Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Heterotrimeric G proteins transmit signals from cell surface receptors to intracellular effector proteins that modulate a wide variety of physiological processes (Neer, 1995). The - and -subunits of G proteins are associated in the inactive GDP-bound form. Receptors activate G proteins by catalyzing replacement of GDP by GTP on the -subunit. Binding of GTP leads to dissociation of the receptor from  · GTP and , each of which can transmit signals to effectors.

Our understanding of the mechanism of receptor-mediated activation of G proteins is incomplete. The predicted membrane-facing side of the heterotrimer includes the amino and carboxyl termini of the -subunit and places the nucleotide binding site too far away to contact the receptor (Fig. 1). Therefore, receptors are thought to stimulate nucleotide exchange through currently undefined conformational changes transmitted from the sites of receptor binding to the nucleotide binding pocket (Bohm et al., 1997; Bourne, 1997).

View larger version (107K): [in this window] [in a new window]   Fig. 1.   Model of the receptor-G protein complex. The G protein heterotrimer is an t/i1 chimera complexed with tt (Lambright et al., 1996). The receptor model is the MII rhodopsin model (Pogozheva et al., 1997). Positioning of the heterotrimer relative to the receptor is based on the model proposed by Bourne (1997). The GTPase domain of the -subunit is light blue. The helical domain of the -subunit is pink. The GDP is yellow. Switch I is dark blue, switch II is green, and switch III is magenta. The 2/4 loop is yellow, the 3/5 loop is orange, and the 4/6 loop is red. The carboxyl terminus (labeled C) of the -subunit is magenta. Selected regions of secondary structure in the -subunit, including the amino-terminal -helix (N), are indicated. The -strands of the -subunit are orange, and the amino-terminal helix and connecting loops are yellow. The -subunit is white. Receptor helices are numbered, and those connected to each other by an intracellular loop are the same color. This figure was drawn using MidasPlus, developed by the Computer Graphics Laboratory at the University of California at San Francisco.

The -subunits consist of two domains: a GTPase domain that resembles that of EF-Tu and Ras, and a helical domain consisting of -helices and connecting loops (Fig. 1). The bound nucleotide is buried between these domains. Three regions in the GTPase domain (switches I-III; Fig. 1) assume different conformations in the structures of guanosine 5'-O-(3-thiotriphosphate) (GTPS)-bound versus GDP-bound -subunits (Noel et al., 1993; Coleman et al., 1994; Lambright et al., 1994; Mixon et al., 1995). These regions are likely to play a role in receptor-mediated activation, because switches I and II contact (Lambright et al., 1996), with which  must associate to be activated by receptors (Fung, 1983), and mutations that disrupt interactions between switch III and the helical domain impair receptor-mediated activation (Grishina and Berlot, 1998; Marsh et al., 1998; Warner et al., 1998).

Previous studies have identified regions of _s that play a role in activation by the ₂-adrenergic receptor (Hildebrandt et al., 1991; Codina and Birnbaumer, 1994; Iiri et al., 1997; Grishina and Berlot, 1998; Marsh et al., 1998; Warner et al., 1998). Of these regions, many are near the nucleotide, and only the extreme carboxyl terminus is likely to be a receptor contact site. However, the carboxyl terminus is not always necessary or sufficient to confer receptor specificity (Lee et al., 1995; Conklin et al., 1996), indicating that additional receptor-binding sites on the -subunit have not been identified. Furthermore, it is not clear how receptor-dependent changes in the position of the carboxyl terminus would affect the conformational switch regions or interactions across the domain interface. Identification of the additional receptor binding site or sites is necessary to establish a connection between the receptor, the conformational switch regions, and the nucleotide binding site.

In this report, we evaluate the roles of three adjacent regions of _s, the 2/4, 3/5, and 4/6 loops, in receptor-mediated activation. The location of these regions on the membrane-facing side of the molecule made them viable candidates for being receptor contact sites (Fig. 1). However, they were not tested previously in a comprehensive scanning mutagenesis study of _s that used receptor-stimulated cAMP accumulation as the read-out for receptor-mediated activation (Marsh et al., 1998), because substitutions in these regions with _i2 residues impair the activation of adenylyl cyclase (Itoh and Gilman, 1991; Berlot and Bourne, 1992).

