Introduction

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A large family of cell surface receptors, conforming to the heptahelical structure, elicit their physiological effects by first coupling to and activating a population of heterotrimeric GTP-binding proteins (G proteins), which then mediate the responses of a plethora of cellular effectors, including enzymes and ion channels. Heterotrimeric G proteins are composed of -, -, and -subunits and are classified by virtue of their -subunit. In the resting state, the G-GDP subunit forms a high-affinity complex with the G heterodimer. Agonist binding to the receptor leads to conformational changes that promote a tighter interaction with specific heterotrimeric G proteins, catalysis of GDP release, and subsequent G protein activation. In the absence of guanine nucleotides, GDP or GTP, agonist binding to the receptor is stabilized by the bound G protein.

The structural basis of receptor-G protein interaction is an active area of study. The heterotrimeric G protein, rather than just the G or G subunit, is required for interaction with the receptor, but studies pointing out the importance of specific regions have been mainly focused to the G subunit (Hamm et al., 1988; Conklin et al., 1993; Rasenick et al., 1994; Lee et al., 1995; Mazzoni and Hamm, 1996; Bae et al., 1997). The most clearly defined contact site with the receptor includes the last 11 carboxyl-terminal amino acids of G subunits (Hamm et al., 1988; Conklin et al., 1993; Dratz et al., 1993; Gilchrist et al., 1998). However, there are also numerous evidences for the participation of other G regions (Hamm et al., 1988; Rasenick et al., 1994; Lee et al., 1995; Mazzoni and Hamm, 1996; Bae et al., 1997) as well as the G subunit (Taylor et al., 1996; Yasuda et al., 1996) in receptor interaction. Thus, the possibility exists that the molecular determinants of receptor-G protein coupling vary somewhat among specific subfamilies of receptors and G proteins.

The A_2A adenosine receptor belongs to the family of G protein-coupled receptors and activates adenylyl cyclase via coupling to G_s proteins (for review, see Palmer and Stiles, 1995; Ongini and Fredholm, 1996). This receptor is distributed in rat and human brain as well as in several peripheral tissues (Ongini and Fredholm, 1996). In brain, A_2A adenosine receptors are widely represented in the striatum where they are involved in dopaminergic pathways (Ongini and Fredholm, 1996) and elicit locomotor depression (Richardson et al., 1997). For this reason, new therapeutic strategies based on blockade of A_2A adenosine receptors are under investigation to treat Parkinson's disease (Richardson et al., 1997). It is now evident that striatopallidal neurons express both A_2A adenosine and D₂ dopamine receptors and these receptors act in an antagonistic manner (Ongini and Fredholm, 1996).

The A_2A adenosine receptor shows a partial insensitivity to modulation of agonist binding by guanine nucleotides (Nanoff et al., 1991; Mazzoni et al., 1993; Luthin et al., 1995). In a previous study on rat striatal membranes, we found that 100 µM GTPS inhibits specific binding of a radiolabeled A_2A-selective agonist, [³H]CGS21680, by only 60% and the inhibition depends on the presence of MgCl₂ (Mazzoni et al., 1993). Luthin et al. (1995) have reported that only a small fraction of A_2A adenosine receptors in rat striatal membranes are coupled to G proteins, suggesting a limited coupling exists between these receptors and G_s. A recent study (Gilchrist et al., 1998) has shown that the A₁ adenosine receptor G_i protein interface presents some peculiar aspects compared with other G protein-coupled receptors (Hamm et al., 1988; Rasenick et al., 1994). Thus, the molecular mechanisms involved in the interaction between A_2A adenosine receptors and G_s proteins are of great interest to define the G protein-coupling properties of adenosine receptors.

In this study, we have examined the ability of synthetic peptides corresponding to selected regions of the G_s carboxyl terminus to affect agonist binding to A_2A adenosine receptors and to disrupt the receptor-mediated activation of G_s. Progressively longer segments of the G_s carboxyl terminus were synthesized and tested for their effects on specific binding of [³H]CGS21680 to rat striatal membranes. These peptides stimulated specific agonist binding. A 21-residue peptide also inhibited receptor-stimulated adenylyl cyclase activity, but it was not able to stabilize the receptor high-affinity state for agonist ligands. However, this G_s peptide, which showed a marked propensity to form an -helical structure in solution, influences the affinity state of the A_2A adenosine receptor. Our findings suggest that the carboxyl-terminal region of G_s takes part in the formation of a complex binding site for the A_2A adenosine receptor.

