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J. Biol. Chem., Vol. 276, Issue 45, 41742-41747, November 9, 2001

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G Protein  Subunit Interaction with a Receptor Regulates Receptor-stimulated Nucleotide Exchange^*

Inaki Azpiazu and N. Gautam^§¶

From the Departments of  Anesthesiology and ^§ Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, May 4, 2001, and in revised form, August 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

The surfaces of heterotrimeric G proteins () in contact with receptors and the molecular events at these sites, which lead to G protein activation, are largely unknown. We show here that a peptide from the C terminus of a G protein  subunit blocks muscarinic receptor-stimulated G protein activation in a sequence-dependent fashion. A G protein mutated at the same site on the  subunit shows enhanced receptor stimulated nucleotide exchange without affecting G protein heterotrimerization. Ineffective contact between the  subunit and receptor increases the rate of receptor-stimulated nucleotide exchange. Specific interaction of the G protein  subunit with the receptor thus helps the complex to act at a distance and control guanine nucleotide exchange in the  subunit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

Although G protein signaling is central to a vast majority of pathways that control the physiology of mammalian cells, the coordinated changes in the conformations of the agonist bound receptor and the G protein subunits that trigger nucleotide release are not known. Peptide and mutant studies using biochemical assays that measure receptor-G protein coupling have implicated three protein domains, the N- and C-terminal domains of the  subunit and the C-terminal domain of the  subunit in receptor interaction (1-3). Functional contact between the  subunit and a receptor is one mechanism that can explain the universal requirement of the complex for receptor-mediated nucleotide exchange in the  subunit. However, the particular role that the  subunit plays at the receptor surface during G protein activation has not been clear. The crystal structure of the G protein heterotrimer indicates that the C termini of the  and  subunits are a considerable distance from the nucleotide-binding site in the  subunit (4, 5). The mechanisms that help the receptor regulate nucleotide exchange by contacting these domains are therefore an outstanding puzzle.

Because a peptide specific to the C terminus of the 1 subunit type stabilizes activated rhodopsin in a sequence-dependent manner (6) and a homologous 5 peptide specifically inhibits muscarinic receptor modulation of a Ca²⁺ current in intact neurons (3), we examined the effect of a geranylgeranylated 5 peptide on the M2 muscarinic receptor activation of a G protein. The peptide specific to the C-terminal 14 residues of the 5 subunit was prenylated because the native 5 subunit is modified with geranylgeranyl at the C-terminal Cys residue that is part of a CAAX motif (2). This peptide inhibited activation of G proteins by reconstituted M2 receptor. A peptide with the same sequence scrambled was inactive. These studies indicated that the  subunit peptide interacts with the receptor in a sequence-specific fashion. To test the effect of mutations in this C-terminal domain of 5 on G protein activation by M2, C-terminal residues of 5 corresponding to the peptides were scrambled. The scrambled sequence was identical to the sequence of the scrambled peptide used in the earlier experiments. Wild type and mutant 15 complexes were purified and bound to o, and the ability of the M2 receptor to activate these heterotrimers was measured in a reconstituted system containing purified proteins. A significant difference in the receptor-stimulated GTPase activity between the wild type and mutant G_o was detected in this system. A phospholipase C enzyme-based assay indicated that efficacy of interaction with the  subunit was unaltered by the mutant  subunit. Together these results indicate that the  subunit interaction with the receptor regulates nucleotide exchange in the  subunit by affecting the positioning of the complex with reference to the  subunit. The results thus identify a mechanism that allows a receptor to regulate nucleotide exchange at a distance in the G protein.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

Synthesis and Prenylation of Peptides-- The amino acid sequences of the peptides were as follows: 5pep-gg (wild type):VSSSTNPFRPQKVC and 5pep-gg-scr (scrambled): PSRTPVNFSQVSKC. Cys residues were retained at the C terminus for chemical geranylgeranylation using geranylgeranyl bromide. Prenylation and purification using fast protein liquid chromatography has been described (7). The integrity of the modified peptides were checked by mass spectrometry and chromatography, and peptide concentrations were estimated by amino acid analysis. Except where specified, all chemicals were from Sigma.