In the crystal structure of _s complexed with the catalytic domains of adenylyl cyclase (Tesmer et al., 1997), the 2/4 and 3/5 regions contact adenylyl cyclase, but the 4/6 loop does not. Therefore, using alanine-scanning mutagenesis, we reevaluate the role of the 4/6 loop in adenylyl cyclase activation as well as test its role in receptor-mediated activation. None of the alanine substitutions cause specific defects in adenylyl cyclase activation or in receptor-mediated activation, suggesting that the loop plays an indirect role in adenylyl cyclase activation and does not mediate receptor binding.

Replacement of _s residues with _i2 homologs in the 3/5 loop, but not the 2/4 loop, decreases receptor-mediated activation and increases affinity for the ₂-adrenergic receptor. The effects of the 3/5 substitutions are not due to a destabilization of the nucleotide-bound state, suggesting that the wild-type residues may be receptor contact sites that are optimized to ensure the reversibility of receptor-G protein association. The correspondence of the 3/5 region to an exchange factor contact site in both EF-Tu and Ras suggests that the mechanisms by which seven-transmembrane receptors and exchange factors catalyze nucleotide exchange may share common elements.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. The mammalian expression vector pcDNA I/Amp was obtained from InVitrogen (Carlsbad, CA). The bacterial expression vector pQE60, plasmid maxi kits, and Ni²⁺ NTA resin were obtained from Qiagen (Santa Clarita, CA). Q Sepharose Fast Flow resin and ECL Western blotting detection reagents were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Isoproterenol, 1-methyl-3-isobutylxanthine, cAMP, ATP, tosylphenylalanyl chloromethyl ketone-treated trypsin (T-8642), and Lubrol-PX were obtained from Sigma Chemical Co. (St. Louis, MO). Complete, EDTA-free protease inhibitor cocktail tablets were obtained from Boehringer Mannheim (Indianapolis, IN). Nitrocellulose filters for the GTPS binding assay were obtained from Millipore (Bedford, MA). Norit-SA3 was obtained from Aldrich (Milwaukee, WI). NuPAGE Bis-Tris 4-12% gels were obtained from NOVEX (San Diego, CA). [2-³H]Adenine was obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). [³⁵S]GTPS, [-³²P]GTP, and [¹²⁵I]iodocyanopindolol (ICYP) were obtained from New England Nuclear (Boston, MA).

Construction of _s Mutant Constructs. For expression in mammalian cells, _s mutant constructs were generated from the rat _s cDNA and contain an epitope, referred to as the EE epitope, that was generated by mutating _s residues DYVPSD (189-194)¹ to EYMPTE (single-letter amino acid code, mutated residues are underlined). For expression in Escherichia coli, _s mutant constructs were generated from the long splice variant of bovine _s containing a carboxyl-terminal hexahistidine tag, which was a generous gift from Alfred Gilman. All mutations were generated by oligonucleotide-directed in vitro mutagenesis using the Bio-Rad Muta-Gene kit except for those in _s(3/5), which were produced by subcloning a mutagenic oligodeoxynucleotide cassette. Subcloning and mutagenesis procedures were verified by restriction enzyme analysis and DNA sequencing.

cAMP Accumulation Assay. To determine the abilities of _s mutant constructs to activate adenylyl cyclase, the constructs were transiently expressed in COS-7 cells using DEAE-dextran under the control of the cytomegalovirus promoter in the expression vector pcDNA 1/Amp, and intracellular cAMP levels in cells labeled with [³H]adenine were determined as described previously (Medina et al., 1996).