	Experimental Procedures

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Materials. [³H]CGS21680 (39.5 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA), and [-³²P]ATP (30 Ci/mmol) and [³H]cAMP (25 Ci/mmol) were from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). [³H]SCH58261 (77 Ci/mmol) and SCH58261 were generous gifts of Dr. E. Ongini (Schering-Plough Research Institute, Milan, Italy). Myokinase, creatine kinase, leupeptin, GTP, and GTPS were obtained from Roche Molecular Biochemicals (Mannheim, Germany). CGS21680 was from Research Biochemicals International (Natick, MA). Bacitracin, benzamidine, and phenylmethylsulfonyl fluoride (PMSF) were products of Fluka Chemie AG (Buchs, Switzerland). Adenosine deaminase, papaverine, and 5'-N-ethylcarboxamidoadenosine (NECA) were from Sigma Chemical Co. (St. Louis, MO). All other reagents were from standard commercial sources and of the highest grade available.

Membrane Preparation. Male Sprague-Dawley rats (150-200 g) were sacrificed by cervical dislocation, and striatal tissue was isolated from the brain by dissection. For radioligand binding assays, membranes were prepared essentially as described previously (Mazzoni et al., 1993). Membrane protein concentration was determined by the method of Lowry et al. (1951) using BSA as a standard. For the adenylyl cyclase assay, striatal tissue was suspended in 10 volumes of ice-cold buffer containing 10 mM HEPES/NaOH (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 1 mM dithiothreitol (DTT) (buffer A) and homogenized with 12 strokes of a tight Teflon-glass homogenizer at 4°C. The homogenate was diluted 6-fold with buffer A and centrifuged at 46,000g for 20 min at 4°C. The pellet was resuspended in 10 volumes of ice-cold buffer A containing 1 mM benzamidine, 200 µg/ml bacitracin, and 2 U/ml adenosine deaminase, and incubated for 30 min at 30°C. The membrane suspension was centrifuged at 46,000g for 20 min at 4°C. The pellet was resuspended in 10 volumes of ice-cold buffer A containing protease inhibitors (as above) and centrifuged at 46,000g for 20 min at 4°C. This washing step was repeated. The final pellet was resuspended in 50 mM HEPES/NaOH, pH 7.5 (buffer B) and used immediately for the adenylyl cyclase assay. Protein concentration was determined using the Coomassie Blue binding method (Bradford, 1976) using BSA as a standard.

Peptide Synthesis. Peptides were synthesized by the continuous-flow solid phase method using Fmoc chemistry on an automatic synthesizer (Milligen 9050; Millipore, Bedford, MA). Crude peptides were purified by reversed HPLC on a preparative Vydac C₁₈ column (2.2 × 25 cm) (Beckman System Gold, San Ramon, CA) using a 15 to 30% gradient of acetonitrile in 0.1% trifluoroacetic acid/distilled water (v/v). After lyophilization, purity was checked by analytical HPLC and electrospray mass spectrometry using a mass spectrometer (VG Quattro; Micromass, Altricham, UK) equipped with a standard Electrospray ion source. Molecular weight calculations were performed by deconvolution using MassLynx software, version 2.00 (Micromass). Table 1 shows the sequence of all peptides used in this study.

View this table: [in this window] [in a new window]   TABLE 1 Amino acid sequence of G synthetic peptides The sequences were taken from Jones and Reed (1987). Peptides were synthesized and purified as described under Experimental Procedures. The identity of each peptide was monitored by electrospray mass spectroscopy.