Purification and Reconstitution of M2 Receptors-- Detailed description of M2 purification, reconstitution, and measurement of G protein stimulation is published elsewhere (7). Briefly, baculoviruses containing His-tagged M2 receptor cDNA (kind gift from Dr. E. M. Ross) were expressed in insect cells. M2 was purified according to a previously published protocol (8). Sf9 cell membranes containing M2 were solubilized in 50 mM Hepes, pH 7, 50 mM NaCl, digitonin (Calbiochem)/sodium cholate added to 1:0.5% final concentration. All procedures were performed at 4 °C. Solubilized receptor was bound to cobalt-chelate beads that were prepared using iminodiacetic acid beads and cobalt chloride. His-M2 eluted from these columns with 200 mM imidazole retain 50% of the N-[methyl-³H]scopolamine binding activity in Sf9 cell membranes. Using modifications of a previous method (8), purified M2 was reconstituted into brain lipids (Folch Fraction VII). The composition of the brain lipid mixture is sphingomyelin (20%), phosphatidyl-ethanolamine (30%), phosphatidyl-serine (20%), and other lipids according to the distributor (Sigma). Typically, 100 µl (1 mg lipid) of the aqueous lipid suspension is added to 18 µl of 10% sodium deoxycholate and 4 µl of 10% sodium cholate. 100-500 µl of pure receptor is mixed with solubilized lipids (~122 µl from above). The mixture is applied to a 10-12-ml column of Sephadex G-50 (fine), equilibrated with solubilization buffer. Vesicle fractions were collected and concentrated. N-[methyl-³H]Scopolamine binding in a standard filter binding assay was used to estimate receptor concentration. The yields were 20-30% relative to purified solubilized receptors.

M2 Stimulation of G Protein Activity-- o subunit protein was expressed in bacteria with yeast myristoyl-transferase and purified as described before (9). 15 and 15scr complexes were produced in Sf9 insect cells by triple infection of His-i2, 1 (kind gifts from Dr. T. Kozasa), and  subunit viruses using a previously published procedure with minor modifications (10). The two complexes were separately purified in complex with His-tagged i2 subunit by binding the complex to nickel-nitrilotriacetic acid resin and eluted the complexes with aluminum fluoride. Yields of the 15 and 15scr were similar. The 5Al and 5 mutants were synthesized as His-tagged proteins, expressed in complex with 1 subunit in Sf9 cells, and purified by directly binding to nickel-nitrilotriacetic acid resin. As a control a wild type 1-His-tagged 5 complex was expressed and purified. Although the yield of 1His-5 was comparable with or higher than that of 15, the yields of the 1His5Ala/ were relatively low. The final purity was examined by gel electrophoresis and Coomassie Blue staining where no significant levels of other proteins were detected. Protein concentration was determined by laser scanning densitometry using standards. G protein heterotrimer was formed by preincubating o with complexes for 30 min at 4 °C. Lipid-reconstituted M2 was incubated with o subunit with or without complexes for 30 min at 4 °C in 20 mM Hepes buffer, pH 8, containing 2 mM MgCl₂, 100 mM NaCl, 10 µM GDP, and 1 mM dithiothreitol. GTPS¹ binding to o was determined using previously published methods (11). In peptide assays, the incubation was performed by mixing with dried peptide or peptide vehicle (3 µM CHAPS) as a control. The final reaction mix contained 1 nM M2, 100 nM o, and 0-10 nM 15 in the buffer above and was equilibrated at 23 °C. The binding reaction was started by addition of 0.2 µM [³⁵S]GTPS and 1 mM carbachol, 1 mM atropine or vehicle. The reaction was stopped with ice-cold reaction buffer containing 200 µM GTPS and 1 mM atropine. [³⁵S]GTPS bound to o was measured in a filter binding assay. For GTPase assays, the conditions were the same as above. o GTPase activity was measured essentially as described (11). The reaction was started with 0.2 µM [-³²P]GTP, 1 mM carbachol, or vehicle and stopped with ice-cold 5% charcoal in 50 mM sodium phosphate, pH 7.0. The samples were centrifuged, and the radioactivity in the supernatant was estimated using scintillation counting. Purified RGS4 was kindly provided by Dr. Maurine Linder.

