-G-protein coupling is reflected as high affinity binding of agonists. The high affinity binding of agonist observed upon reconstitution of the membrane-bound 5-HT1 receptor with Giαβγ was inhibited by addition of the GPR peptide (Fig. 4). This action of the GPR peptide was not observed with the R23F peptide and was selective for Gi versus Go (Fig. 4). The influence of the single amino acid substitution on the bioactivity of the GPR both within the context of a short peptide and a GST fusion protein containing an additional 74 amino acids of AGS3 sequence strongly suggest a relatively discrete and specific surface interaction with Giα (Fig. 2) (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). 2M. Bernard, Y. K. Peterson, P. Chung, and S. M. Lanier, submitted for publication., 3N. Pizzinat, A. Takesono, and S. M. Lanier, submitted for publication., 4Y. K. Peterson and S. M. Lanier, unpublished observations., 5M. Natochin, B. Lester, Y. K. Peterson, M. L. Bernard, S. M. Lanier, and N. O. Artemyev, submitted for publication., 6H. Ma, M. L. Bernard, S. M. Lanier, and S. G. Graber, unpublished observations. Helical wheel projections and 3D models indicated that when the GPR consensus peptide is fixed in an α helical conformation, the Phe8, Ala12, Gln15, Met19, and Arg23 residues are on the same face of the helix. On this face of the helix is a hydrophobic sector defined by Phe8, Ala12, and Met19 that is bound by polar residues, which may be involved in charge pairing to residues in Giα. As was the case for the R23F substitution, disruption of this hydrophobic sector by substitution of Arg for Phe8 also resulted in a loss of activity for the GST-AGS3-GPR fusion protein in GTPγS binding and protein interaction assays (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar).2 Thus, either extension (R23F substitution) or shortening (F8R) of the hydrophobic sector on this face of the helix resulted in a loss of bioactivity for the GPR motif. In contrast, strengthening of this hydrophobic sector by substitution of Ala for Gln15 did not alter the activity of the GPR peptide. These data indicate an important role for a spatially constrained hydrophobic stretch of ∼16.6 Å that is key for peptide interaction with Giα. The inability of receptor to productively couple to GαGDP-GPR is of interest. The GαGDPconformation stabilized by the GPR peptide may differ from that stabilized by Gβγ in such a manner that the receptor cannot recognize Gα. Indeed, the orientations of the amino and carboxyl domains of Giα1, which are important interaction sites with receptor, are quite different in the GiαGDP and GiαGDPβγ structures (11Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (266) Google Scholar, 12Wall M.A. Coleman D.E. Lee E. Iniguezlluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1002) Google Scholar, 13Sprang S.R. Ann. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (875) Google Scholar). In addition to such differences in the structural orientation of Giα domains interacting with receptor, it is likely that receptor contact points on Gβγ also play a role in receptor-mediated activation of guanine nucleotide exchange (14Wu G. Hildebrandt J. Benovic J.L. Lanier S.M. J. Biol. Chem. 1998; 273: 7197-7200Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15Kleuss C. Scherubl H. Hescheler J. Schultz G. Wittig B. Science. 1992; 358: 424-426Google Scholar, 16Taylor J.M. Jacob-Mosier G.G. Lawton R.G. Remmers A.E. Neubig R.R. J. Biol. Chem. 1994; 269: 27618-27624Abstract Full Text PDF PubMed Google Scholar, 17Hildebrandt J.D. Biochem. Pharmacol. 1997; 54: 325-339Crossref PubMed Scopus (126) Google Scholar, 18Richardson M. Robishaw J.D. J. Biol. Chem. 1999; 274: 13525-13533Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Alternatively, the receptor may indeed interact with the GαGDP-GPR complex, but this interaction stabilizes a receptor conformation with low affinity for agonist (19Nanoff C. Freissmuth M. Physiol. Res. 1997; 46: 79-87PubMed Google Scholar). Ultimately, one may think of the GαGDP-GPR complex as a type of dimeric G-protein, and it is not clear what might provide signal input to such a complex. Although the GPR motif is present in several proteins that interact with Gα and/or regulate nucleotide binding/hydrolysis (1Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 2Siderovski D.P. Diverse-Pierlussi M.A. De Vries L. Trends Biochem. Sci. 1999; 24: 340-341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), these proteins have different and often opposing effects on the activation state of G-protein (20Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 21Luo Y. Denker B.M. J. Biol. Chem. 1999; 274: 10685-10688Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).2,5 Pcp2, which contains two GPR motifs based upon this consensus sequence, actually appears to increase the dissociation of GDP from Goα (21Luo Y. Denker B.M. J. Biol. Chem. 1999; 274: 10685-10688Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Thus, there are either subtle differences in this motif or other residues outside of this motif that play a key role in the specific functional output gendered by interaction of the GPR motif with Gα. Of note is the selective effects of the AGS3-GPR peptide for Giα versusGoα in both nucleotide binding assays and the analysis of receptor coupling to G-proteins. Further dissection of the structural basis for this selectivity will provide clues as to the site of interaction of the GPR peptide with Giα and the mechanism by which it stabilizes the GDP-bound conformation. One prominent area of sequence divergence between Goα and Giα encompasses switch IV, a region implicated in the formation of Gi1αGDP multimers (11Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (266) Google Scholar). The role of AGS3 as a GDI is an unexpected concept for heterotrimeric G-proteins, although such proteins serve similar regulator roles for Ras-related G-proteins. Proteins containing the AGS3-GPR motif may promote dissociation of Gα and Gβγ in the absence of nucleotide exchange and present an opportunity for selective control of Giα- and Gβγ−regulated effector systems. GPR-containing proteins likely play a role in regulating basal activity of G-protein signaling systems in the cell and provide alternative modes of signal input to G-protein signaling systems that may either augment, complement, or antagonize G-protein activation by GPCRs. We thank Emir Duzic, Mary Cismowski, Nathalie Pizzinat, Michael Natochin, Nikolai Artemyev, and John Hildebrandt for valuable discussions. We appreciate the technical assistance of Jane Jourdan, Lori Flood, and Peter Chung." @default.
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• W2048073308 title "Stabilization of the GDP-bound Conformation of Giα by a Peptide Derived from the G-protein Regulatory Motif of AGS3" @default.
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