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Radioligand Binding Assays. Routine [³H]CGS21680 binding assays were performed as described previously (Mazzoni et al., 1993). Striatal membranes (~130 µg of proteins) were incubated with 3 nM [³H]CGS21680 in buffer C containing protease inhibitors (1 mM benzamidine, 100 µM PMSF, and 10 µM leupeptin) and adenosine deaminase (1 U/ml) in the presence and absence of G_i(344-354) or G_s peptides (~300 µM) for 2 h at 25°C. Equivalent experiments were performed in the presence of 100 µM GTPS. Binding was terminated by vacuum filtration over Whatman GF/C glass fiber filters (Whatman Int. Ltd, Springfield Mill, Maidstone, UK), with five washes (3 ml each) of ice-cold buffer C. Nonspecific binding was determined in the presence of 100 µM NECA. In the presence and absence of GTPS, specific binding represented 70% and 90% of total binding, respectively.

Adenylyl Cyclase Assay. Adenylyl cyclase activity was assayed by monitoring the conversion of [-³²P]ATP to [-³²P]cAMP, using a previously reported method (Johnson et al., 1994). The method involved addition of [-³²P]ATP to membranes in the presence of an A_2A adenosine receptor agonist (NECA or CGS21680) and GTP or forskolin (FSK) to stimulate adenylyl cyclase and papaverine as a phosphodiesterase inhibitor. Briefly, to study G_s-mediated adenylyl cyclase activation, the enzymatic activity was routinely assayed in a 100-µl reaction mixture containing 50 mM HEPES/NaOH buffer, pH 7.5, 5 mM MgCl₂, 1 mM DTT, 1 mM EGTA, 0.1 mg/ml creatine phosphokinase, 0.1 mg/ml bacitracin, 0.5 mg/ml creatine phosphate, 0.1 mM ATP, 0.05 mM cAMP, 2 U/ml adenosine deaminase, 0.9 µCi of [-³²P]ATP, and 0.1 mM papaverine. The concentration of GTP and A_2A adenosine receptor agonists were 100 and 10 µM, respectively. G peptides were used at a concentration of 300 µM. The incubation was started by the addition of membranes (~25 µg of proteins) and was carried out at 30°C. To study FSK-mediated activation of adenylyl cyclase, membranes (~10 µg of proteins) were incubated at 23°C in 100 µl of medium containing the same buffer and reagents as above except that EGTA was omitted and MgCl₂ was 1 mM. The reactions were stopped after 10 min by placing assay tubes into an ice bath and adding 0.6 ml of a cold stop solution containing 120 mM Zn(C₂H₃O₂)₂/[³H]cAMP (~12,000 cpm/sample) and then 0.5 ml of 144 mM Na₂CO₃. The total radiolabeled cAMP was isolated on columns of Dowex 50 ion-exchange resin and alumina as described previously (Johnson et al., 1994).

Data Analysis. A nonlinear multipurpose curve-fitting computer program (EBDA/LIGAND; Elsevier-Biosoft, Cambridge, UK) was used for analysis of saturation data. A partial F test was utilized to determine whether the binding data were best fitted by a one- or two-site model. Saturation curves were also fitted by a nonlinear regression analysis of the GraphPad Prism version 3.0 program (GraphPad Software). Data from concentration-response or displacement curves were analyzed by a least-squares curve-fitting computer program (GraphPad Prism, version 3.0), and the EC₅₀ values were derived. The K_i values for competition binding assays were calculated from the EC₅₀ values by the Cheng and Prusoff equation (Cheng and Prusoff, 1973). Values represent the mean ± S.E. of at least three experiments except when otherwise stated. The statistical significance of the differences between means was determined by Student's t test using GraphPad Prism 3.0.

NMR and Structure Calculations. The sample for NMR spectroscopy was prepared by dissolving the appropriate amount of G_s(374-394)C³⁷⁹A in 0.5 µl of ¹H₂O phosphate buffer (pH 6.6) to obtain a 1 mM solution. The sample was lyophilized and redissolved in aqueous solution with 50% (v/v) hexafluoroacetone trihydrate (HFA).