Inhibition of -stimulated PLC- 3 Activity by o-- o inhibition of complex-stimulated PLC- activity was measured as described before (12). o and 15 or 15scr complexes were preincubated for 30 min at 4 °C. The reaction mixture contained G protein, PLC-, and lipids. Freeze dried lipid mixtures were ultrasonicated before use. Final concentrations were 150 µM phosphatidyl ethanolamine, 50 µM [³H]phosphatidyl inositol diphosphate, 1.25 nM PLC-3, and 4 nM 15 (wild type or mutant). Ca²⁺ was added to initiate the reaction. The reaction was performed at 30 °C for 30 min. The reaction was stopped with trichloroacetic acid with bovine serum albumin, and the radioactivity in the supernatant was measured. No more than 10-15% of the substrate, [³H]phosphatidyl inositol diphosphate, was used during the reactions. Purified recombinant PLC- was a kind gift from Dr. A. Smrcka.

Construction of 5 Mutants-- The rat 5 cDNA was mutated by replacing the 3' end of the 5 cDNA (beginning from a BsmI site at base 156) with a double-stranded DNA cassette encoding the scrambled sequence. Alanine substitutions and the deletion of 10 residues upstream of the codon encoding the C-terminal Cys were performed using polymerase chain reaction based methods in a 5 cDNA that was His-tagged. Mutant cDNAs were transferred to the baculovirus using the pFastBac system (Invitrogen-Life Technologies, Inc.).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS REFERENCES

Effect of 5 Subunit Peptide on M2-dependent G Protein Activation-- A peptide specific to the C-terminal 14 residues of 5 was geranylgeranylated (5pep-gg), and the ability of the peptide to compete with a G protein (o15) for interaction with M2 was examined. A reconstituted system containing purified M2 and G protein subunits was set up (described under "Materials and Methods"; Fig. 1, A and B). Purified reconstituted M2 had a K_d for N-methyl-scopolamine of 250 pM (12) and activated G_o in an agonist- (Fig. 1C) and -dependent fashion (data not shown). M2-stimulated G_o activation was significantly inhibited by 5pep-gg but not by a peptide with the same amino acids sequence scrambled, 5pep-gg-scr (Fig. 1C). Because these peptides have no effect on the G protein heterotrimer (3), these results indicate that 5pep-gg competes with the G protein for a site on the M2 receptor in a sequence-specific manner.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Purified M2 receptor (0.2 µg) (A) and G protein complexes (B) separated by SDS-PAGE and stained with Coomassie Blue. Lanes contain the following protein samples with optical densities determined by laser densitometry in parenthesis: lane 1, 0.2 µg of 15 (93); lane 2, 0.2 µg of 15scr (97); lane 3, 0.4 µg of 15 (171); lane 4, 15scr (183). C, 5pep-gg inhibits M2 activation of added G protein (o15) in a reconstituted system with purified proteins. M2-stimulated GTPS binding to o was measured with 1 nM M2, 100 nM o, and 10 nM 15 in the presence of 3 µM 5pep-gg or 5pep-gg-scr. 3 µM peptide in 3 µM CHAPS was preincubated with reconstituted M2-Go as above. Controls were performed by adding only peptide vehicle to M2/Go with agonist or without agonist. The means ± S.E. from five independent experiments performed in duplicate are shown. The asterisks denote that GTPS binding to o in the presence of the wild type 5pep-gg is significantly different (p < 0.05) from binding in the presence of the mutant 5pep-gg-scr peptide.

Effect of Mutating the C-terminal Domain of the 5 Subunit on G_o Heterotrimer Activation by the M2 Receptor-- Competition of the 5 peptide with the G protein indicated that the G protein 5 C-terminal domain interacts with the M2 receptor. To obtain further evidence to support this mechanism, mutant forms of the  subunit were synthesized with altered C-terminal sequences. If effective interaction of the 5 subunit C terminus with the M2 receptor is a requirement for activation of a G protein, one or more of these mutant forms of 5 were expected to alter the activation properties of the G protein. Three different mutants of the 5 subunit were expressed and purified from insect cells in complex with the 1 subunit type (Fig. 2A). These mutants were as follows: (i) 5scr: the last 13 residues upstream of the Cys in the CAAX box were scrambled identical to the sequence of 5pep-gg-scr; (ii) 5: 10 residues upstream of the Cys residue were deleted; and (iii) 5Ala: a short sequence of three residues, NPFR, which is conserved in all G protein  subunits, was substituted with Ala residues.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of  subunit mutation on Go activation by M2. A, amino acid sequences at the C terminus of the mutant forms of the 5 subunit. 5WT, wild type; 5scr, wild type amino acid sequence scrambled; 5Ala, the wild type sequence NPFK has been substituted with Ala residues; 5, deletion of 10 residues upstream of the C-terminal Cys. B and C, M2-stimulated GTPS binding to o15 or o15scr. GTPS binding with 1 nM M2 and 100 nM o was assayed for 2 min. The reaction is linear at this time point as in Fig. 1. B, at varying concentrations of at 1 mM carbachol. C, at varying concentrations of carbachol at 7 nM complex. The points represent the means ± S.E. (n = 3 in B and n = 2 in C).