Membrane Preparations from COS-7 Cells and Trypsin Assay. COS-7 cells were transiently transfected with _s mutant constructs using DEAE dextran as described above. Membranes were prepared 48 h after transfection as described previously (Medina et al., 1996). The trypsin resistance assay was performed as described previously (Berlot and Bourne, 1992). Samples were resolved by SDS-polyacrylamide gel electrophoresis (10%), transferred to nitrocellulose, and probed with the anti-EE monoclonal antibody as described previously (Medina et al., 1996). The antigen-antibody complexes were detected using an anti-mouse horseradish peroxidase-linked antibody according to the ECL Western blotting protocol.

Preparation of Stable Cell Lines. The _s constructs were expressed in cyc kin S49 lymphoma cells, which lack endogenous _s (cyc) and in which cAMP-dependent protein kinase is inactivated (kin), and cell membranes were prepared as described previously (Grishina and Berlot, 1998).

Adenylyl Cyclase Assay. Adenylyl cyclase activity in membranes of cyc kin S49 lymphoma cell lines expressing _s constructs was measured and used to determine EC₅₀ values for stimulation of adenylyl cyclase by GTPS in the presence and absence of isoproterenol as described previously (Grishina and Berlot, 1998).

Receptor Binding Assay. Competition between isoproterenol and [¹²⁵I]ICYP for binding to ₂-adrenergic receptors in membranes of cyc kin S49 lymphoma cell lines expressing _s constructs was measured as described by Grishina and Berlot (1998). The experimental data were analyzed for competition at two sites by nonlinear least-squares curve fitting as described by Grishina and Berlot (1998). K_L and K_H, the low- and high-affinity dissociation constants, were allowed to vary under the two conditions.

Expression and Purification of _s from E. coli. Both _s and _s(3/5) in the plasmid pQE60 were expressed in E. coli strain JM109. Cultures were grown, _s expression was induced, and lysates were produced as described previously (Lee et al., 1994), except that Complete, EDTA-free protease inhibitor cocktail tablets were included in the lysis buffer. The supernatant from a 30-min, 25,000g centrifugation was applied to a Ni²⁺ NTA column that had been equilibrated with buffer A (50 mM Tris, pH 8.0, 20 mM 2-mercaptoethanol, 50 µM GDP, 1 mM phenylmethylsulfonyl fluoride, and 1 Complete, EDTA-free protease inhibitor cocktail tablet/50 ml). The column was washed sequentially with buffer A containing 500 mM NaCl and buffer A containing 50 mM NaCl and 10 mM imidazole before elution with buffer A containing 50 mM NaCl, 150 mM imidazole, and 10% glycerol. The protein was concentrated and exchanged into buffer B (50 mM Tris, pH 8.0, 1 mM EDTA, 2 mM dithiothreitol (DTT), and 10% glycerol) containing 50 µM GDP. The protein was then applied to a Q Sepharose Fast Flow column. The column was washed with buffer C (50 mM Tris, pH 8.0, 1 mM EDTA, 5 mM MgCl₂, 14.5 mM 2-mercaptoethanol, 25 µM GDP, and 10% glycerol), and _s was eluted with buffer C containing a linearly increasing gradient of NaCl (0-300 mM). Peak fractions were concentrated, exchanged into buffer B containing 10 µM GDP, snap-frozen in liquid nitrogen, and stored at 80°C at a concentration greater than 2 mg/ml.

GTPS Binding Assays. To measure association of GTPS, 100 nM _s or _s(3/5) was incubated at 20°C with 1 µM [³⁵S]GTPS (5 × 10⁴ cpm/pmol) in a buffer containing 25 mM HEPES (pH 8.0), 10 mM MgCl₂, 1 mM EDTA, 100 mM NaCl, and 1 mM DTT. At various times, aliquots (50 µl; 5 pmol) were withdrawn and immediately filtered under vacuum on nitrocellulose filters. The filters were rinsed twice with 10 ml of ice-cold Stop Buffer (25 mM Tris, pH 8.0, 100 mM NaCl, and 25 mM MgCl₂). Apparent on rates of GTPS binding (k_app) were calculated by a nonlinear least-squares fit to the equation:
To measure dissociation of GTPS, 100 nM _s or _s(3/5) was incubated with 1 µM [³⁵S]GTPS as described above for 50 min. Dissociation of [³⁵S]GTPS was initiated by the addition of unlabeled GTPS to a final concentration of 100 µM. At various times, aliquots (50 µl; 5 pmol) were withdrawn and added to 2 ml of ice-cold Stop Buffer and maintained on ice until all samples were collected. Samples were then filtered on nitrocellulose filters as described above.