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	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Synthetic peptides of various length from the G_s carboxyl terminus were used as probes of contact regions between the A_2A adenosine receptor and G_s. For other receptor-G protein systems, peptides corresponding to the last 11 residues of the G carboxyl terminus can serve as inhibitors of signal transduction (Hamm et al., 1988; Rasenick et al., 1994; Gilchrist et al., 1998) and stabilize the high-affinity state of the receptor (Hamm et al., 1988). However, the ability of these peptides to stabilize receptor affinity for agonists is not observed for all receptor G protein systems (Gilchrist et al., 1998). We examined the importance of G_s peptide size in relation to its competence to affect A_2A receptor-G_s protein interaction. Table 1 shows the amino acid sequences of the synthetic peptides used in this study. In G_s(378-394), G_s(376-394), and G_s(374-394), Cys³⁷⁹ was substituted with Ala to prevent peptide dimerization.

Effects of G_s Carboxyl-Terminal Peptides on Agonist Binding to A_2A Adenosine Receptors. G_s carboxyl-terminal peptides stimulated specific binding of [³H]CGS21680 to A_2A adenosine receptors in rat striatal membranes (Fig. 1), whereas nonspecific binding was not altered. Both in the presence (Fig. 1b) and absence (Fig. 1a) of GTPS, all peptides tested caused an evident increase of specific binding as compared with respective controls. However, in the presence of the GTP analog, this effect was generally more evident (Fig. 1b). The most effective peptides were G_s(378-394)C³⁷⁹A, G_s(376-394)C³⁷⁹A, and G_s(374-394)C³⁷⁹A, whereas the shortest peptide, G_s(384-394), was less active. In accordance to the crystal structure of G_s (Sunahara et al., 1997), the bioactive conformation of its carboxyl-terminal portion is an -helix (5) spanning from Asp³⁶⁸ to Leu³⁹⁴. Thus, 17-, 19-, and 21-residue peptides may have a stronger propensity to assume an -helical conformation than do the shortest G_s peptides.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of Gs synthetic peptides on [3H]CGS21680 binding to rat striatal membranes. Membranes (130 µg of proteins) and [3H]CGS21680 (3 nM) were incubated in 0.5 ml of buffer C containing protease inhibitors and adenosine deaminase in the presence and absence of Gs or Gi1/2 peptides (300 µM) as described under Experimental Procedures. Peptide activity was tested in the presence (b) and absence (a) of 100 µM GTPS. Control in the presence and absence of the guanine nucleotide was 39 ± 2 and 154 ± 6 fmol/mg of protein, respectively. Values are mean ± S.E. of four independent experiments, each performed in duplicate. Values that are significantly different from control values, as determined by paired Student's t test, are indicated (***P < .001; **P < .01; *P < .05).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Dose-dependent effect of peptide Gs(374-394)C379A on [3H]CGS21680 binding to rat striatal membranes. Membranes (110 µg of proteins) were incubated with 3.6 nM [3H]CGS21680 and various concentrations of Gs(374-394)C379A in the presence of 100 µM GTPS as described under Experimental Procedures. Control was 31 ± 3 fmol/mg of protein. The EC50 value for stimulation of specific binding was 14.9 ± 1.6 µM. Values are mean ± S.E. of three independent experiments, each performed in duplicate. The EC50 value was determined by fitting the data as a sigmoidal dose-response curve with the GraphPad Prism version 3.0 computer program.

Effects of G_s(374-394)C³⁷⁹A on [³H]CGS21680 Binding Parameters to A_2A Adenosine Receptors. To evaluate whether peptide G_s(374-394)C³⁷⁹A was able to stabilize the high-affinity state of the A_2A adenosine receptor and thus to mimic G_s, saturation binding studies were carried out in the presence and absence of a fixed concentration of the peptide. Analysis of data using nonlinear, curve-fitting programs revealed that both saturation curves were better represented by one-site models (Fig. 3a). In addition, Scatchard transformation of these data produced linear plots (Fig. 3b). This suggested the existence of a single class of binding sites in both conditions at the concentrations of ligand used. In the absence of the G_s peptide, the K_D and B_max values were 10.3 ± 0.3 nM and 611 ± 77 fmol/mg of protein (n = 3), whereas in the presence of peptide G_s(374-394)C³⁷⁹A the K_D and B_max values were 9.1 ± 0.02 nM and 897 ± 4 fmol/mg of protein (n = 3), respectively. The addition of the G_s peptide caused an increase of the maximal number of binding sites (P < .05) without significantly affecting the receptor affinity state.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Saturation isotherms (a) and Scatchard plots (b) of specific [3H]CGS21680 binding to rat striatal membranes in the presence and absence of peptide Gs(374-394)C379A. Membranes (75 µg of proteins) were incubated with eight different concentrations of [3H]CGS21680 ranging from 0.5 to 108 nM in the presence and absence of Gs(374-394)C379A as described under Experimental Procedures. In the absence of the Gs peptide, the derived KD and Bmax values were 10.9 nM and 680 fmol/mg of protein, whereas in the presence of the peptide, the KD and Bmax values were 9.1 nM and 890 fmol/mg of protein, respectively. Nonlinear, curve-fitting computer programs (EBDA/LIGAND and GraphPad Prism version 3.0) were used to analyze equilibrium binding data and to calculate binding parameters. The illustrated data are taken from single experiments carried out in duplicate. The results are representative of experiments repeated two additional times.