His-tagged forms of 5Ala and 5 mutants were co-expressed with the 1 subunit and purified as 15 and 15Ala complexes. A wild type 1His-5 complex was also expressed and purified as a control. Gel exclusion chromatography indicated that the two mutants formed effective complexes with the 1 subunit. However, in contrast to the wild type 1His-5, the mutant 1His-5Ala and 1His-5 did not activate PLC-3 and did not form a heterotrimer with the o subunit effectively (measured using the ability of pertussis toxin to ADP-ribosylate the o subunit in the presence of the complex (data not shown)). They were therefore not used in the experiments described below.

The 5scr mutant was co-expressed in the native state with 1 and His tagged i2 subunits. The heterotrimer was bound to a nickel-nitrilotriacetic acid column. The 15scr complex was eluted using imidazole. As a control wild type 15 was synthesized using a similar approach (Fig. 1B). The purified 15scr activated PLC-3 and formed heterotrimers effectively similar to wild type 15 (described below). All experiments were therefore performed with this mutant.

The mutant 15scr and wild type 15 in complex with o were first compared for their relative levels of activation by the M2 receptor. At a receptor to o subunit ratio of (1:100), no significant difference in receptor-stimulated GTPS binding was detected between the wild type and mutant heterotrimers at various concentrations of the complex (Fig. 2B). Assaying M2 activation of o15 and o15scr at various concentrations of carbachol also did not indicate differential activation (Fig. 2C).

In contrast to M2-stimulated GTPS binding where each  subunit can bind utmost one molecule of GTPS, M2-stimulated GTPase activity can result in several cycles of activation of a G protein  subunit resulting in the formation of many molecules of P_i for each G protein. Because of this amplification, GTPase assays could be performed with significantly lower concentrations of the o subunit compared with the GTPS binding assay, thus approaching a ratio of [receptor] to [G protein] of 1:1. Although the K_d for G_o binding to M2 is not known, the K_d for G_t binding to rhodopsin is 1 nM in the absence of nucleotide (13). The conditions in the GTPase assay were thus potentially closer to the K_d for G_o binding to M2 and likely to reveal differences between wild type and mutant 15 complexes during M2 activation. To enhance sensitivity, we examined the receptor-stimulated GTPase activity in the presence of saturating concentrations of RGS protein, RGS4 (7). RGS4 acts as a GTPase-activating protein for the G_o/i family (14). RGS4 potentiated M2-stimulated GTPase activity over 10-fold (7). When the time course of M2-stimulated GTPase activity was measured in the presence of RGS4, the M2 receptor activated o15scr significantly more than the wild type (Fig. 3). In the absence of the agonist or the complex, M2 did not effectively stimulate G_o GTPase activity (footnote a in Table I). G_o containing the 5scr mutant was also more active at several ratios of G_o:M2 (Table I). Under these conditions the GTPase activity is a measure of the rate of receptor-stimulated nucleotide exchange (15). The results thus indicate that the mutant has a higher rate of receptor-stimulated nucleotide exchange compared with the wild type.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Time course of M2-stimulated GTPase activity with 1 nM M2 and different concentrations of o15 or o15scr. Activity in the presence of 100 nM RGS4 is shown. o and are equimolar. The points are the means ± S.E. from three independent experiments performed in duplicate. The differences in values are significant at p < 0.05. Moles of 32Pi produced during the reaction was 10-40% of total moles of GTP present. Nonspecific 32Pi was measured by the addition of 200 µM GTP to the reaction mix. This value was subtracted from the values obtained in experimental samples.