Single Turnover GTPase Assay. One-hundred nanomolar _s or _s(3/5) was incubated at 20°C with 1 µM [³²P]GTP (2 × 10⁴ cpm/pmol) in a buffer containing 50 mM HEPES (pH 8.0), 1 mM EDTA, and 1 mM DTT. After 30 min, the first aliquot (50 µl; 5 pmol) was withdrawn, and MgCl₂ and GTP were added to final concentrations of 10 mM and 100 µM, respectively. Aliquots were withdrawn at various times and added to 750 µl of ice-cold 5% (w/v) Norit-SA3 in 50 mM NaH₂PO₄. Samples were microcentrifuged, and [³²P]Pi released in the supernatant was determined by liquid scintillation counting. Catalytic rates of GTP hydrolysis (k_cat) were calculated by a nonlinear least-squares fit to the equation:
where P is the amount of phosphate released at time t, P_max is the maximum amount of phosphate released, and P₀ is the amount of phosphate released during the 30-min preincubation in the absence of MgCl₂. P_max for _s and _s(3/5) was 1.8 and 2.7 pmol, respectively.

Trypsin Assay Using Purified _s. _s or _s(3/5) (2.7 µM) was incubated at 30°C for 30 min in a buffer containing 20 mM HEPES (pH 8.0), 2 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 0.025% Lubrol-PX, and activators as indicated. Tosylphenylalanyl chloromethyl ketone-treated trypsin was then added to a final concentration of 0.4 mg/ml, and the mixture was incubated for 5 min at 30°C. The digestion was terminated by adding soybean trypsin inhibitor to a final concentration of 1 mg/ml. The samples were resolved by polyacrylamide gel electrophoresis on a NuPAGE Bis-Tris 4-12% gel. Proteins were visualized by staining with Coomassie Blue.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Role of 4/6 Loop in Adenylyl Cyclase Activation. Substitutions of _i2 residues for _s residues in the 4/6 loop were shown previously to decrease activation of adenylyl cyclase by _s (Berlot and Bourne, 1992). However, in the crystal structure of _s complexed with the catalytic domains of adenylyl cyclase (Tesmer et al., 1997), the 4/6 loop does not contact adenylyl cyclase. In the structure of _s (Sunahara et al., 1997), the 3/5 and 4/6 loops are closer together than in the corresponding structures of _t (Noel et al., 1993) and _i1 (Coleman et al., 1994), in part because of an interaction between Trp277 in 3/5 and His357 in 4/6. The proximity of these loops suggested that the effects of mutations in the 4/6 loop might be due to changes in the conformation of the 3/5 loop, which contacts adenylyl cyclase (Tesmer et al., 1997) and is important for its activation (Itoh and Gilman, 1991; Berlot and Bourne, 1992). To test this hypothesis, we determined the effects of substituting alanine residues in the 4/6 loop (Fig. 2A). The ability of mutant _s constructs to stimulate cAMP accumulation was measured after transient transfection in COS-7 cells. All constructs contained a GTPase-inhibiting mutation, referred to as the RC mutation, that replaces Arg201 with cysteine and causes constitutive activation, facilitating detection of adenylyl cyclase-activating ability above that of _s endogenous to COS-7 cells, as described previously (Berlot and Bourne, 1992).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Adenylyl cyclase activation by s constructs with substitutions in the 4/6 loop. A, panel of s mutant constructs with substitutions in the 4/6 region. The top sequence is that of s. Below that is the sequence of i2. Residues that are identical with s residues are represented by dashes. s(4/6)1 and s(4/6)2 are mutant s constructs with decreased abilities to activate adenylyl cyclase (Berlot and Bourne, 1992). The i2 substitutions in s(4/6)2 were based on an alignment of the s and i2 sequences that differs from the current alignment, which is based on the crystal structures of s (Sunahara et al., 1997) and i1 (Coleman et al., 1994). The remaining constructs contain alanine substitutions as indicated. B, top, cAMP accumulation in 106 COS-7 cells transfected with 1.5 µg of vector alone or vector containing sRC or the indicated mutant constructs. cAMP levels in [3H]adenine-labeled cells were determined as described in Experimental Procedures. Each value represents the mean ± S.E. of three independent experiments. Bottom, expression and trypsin sensitivity of these constructs. COS-7 cells (6.25 × 106) were transfected with 3 µg plasmid/106 cells of vector alone or vector containing sRC or the indicated constructs, and membranes were prepared, treated with trypsin, and immunoblotted as described in Experimental Procedures. The first lane in each set is the control (no trypsin). The second and third lanes show the result of trypsin digestion in the presence or absence, respectively, of GTPS. Similar results were obtained in two additional experiments.