Influence of G_s(374-394)C³⁷⁹A on Displacement of [³H]SCH58261 Binding by NECA. To detect receptor affinity states and test the hypothesis that the 21-residue G_s peptide stabilized an intermediate affinity state of rat A_2A adenosine receptors, we examined the effect of the peptide on the ability of an agonist, NECA, to compete specific binding of an A_2A-selective antagonist, [³H]SCH58261, in the presence and absence of GTPS.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of Gs(374-394)C379A on displacement of [3H]SCH58261 binding by NECA or SCH58261. Rat striatal membranes (70 µg of proteins) were incubated with 0.2 nM [3H]SCH58261 and various concentrations of either NECA (0.5-1000 nM) (a and b) or SCH58261 (0.025-5 nM) (c) in the presence (b and c) and absence (a and c) of GTPS (100 µM) with and without Gs(374-394)C379A (300 µM) as described under Experimental Procedures. In the absence of both the guanine nucleotide and Gs peptide, control was 106 ± 4 fmol/mg of protein (n = 5), whereas in the presence of the peptide, control was 114 ± 2 fmol/mg of protein (n = 3). In the presence of GTPS with and without the Gs peptide controls were 111 ± 4 (n = 4) and 96 ± 5 (n = 4) fmol/mg of protein, respectively. Values are mean ± S.E. of at least three independent experiments, each per formed in duplicate. The EC50 values were determined by fitting the dataas one- or two-site competition curves with the GraphPad Prism version 3.0 computer program. The Ki values were calculated from the EC50 values by the Cheng and Prusoff equation (Cheng and Prusoff, 1973). a, displacement of [3H]SCH58261 binding by NECA in the absence of GTPS with and without the Gs peptide. In the absence of the peptide, data were significantly better represented by a two-site model (P < .01), whereas data obtained in the presence of the peptide were fitted by a one-site model. b, displacement of [3H]SCH58261 binding by NECA in the presence of GTPS with and without the Gs peptide. Both curves were represented by one-site models. c, displacement of [3H]SCH58261 binding by SCH58261. In all three conditions, data were fitted by one-site models.