View this table: [in this window] [in a new window]   Table I M2 activation of o15 wild type and o15-scrambled

In the absence of the RGS protein, the mutant and wild type G_o proteins still showed differential receptor-stimulated GTPase activities (Table I). This result indicates that differential receptor-stimulated GTPase activities of o15scr mutant and wild type are not due to differential effects of the RGS4 protein on receptor activation of the mutant and wild type.

Alternative explanations were excluded for this differential activity: (i) Both complexes were not contaminated with  subunit because M2 stimulated GTPase activity of the complexes alone was not detectable. (ii) The difference in activity was not due to variation in protein concentrations. When M2-stimulated GTPase activity was measured at various concentrations of 15 (but constant concentrations of o and M2), a 2-3-fold increase in complex elicited the magnitude of increase in activity seen between the mutant and wild type. Thus a 2-3-fold difference in concentration between 15 and 15scr must remain undetected. However, we could clearly detect 2-fold differences in the concentration of subunits by densitometry of Coomassie Blue-stained proteins in SDS-PAGE gels (Fig. 1B). (iii) The difference was not due to differences in functional proportion of complexes because in the GTPS binding assay, a 2-fold difference in concentration elicits an equivalent increase in GTPS binding (Fig. 2A). (iv) Thin layer chromatography of complex samples indicated that both samples contained the same concentrations of detergent. (v) Buffer components in the complexes did not contribute to the differential activity because the addition of heat-denatured mutant sample to the wild type and vice versa had no effect. (vi) The differences in M2 activation of wild type and mutant o15 and o15scr also cannot be due to differences in the proportion of prenylated 5 subunit because the 5 and 5scr proteins were purified using a His-tagged i subunit. Prenylation is essential for complex interaction with the  subunit. (vii) New stocks of complexes that were independently expressed, purified, and assayed again showed the same difference in M2-stimulated o GTPase activity.

Wild Type and Mutant 15 Have Similar Affinities for o-- To examine the affinities of 15 and 15scr for o in the 1-10 nM concentration range used in the GTPase assays, we used a recently developed assay (12) that relies on the overlap in binding sites for  subunits and PLC-2/3 on the complex. Thus binding of o to the complex inhibits PLC-3 stimulation by the complex. There was no significant difference in the inhibition of 15 and 15scr, indicating that o affinity for both is the same (Fig. 4). This result also further confirmed that both the wild type and mutant  subunits are prenylated to the same extent because prenylation is essential for complex activation of PLC-. The difference in GTPase activity between mutant and wild type thus arises from differential receptor interaction.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   o heterotrimerization with 15 and 15scr. Inhibition of 15 and 15scr stimulated PLC-3 activity by o. Activities were normalized to that in the presence of 4 nM 5 wild type and scrambled. The means ± S.E. from four independent experiments are shown.

The results from the experiment above (Fig. 1C), where 5 peptide interaction with M2 was tested, indicated that the 5 scrambled peptide does not effectively interact with the receptor. An earlier report also indicated that in contrast to the wild type, the scrambled 5 peptide had little effect on a muscarinic receptor modulation of a Ca²⁺ current or on a the muscarinic receptor modulation of an excitatory postsynaptic current (3). Viewed in this context, the higher M2-stimulated GTPase activity in the o15scr mutant indicates that weak interaction of the mutant  subunit C terminus with M2 leads to a higher nucleotide exchange rate.

The receptor-G protein activation cycle involves three broad steps: (i) G protein binding to receptor; (ii) receptor-initiated nucleotide exchange in the G protein and dissociation of the ternary complex; and (iii) deactivation of G by RGS protein and reassociation with the complex. The difference in receptor-stimulated GTPase rates between the G_o wild type and mutant must arise at one of these steps. We infer that differences between mutant and wild type proteins occurs during receptor-initiated nucleotide exchange in the G protein. Other steps in the receptor-G protein activation cycle cannot be affected because the results presented indicate that heterotrimer formation of the mutant is unaltered and that RGS4 has no influence on the differential activation of the mutant. The affected step cannot be initial binding of G protein with receptor because this would lead to lower GTPase rates in the mutant compared with wild type.