Role of 4/6 Loop in Receptor-Mediated Activation. Because _s mutants with alanine substitutions in the 4/6 loop did not exhibit defects in ability to activate adenylyl cyclase when expression level was controlled for, we tested their responses to ₂-adrenergic receptors by measuring receptor-dependent cAMP accumulation in transiently transfected cyc S49 lymphoma cells, which lack endogenous _s (Harris et al., 1985). We used receptor-independent cAMP accumulation due to versions of the _s mutants containing the RC mutation (the RC versions) to normalize for expression level, as described previously (Grishina and Berlot, 1998; Marsh et al., 1998). For all of the constructs shown, the plasmid doses required to produce similar receptor-independent activities of the RC versions in cyc cells (Fig. 3B) were consistent with their activities and expression levels in COS-7 cells (Fig. 2B). Y358A/Y360A_s could not be evaluated in this assay due to the unexpectedly low activity of its RC version in cyc cells.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Receptor-mediated activation of s proteins with mutations in the 4/6 loop. A, cAMP accumulation in cyc cells transiently transfected with the indicated mutants in the s context. Cells were electroporated with 30 µg of vector alone and of vector containing s and S349As, 45 µg of vector containing D354A/R356As and C359A/P361As, and 60 µg of vector containing H357As. At these plasmid doses, similar amounts of receptor-independent cAMP accumulation were produced by versions of the constructs containing the RC mutation (B). cAMP values from unstimulated cells and from cells stimulated with 0.1 mM isoproterenol are dark gray and light gray, respectively. B, receptor-independent cAMP accumulation in cyc cells containing the indicated mutants in the sRC context. For each mutant, the same amount of plasmid was used as is indicated in A for the corresponding s mutant. cAMP levels in [3H]adenine-labeled cells were determined as described in Experimental Procedures. All values represent the mean ± S.E. of three independent experiments.

Mutations in 3/5 Region but Not 2/4 Region of _s Decrease Receptor-Mediated Activation. To test the roles of the 2/4 and 3/5 loop regions in receptor-mediated activation, we examined an _s construct referred to as _s(2/4), containing three substitutions of _i2 homologs for _s residues (Q236H/N239E/D240G) in the 2 helix and 2/4 loop, and an _s construct referred to as _s(3/5), containing five _i2 homolog substitutions (N271K/K274D/R280K/T284D/I285T) in the 3 helix and 3/5 loop, after stable expression in cyc S49 lymphoma cells. (Figure 9 shows the locations of these substitutions in the secondary structure of _s.)