Functional Effects of G_s(374-394)C³⁷⁹A on A_2A Adenosine Receptor Signal Transduction. The effects of peptides G_s(374-394)C³⁷⁹A, G_s(384-394), and G_i1/2(344-354) on adenylyl cyclase stimulated by agonist activation of A_2A adenosine receptors were evaluated (Fig. 5a). The addition of peptide G_s(374-394)C³⁷⁹A decreased the basal cAMP production, whereas G_s(384-394) and a control peptide, G_i1/2(344-354), had no effect. However, statistical analysis showed that, in the presence of G_s(374-394)C³⁷⁹A, cAMP production was not significantly different from basal values.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Modulation of A2A agonist- and FSK-stimulated adenylyl cyclase by Gs and Gi1/2 peptides in rat striatal membranes. For basal adenylyl cyclase activity (a) rat striatal membranes (25 µg of proteins) were incubated with [-32P]ATP (0.9 µCi) in the absence of GTP with and without Gs or Gi1/2 peptide (300 µM), whereas for Gs-mediated stimulation of adenylyl cyclase activity (a), membranes (25 µg of proteins) were incubated with [-32P]ATP in the presence of GTP (100 µM) or GTP plus an agonist (10 µM NECA or CGS21680) with and without G peptides (300 µM) as described under Experimental Procedures. The basal adenylyl cyclase activity was 72 ± 5 pmol/10 min/mg of protein (n = 7). Values are mean ± S.E. of at least three independent experiments, each performed in triplicate. Values that are significantly different from control values, as determined by paired Student's t test, are indicated (***P < .001; **P < .01; *P < .05). For basal and FSK-stimulated adenylyl cyclase activity (b) rat striatal membranes (10 µg of proteins) were incubated with [-32P]ATP (0.9 µCi) in the presence and absence (basal) of FSK (10 µM) with or without G peptides (300 µM) as described under Experimental Procedures. The basal adenylyl cyclase activity was 2 ± 0.8 pmol/10 min/mg of protein (n = 3), whereas FSK-stimulated activity was 18 ± 0.3 pmol/10 min/mg of protein (n = 3). Values are mean ± S.E. of at least three independent experiments each performed in triplicate.

NMR and Structure Calculations. The conformational properties of G_s(374-394)C³⁷⁹A were extensively studied by NMR spectroscopy and molecular modeling. The usual high conformational freedom of linear short peptides in water solution leads to inextricable mixtures of isoenergetic confomers. The use of a solvent system having suitable viscosity and polarity properties allows the most energetically stable confomers to prevail, thus making the NMR spectra more interpretable (Amodeo et al., 1991). Therefore, we recorded spectra in a mixture of water:HFA (50:50, v/v). HFA, which is a typical structure stabilizing cosolvent, acts by favoring the intramolecular hydrogen bonds and consequently folded conformations (Rajan et al., 1997).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   NOESY spectrum of Gs(374-394)C379A. Backbone amidic-NH (i,i + 1) (NH/NH) (a) and NH/alkyl side chain (b) regions of the 150-ms NOESY spectrum of the Gs peptide in water:HFA (50:50, v/v) at 300 K. Sequential NH/NH connectivities are labeled.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Alignment of C atoms of Gs(374-394)C379A with corresponding atoms of Gs(384-394) and the Gs fragment Arg374-Leu393. a, Gs(374-394)C379A is represented by a blue ribbon, whereas Gs(384-394) is shown as a red ribbon. The Gs(384-394) structure was obtained as reported by Albrizio et al. (2000). The helical arrangement of Arg380 to Leu393 residues is highlighted by a yellow tube ribbon. b, Gs(374-394)C379A is represented by a blue ribbon, whereas the Gs fragment Arg374 to Leu393 is shown as a red ribbon. The -helical structure of the Gs carboxyl terminus (red) was retrieved from the X-ray structure of the parent protein (Sunahara et al., 1997). The yellow tube ribbon represents the helix exhibited by both Gs(374-394)C379A and Gs carboxyl-terminal fragment.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The structural determinants of receptor-G protein interactions have recently received significant attention. Because the A_2A adenosine receptor appears to be loosely coupled to G_s in rat striatal membranes (Luthin et al., 1995), a study of their interaction mechanisms is important in understanding certain peculiar characteristics of this G protein-coupled receptor system. Therefore, we synthesized and used a series of peptides corresponding to various segments of the G_s carboxyl terminus in an attempt to identify specific requirements for interaction of the subunit with the A_2A adenosine receptor.

This study shows that synthetic peptides corresponding to progressively longer segments of the G_s carboxyl terminus modulate agonist binding to the A_2A adenosine receptor in rat striatal membranes. The ability of these peptides to stimulate agonist binding is related to their size, i.e., amino acid numbers. Thus, the 11-residue peptide, G_s(384-394), is one of the least effective, whereas peptides containing 17 to 21 residues, G_s(378-394)C³⁷⁹A, G_s(376-394)C³⁷⁹A, and G_s(374-394)C³⁷⁹A, are the most active. The specificity of longer peptide effects, at the level of the receptor-G_s interface, is supported by the evidence that they show a greater ability to stimulate agonist binding in the presence of GTPS.