A model that explains the results here must take into account the effect of the  subunit mutation on receptor-stimulated nucleotide exchange. It should also take into account (i) the inability of the scrambled 5 peptide to interact with the receptor and (ii) the higher receptor-stimulated GTPase in mutant G_o containing 5scr.

The crystal structure of the G protein heterotrimer (G_t) when compared with the structures of Gt bound to GTP or GDP indicates that the domains on the  subunit that undergo the most significant changes in conformation during activation are the same domains that contact the  subunit (4). It was suggested based on this that the complex occludes nucleotide release from the  subunit when the G protein is bound to the receptor (4). Receptor-stimulated nucleotide exchange will therefore require the receptor to shift the complex away from the  subunit (Fig. 5A). This was later detailed as a model for receptor activation of a G protein (16). In this model it was proposed that two mechanisms account for the ability of the receptor to stimulate nucleotide exchange in the G protein  subunit: (i) the  subunit C terminus contacts a receptor, and receptor activation leads to conformational changes being triggered through the C-terminal domain to the 6/5 loop of the  subunit, which is in contact with GDP; (ii) receptor loop(s) enter the cavity between the  subunit and complex, prising them apart and creating an opening through which GDP leaks out. It was proposed in this model that only the  subunit C terminus contacts the receptor and that the C terminus of the  subunit interacts with membranes, despite evidence to the contrary (6, 17). Recently, this model has been modified to include the evidence indicating  subunit interaction with a receptor (3, 6, 12, 17) to propose that the  subunit C terminus also interacts with the receptor and that the receptor loops, instead of entering the cavity between the  subunit and complex, actually prise the subunits apart through their interaction with the  and  subunit C termini (18). This model is now similar to our earlier proposal that interaction with the  subunit and  subunit C termini with a receptor is a requirement for G protein activation (2, 12). Such a model would predict that mutating the  subunit C terminus would result in weaker activation of the G protein by the receptor. However, earlier results indicate that this may be a simplistic expectation. Although a 5 but not a 7 subunit-specific peptide inhibits muscarinic receptor-stimulated signaling (3), the M2 muscarinic receptor stimulates higher nucleotide exchange in G_o containing 7 compared with 5 (12). These results suggested that contrary to expectations, poor interaction of the  subunit with a receptor can result in more robust G protein activation. This result is not surprising if potential mechanisms underlying nucleotide exchange are considered based on the crystal structures of a G protein. Crystal structures of the heterotrimer and active and inactive  subunits indicate that guanine nucleotide release requires the complex to move away from the  subunit (4). Any orientation of the complex that results in exposing the bound GDP will therefore result in more rapid nucleotide release. If the  subunit does not effectively interact with the receptor and anchor the complex, the complex may be inappropriately oriented with reference to the  subunit. The inappropriately oriented complex will enhance nucleotide exchange by exposing the bound nucleotide in the  subunit. Fig. 5B shows one such potential orientation of a complex where the  subunit C terminus does not effectively anchor the complex by interacting with the receptor. As shown in Fig. 5B, nucleotide exchange in the  subunit will be facilitated by this orientation of the complex.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Model of G protein interaction with receptor. A, The  subunit C terminus ( C terminus) interaction with the receptor (gray arrow) allows the complex to orient itself appropriately with reference to the  subunit. Receptor-initiated nucleotide exchange from the  subunit (GDP and GTP arrows) is regulated by the appropriate orientation of the complex with reference to the  subunit.  subunit C terminus ( C terminus) contact with the receptor is denoted with a gray arrow. The space-filling model of GDP bound to the  subunit is shown. B, ineffective interaction of the  subunit C terminus with the receptor affects orientation of the complex with reference to the  subunit and increases the rate of nucleotide exchange during activation (gray arrows). G protein structure is from Lambright et al. (4).