View larger version (19K): [in this window] [in a new window]   Fig. 4.   s(3/5) exhibits a defect in receptor-mediated activation. Adenylyl cyclase activities in membranes of cyc cells stably expressing s, clone 3 (A), s(2/4), clone 1 (B), or s(3/5), clone 1 (C) were determined in the presence of the indicated concentrations of GTPS in the presence () or absence () of 100 µM isoproterenol (Iso). Expression levels of these clones are shown in Fig. 5. Data points represent the mean values from three independent experiments and are expressed as the percentage of the maximum observed adenylyl cyclase activity. EC50 values were calculated as described previously (Grishina and Berlot, 1998).

Mutations in 3/5 Region but Not 2/4 Region of _s Increase Apparent Affinity of _s for ₂-Adrenergic Receptor. To determine whether the decreased receptor-mediated activation of _s(3/5) was due to altered receptor binding, we used a competitive binding assay that measures an _s-dependent increase in the affinity of the ₂-adrenergic receptor for the agonist isoproterenol (Grishina and Berlot, 1998). The high-affinity isoproterenol-binding state of the receptor, which requires the presence of G_s in the nucleotide-free state, reflects receptor-G_s interaction. Isoproterenol binding is measured in competition with the antagonist ICYP, which binds to the receptor with the same affinity in the presence and absence of G_s.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Competition between isoproterenol and [125I]ICYP for binding to the 2-adrenergic receptor. A, immunoblot showing expression levels of s, s(2/4), and s(3/5) in stable cyc cell lines. Membranes of s, clone 3, (B), s(2/4), clone 1 (C), or s(3/5), clone 1 (D), were incubated with [125I]ICYP (75 pM) and the indicated concentrations of isoproterenol in the presence () or absence () of 300 µM GTP. Values represent the mean values of two independent experiments. The solid lines represent a nonlinear least-squares fit to the data, as described previously (Grishina and Berlot, 1998). KL and KH are the low- and high-affinity dissociation constants, respectively, and % RH is the percentage of receptors in the high-affinity form. In C and D, the binding curves for membranes from s-expressing cells, from B, are redrawn as dotted lines. Similar results for each construct were obtained in two additional experiments using the other cell lines in A.

_s(3/5) Exhibits Normal Guanine Nucleotide Handling Properties. There is precedent for mutations that both decrease receptor-mediated activation of _s and impair guanine nucleotide binding (Iiri et al., 1997; Warner et al., 1998) and/or hydrolysis (Warner and Weinstein, 1999). In addition, because the affinity of G proteins for receptors is greatest in the nucleotide-free state, changes in nucleotide binding could affect apparent receptor affinity in the presence of nucleotide. Therefore, using purified _s and _s(3/5), we investigated whether the mutations in _s(3/5) are associated with intrinsic defects in guanine nucleotide handling.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Biochemical properties of s and s(3/5). A, rates of GTPS binding. s () or s(3/5) () (100 nM each) was incubated at 20°C with 1 µM [35S]GTPS (5 × 104 cpm/pmol). At the times indicated, aliquots (50 µl; 5 pmol) were withdrawn and filtered on nitrocellulose filters as described in Experimental Procedures. Apparent on rates of GTPS binding (kapp) were calculated as described in Experimental Procedures. Data points represent the mean ± S.E. of six experiments. B, dissociation of GTPS. s () or s(3/5) () (100 nM each) was incubated at 20°C with 1 µM [35S]GTPS as described in A for 50 min. Dissociation of [35S]GTPS was initiated by the addition of unlabeled GTPS to a final concentration of 100 µM. At the times indicated, aliquots (50 µl; 5 pmol) were withdrawn and filtered on nitrocellulose filters as described in Experimental Procedures. Data points represent the mean ± S.E. of four experiments. The values of koff for both s and s(3/5) were indistinguishable from 0. C, kcat for the hydrolysis of GTP. s () or s(3/5) () (100 nM each) was incubated at 20°C with 1 µM [32P]GTP (2 × 104 cpm/pmol) for 30 min in the presence of 1 mM EDTA. After withdrawal of the first aliquot, MgCl2 and GTP were added to final concentrations of 10 mM and 100 µM, respectively. At the times indicated, aliquots (50 µl; 5 pmol) were withdrawn, and [32P]Pi released was determined as described in Experimental Procedures. Catalytic rates of GTP hydrolysis (kcat) were calculated as described in Experimental Procedures. Data points represent the mean ± S.E. of six experiments.