A role of the carboxyl-terminal -helix (5) in specific receptor recognition has been indicated by the crystal structure resolution of G_s·GTPS showing a continuous helix from Asp³⁶⁸ to Leu³⁹⁴ (Sunahara et al., 1997). Our NMR analysis demonstrates a marked propensity of the 11- (Albrizio et al., 2000) and 21-residue peptides to form an -helical structure in solution. Although for the shortest peptide, a defined structure is represented by a turn of -helix between Arg³⁸⁹ and Leu³⁹⁴, for the longest peptide, the -helical structure spans from Asp³⁸¹ to Leu³⁹⁴. This shows good overlapping with -5 of the G_s subunit (Sunahara et al., 1997). These results are in agreement with our observation that the longest peptides have a higher ability to stimulate [³H]CGS21680 binding and support the important role of carboxyl-terminal conformation for G_s interaction with the A_2A adenosine receptor. Indeed, the importance of this -helix is also pointed out by the notion that G_s is the only G subunit so far crystallized that shows a defined structure at the level of the last carboxyl-terminal residues (Noel et al., 1993; Coleman et al., 1994; Sunahara et al., 1997). An 11-residue synthetic peptide from the G_t carboxyl terminus does not acquire an -helical structure, but it displays a -turn around Gly³⁴⁸ (Glu³⁹² in G_s) in the presence of the inactive receptor, dark-rhodopsin (Rh) (Dratz et al., 1993). This conformation is lost when the peptide is bound to light-activated rhodopsin (metarhodopsin II or Rh*) (Dratz et al., 1993). Thus, an -helix appears to be a specific bioactive determinant for interaction of G_s with its cognate receptors.

In the presence of GTPS, which uncouples G proteins from receptors, a G_i1/2 carboxyl-terminal peptide, G_i1/2(344-354), is as effective as the shortest G_s peptides in stimulating agonist binding. This finding may suggest that in some conditions the A_2A adenosine receptor is able to interact with G_i proteins. Daaka et al. (1997) have shown that a classical G_s coupled receptor, the ₂-adrenergic receptor, switches to activate G_i proteins after phosphorylation by protein kinase A. A similar event may also occur for agonist-activated A_2A adenosine receptors in rat striatum.

In rat striatal membranes, agonist-activated A_2A adenosine receptors interact tightly with heterotrimeric G_s proteins in the presence of 5 to 10 mM MgCl₂ and absence of guanine nucleotides (Mazzoni et al., 1993). This interaction stabilizes the high-affinity state of A_2A adenosine receptors. However, it is now evident (Luthin et al., 1995) that in this condition there are still some A_2A adenosine receptors uncoupled from G_s. The G_s carboxyl-terminal peptides may bind to these receptors in the low affinity state and stimulate [³H]CGS21680 binding (Fig. 1a). In the presence of GTPS (Mazzoni et al., 1993), most receptors are uncoupled from G_s and are, therefore, in the low affinity state. The addition of the G_s peptide may restore the high-affinity state, mimicking the effect of G_s. This possibility has been tested for the longest G_s peptide, G_s(374-394)C³⁷⁹A, which shows dose dependence and saturability of its stimulatory activity. Indeed, G_s(374-394)C³⁷⁹A evokes a larger stimulation of specific agonist binding in the uncoupling condition, but it does not stabilize the high-affinity state of the receptor either in the presence or absence (Fig. 3) of the guanine nucleotide. An intermediate receptor affinity state for the agonist may be reached.