This model predicts that a mutant  subunit that interacts weakly with a receptor will encourage higher nucleotide exchange in the associated  subunit compared with a wild type  subunit that interacts strongly with the receptor. This prediction is borne out by the results presented here. Peptide evidence indicates that the 5scr mutant interacts poorly with the receptor compared with the wild type. However, G_o containing the 5scr mutant shows a higher rate of receptor-stimulated nucleotide exchange compared with the wild type. The model also predicts that a  subunit type with a lower affinity for the receptor will allow higher rates of receptor-stimulated nucleotide exchange in the associated  subunit compared with a different  subunit type with a higher affinity for the receptor. This prediction is also supported by previous results. Only the C-terminal peptide specific to 5 but not 7 disrupts muscarinic receptor regulation of Ca²⁺ current in neurons (3). This result indicated that the 5 subunit type but not 7 interacts with the M2/M4 receptor types. However, o17 shows significantly higher M2-stimulated nucleotide exchange compared with o15 (12). Thus, consistent with the results obtained with the 5scr mutant, a G protein containing a  subunit type that does not interact effectively with a receptor shows enhanced receptor-stimulated GTPase activity in comparison with the wild type.

Although o15scr contains a mutant  subunit that does not effectively interact with the receptor, it still possesses higher receptor-stimulated nucleotide exchange compared with wild type. This result does not imply that a model for receptor activation of a G protein that invokes receptor loops contacting the  and  subunit C termini and prising the complex away from the  subunit is incorrect. It does suggest that mutational approaches with purified proteins may not provide direct evidence for such a model because any mutant that disturbs the orientation of the complex with reference to the  subunit can potentially encourage nucleotide exchange.

As mentioned before, there is evidence for the  subunit N and C termini interacting with the receptor. Inspection of the crystal structure for the heterotrimeric G protein indicates that the  subunit N terminus, C terminus and the  subunit C terminus lie roughly along the same axis and can be oriented toward the plane of the membrane (Fig. 5). The distance between the C termini of  and  subunits cannot be estimated precisely in the crystal structure because at least seven residues at the  subunit C terminus are not resolved, and the  subunit is devoid of the prenyl moiety as well as three residues at its C terminus (4). The NMR structure of an 11-amino acid peptide specific to the  subunit C terminus indicates that this domain forms a constrained structure in the presence of a receptor (19). It is thus likely that the distance between the  subunit C terminus that includes the prenyl group and the C terminus of the  subunit is less than 40 Å. The crystal structure of inactive rhodopsin that has been determined recently indicates that the intracellular portions of rhodopsin are folded such that the longest distance across the intracellular domains is more than 40 Å (20). This indicates that the exposed surface of the receptor will be sufficient for both the  and  subunit C termini to contact the receptor simultaneously. However, this may not be a requirement if the two domains contact the receptor in a temporal sequence. We propose that by interacting with the receptor, the  subunit appropriately positions the complex with reference to the  subunit. This can allow the receptor to regulate nucleotide exchange at a site in the  subunit that does not contact the receptor directly.

	ACKNOWLEDGEMENT

We thank Vani Kalyanaraman for synthesizing mutant  subunit cDNAs.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM46963.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 is to be addressed: Box 8054, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-362-8568; E-mail: gautam@morpheus.wustl.edu.

Published, JBC Papers in Press, September 6, 2001, DOI 10.1074/jbc.M104034200

	ABBREVIATIONS

The abbreviations used are: GTPS, guanosine 5'-3-O-(thio)triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PLC, phospholipase C.

	REFERENCES

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				M. Akgoz, V. Kalyanaraman, and N. Gautam Receptor-mediated Reversible Translocation of the G Protein {beta}{gamma} Complex from the Plasma Membrane to the Golgi Complex J. Biol. Chem., December 3, 2004; 279(49): 51541 - 51544. [Abstract] [Full Text] [PDF]

				Q. Zhang, A. Dickson, and C. A. Doupnik G{beta}{gamma}-activated Inwardly Rectifying K+ (GIRK) Channel Activation Kinetics via G{alpha}i and G{alpha}o-coupled Receptors Are Determined by G{alpha}-specific Interdomain Interactions That Affect GDP Release Rates J. Biol. Chem., July 9, 2004; 279(28): 29787 - 29796. [Abstract] [Full Text] [PDF]

I. Azpiazu and N. Gautam A Fluorescence Resonance Energy Transfer-based Sensor Indicates that Receptor Access to a G Protein Is Unrestricted in a Living Mammalian Cell J. Biol. Chem., June 25, 2004; 279(26): 27709 - 27718. [Abstract]