_s(3/5) Exhibits Normal Activation by AlF₄. Some _s mutations (Hildebrandt et al., 1991; Codina and Birnbaumer, 1994; Iiri et al., 1997; Grishina and Berlot, 1998; Warner et al., 1998) that decrease receptor-mediated activation also decrease activation by AlF₄, which activates -subunits by mimicking the -phosphate of GTP in the postulated transition state intermediate of the GTPase reaction (Coleman et al., 1994; Sondek et al., 1994). In contrast, AlF₄ induced the trypsin-resistant activated conformation in _s(3/5) to the same extent as GTPS did and to the same extent as in _s (Fig. 7). Because activation by AlF₄ requires that the nucleotide binding site contain GDP and be in an appropriate conformation, this result further supports the conclusion that the mutations in _s(3/5) specifically alter receptor-mediated activation and receptor binding without causing global structural distortions in _s.

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Effects of GTPS and GDP/AlF4 on tryptic cleavage; 2.7 µM s or s(3/5) was incubated with 12.5 µM GTPS, 12.5 µM GDP plus 25 µM AlCl3 and 12.5 mM NaF, or 12.5 µM GDP and then treated with trypsin as described in Experimental Procedures. The s and trypsin-resistant fragments of s were resolved by polyacrylamide gel electrophoresis and visualized by staining with Coomassie Blue. Similar results were obtained in two additional experiments.

Mapping of Residues Important for the G_s-₂-Adrenergic Receptor Interaction onto a Receptor-G Protein Model. We visualized the results of this and previous studies of the _s residues important for interaction with the ₂-adrenergic receptor using a model of the receptor-G protein complex with essentially the same constraints as the model proposed by Bourne (1997). The amino terminus of the -subunit and the carboxyl terminus of the -subunit, which contain lipid modifications important for membrane attachment, face the membrane. The third intracellular loop of the receptor is located at the interface between  and , near regions shown by mutagenesis and peptide studies to be important for receptor interaction (Bourne, 1997): the amino and carboxyl termini of  and the carboxyl terminus of .

View larger version (68K): [in this window] [in a new window]   Fig. 8.   Mapping of residues important for Gs-2-adrenergic receptor interaction onto a receptor-G protein model. s residues are mapped onto the model of the receptor-G protein complex shown in Fig. 1A, ribbon diagram. The -strands of the -subunit are orange, and the amino-terminal helix and connecting loops are yellow. The -subunit is white. The GDP is yellow. s residues in which substitutions with the homologous i2 residues were shown previously to leave receptor-mediated activation intact (Masters et al., 1988; Grishina and Berlot, 1998; Marsh et al., 1998) are green. s residues that have not been tested for their roles in receptor interaction are light blue. The residues mutated in s(3/5) are red spheres (3/5 loop) or pink spheres (3 helix). Additional residues in which mutations alter receptor interaction (Hildebrandt et al., 1991; Codina and Birnbaumer, 1994; Iiri et al., 1997; Grishina and Berlot, 1998; Marsh et al., 1998) are red, except for the carboxyl terminus (labeled C), which is magenta. Residues in the 2/4 and 4/6 loops, in which substitutions with i2 homologs and alanine residues, respectively, do not alter receptor interaction, are green spheres. The numbers on the spheres represent s residue numbers. Receptor helices are numbered, and those connected to each other by an intracellular loop are the same color. B, space-filling model viewing the heterotrimer surface that faces the receptor. The receptor helices are outlined in black, and the intracellular loops (i.c.1-3) and carboxyl-terminal tail are indicated. Colors are as in A except that  is entirely orange. These figures were drawn using MidasPlus, developed by the Computer Graphics Laboratory at the University of California at San Francisco.