When the effect of G_s(374-394)C³⁷⁹A on agonist displacement of [³H]SCH58261 binding has been examined, a stabilization of an intermediate affinity state of A_2A adenosine receptors is observed. The addition of the G_s peptide appears to modulate the low and high-affinity states of the receptor leading to a single intermediate affinity state. In the presence of GTPS, the agonist competition curve is shifted to the right and fitted by a one-site model as compared with that in the absence of the nucleotide (Fig. 4b), whereas the addition of the G_s peptide causes a left shift of the displacement curve. The peptide is responsible for a 2-fold decrease of the K_i value. This K_i value is similar to that calculated for the low affinity state of the receptor in basal displacement conditions. The peptide appears to affect the receptor conformation in a subtle way. The observation also implicates the existence of multiple distinct receptor conformations, which differ in their interaction with G_s proteins. A similar possibility has been suggested for rhodopsin (Arnis and Hofmann, 1995), the human thyroid-stimulating hormone (Gudermann et al., 1996), and A₁ adenosine receptors (Gilchrist et al., 1998). Our data suggest that G_s has an important role in modulating receptor affinity for agonist ligands. The A_2A adenosine receptor affinity states seem to be regulated by the type of interaction with the G_s subunit. However, an alternative or complementary explanation is possible. Disruption of G_s activation by the 21-residue peptide (see below) compromises agonist-induced GTPS binding to G_s and, consequently, the shift of receptor affinity without affecting receptor-G_s coupling. The indirect effect is that the binding affinity for agonist ligands does not decrease while A_2A receptors are still coupled to heterotrimeric G_s proteins. In support of this interpretation is a recent observation (Wade et al., 1999) that basic residues within the carboxyl-terminal portion of the third intracellular loop of the _2a-adrenergic receptor are important for G_i activation but not required for receptor-G_i coupling. Similarly, different determinants of the G subunits can be involved in receptor-G protein coupling and activation.

The G_s(374-394)C³⁷⁹A peptide effectively inhibits receptor-mediated adenylyl cyclase activation in rat striatal membranes. The inhibition appears to be specific because the peptide shows no significant effect on basal and FSK-stimulated cAMP production. This observation supports the notion that the carboxyl-terminal region of G_s is critical for signal transduction from the activated A_2A adenosine receptor to G_s, but it is not so decisive for mimicking the effects of G_s on the receptor. In addition, disruption of signal transduction appears to require a 21-residue segment rather than the extreme carboxyl terminus. Thus, the peptide secondary structure is a determinant factor in supporting its activities. Recently, Gilchrist et al. (1998) have examined the effects of 11-residue G_i carboxyl-terminal peptides and analogs on agonist binding to A₁ adenosine receptors and activation of the effector pathway. Although the G_i peptide and some analogs completely block receptor-activated K⁺ current, they do not stabilize the high-affinity state of the receptor. The similarity is obvious and may underlie a specific molecular mechanism of interaction between adenosine receptors and G proteins.

Some studies have pointed out the importance of other regions of the G subunits beside the extreme carboxyl terminus in directing the G protein coupling to their cognate receptors. Both tryptic digestion experiments (Mazzoni and Hamm, 1996) and a site-directed mutagenesis investigation (Onrust et al., 1997) have demonstrated that the 4-6 loop of G_t is a point of rhodopsin contact. Furthermore, using various G_t/G_i1 chimeric proteins, Bae et al. (1997) have shown that the 4-helix and 4-6 loop are critical for specific 5-HT_1B receptor-G_i1 interaction and are required for G protein activation by the receptor. In addition, this study has also involved a secondary role for the amino-terminal half of G_i1 in 5-HT_1B receptor coupling.

Our study provides additional evidence that the molecular mechanisms of interaction have similar features in different receptor-G protein systems, but the role and importance of each contact site change depending on the receptor type. In the case of the A_2A adenosine receptor, the -helix conformation of the G_s carboxyl terminus seems to be important for signal transduction. However, other parts of the G_s molecule are probably involved in determining the allosteric modulation of receptor affinity for agonists. Thus, as suggested for the A₁ adenosine receptor, the interacting site on G subunits may be a mosaic with each piece playing a distinct role depending on the type of receptor and G protein. Whereas the G_s carboxyl terminus is pivotal for receptor-mediated activation of G_s, multiple interactions may be required to stabilize the high-affinity state of the receptor.

In conclusion we have found that the -helical conformation of the G_s carboxyl terminus is involved in supporting its ability to interact with the A_2A adenosine receptor, but this part of the molecule does not stabilize the high-affinity state of the receptor. The 21-residue carboxyl-terminal peptide, which is able to disrupt signal transduction, causes a direct conformational change of the receptor with stabilization of an intermediate affinity state or induces an indirect effect by preventing GTPS binding to G_s.