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. 1997 May 27;94(11):5656-61.
doi: 10.1073/pnas.94.11.5656.

Conditional activation defect of a human Gsalpha mutant

T Iiri  1 , Z Farfel, H R Bourne
Affiliations

Conditional activation defect of a human Gsalpha mutant

T Iiri et al. Proc Natl Acad Sci U S A. .

Abstract

Hormonal signals activate trimeric G proteins by promoting exchange of GTP for GDP bound to the G protein's alpha subunit (Galpha). Here we describe a point mutation that impairs this activation mechanism in the alpha subunit of Gs, producing an inherited disorder of hormone responsiveness. Biochemical analysis reveals an activation defect that is paradoxically intensified by hormonal and other stimuli. By substituting histidine for a conserved arginine residue, the mutation removes an internal salt bridge (to a conserved glutamate) that normally acts as an intramolecular hasp to maintain tight binding of the gamma-phosphate of GTP. In its basal, unperturbed state, the mutant alphas binds guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]), a GTP analog, slightly less tightly than does normal alphas, but (in the GTP[gammaS]-bound form) can stimulate adenylyl cyclase. The activation defect becomes prominent only under conditions that destabilize binding of guanine nucleotide (receptor stimulation) or impair the ability of alphas to bind the gamma-phosphate of GTP (cholera toxin, AlF4- ion). Although GDP release is usually the rate-limiting step in nucleotide exchange, the biochemical phenotype of this mutant alphas indicates that efficient G protein activation by receptors and other stimuli depends on the ability of Galpha to clasp tightly the GTP molecule that enters the binding site.

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Figures

Figure 1
Figure 1
cAMP accumulation and β-adrenergic receptor binding in S49 cyc− cells transfected with wild type or mutant αs. (A) cAMP accumulation in intact cells. S49 cyc− cells stably transfected with HA-αs or HA-αs-R231H were incubated at 37°C for 30 min with 100 nM 3-isobutyl-1-methylxanthine and 10 μM isoproterenol (▪) or no drug (□ and ▨) and cAMP accumulation was measured (21). Cholera toxin (1 μg/ml; ▨) was added to the culture medium 3 h before adding IBMX. Values represent mean ± SD of triplicate determinations. (B) Competition between isoproterenol and [125I]pindolol for binding the β-adrenergic receptor. Membranes (0.15 mg/ml) of S49 cyc− cells stably transfected with HA-αs or HA-αs-R231H were incubated with [125I]pindolol (72 pM) and the indicated concentrations of isoproterenol in the presence (open symbols) or absence (filled symbols) of 30 μM GTP[γS]; incubations and binding assays were performed as described (22).
Figure 2
Figure 2
Immunofluorescence of wt or mutant αs. HEK293 cells stably expressing the β2-AR and HA-αs or HA-αs-R231H were incubated with (C, D, G, and H) or without (A, B, E, and F) 10 μM isoproterenol for 20 min and then fixed with 4% formaldehyde. αs was detected by incubation with mAb 12CA5, followed by fluorescein isothiocyanate-conjugated anti-mouse antibody (15). The β2-AR was detected by incubation with a polyclonal rabbit antiserum against its C terminus (14), followed by Texas red-conjugated anti-rabbit antibody (15). Images were visualized as described (14, 15).
Figure 3
Figure 3
Biochemical properties of recombinant wt and mutant αs. (A) Rates of GTP[γS] binding. αs or αs-R231H (• and ▴, respectively; 50 nM each) were incubated at 22°C with 1 μM [35S]GTP[γS] (3 × 105 cpm/pmol). At the times indicated, the reaction was terminated and GTP[γS] binding was quantitated by filtration on nitrocellulose filters (13, 20) and apparent on-rates of GTP[γS] binding (kapp) were calculated (19). (B) Rates of dissociation of GTP[γS]. αs or αs-R231H (• and ▴, respectively; 50 nM each) were incubated at 22°C with 1 μM [35S]GTP[γS] as described in A for 45 min. Dissociation of bound [35S]GTP[γS] was assessed by adding 200 μM unlabeled GTP[γS] (at time zero in B). At the times indicated, the reaction was terminated and GTP[γS] binding was quantitated as described in A. (C) cAMP synthesis stimulated by different concentrations of αs or αs-R231H in the presence of GTP[γS]. Reactions were conducted at 22°C for 15 min in 50 μl volumes containing 15 μg cyc− membranes, as described (13). Before the assay, the αs proteins were incubated with 100 μM GTP[γS] for 60 min.
Figure 4
Figure 4
Effect of activation on tryptic cleavage of wt and mutant αs. (A) Receptor dependent activation of wt and mutant αs. Membranes (0.2 mg/ml) of COS-7 cells expressing recombinant HA-αs (•) or HA-αs-R231H (▴) plus the β2-AR and G protein β2 and γ2 subunits were incubated at 22°C with (filled symbols) or without (open symbols) 10 μM isoproterenol plus 10 μM GTP[γS]. At the times indicated, the reaction was terminated and samples were treated with trypsin (0.6 mg/ml) as described in Materials and Methods. Trypsin-resistant fragments of αs were visualized and quantitated by Western blot analysis using 12CA5 antibody (12). (B) Effect of modification by cholera toxin on protection by GTP[γS] against cleavage by trypsin. HEK293 cells stably transfected with HA-αs or HA-αs-R231H were cultured in the absence or in the presence of 1 μg/ml of cholera toxin for 3 h. Membranes were incubated with 10 μM GTP[γS] at 22°C for 10 min. Samples were incubated with trypsin (10 μg/ml) and trypsin-resistant fragments of αs (indicated by arrow) were visualized by Western blot analysis as described in A. (C) Effect of GTP[γS] and GDP/AlF4− on tryptic cleavage. αs or αs-R231H (0.7 μM each) were incubated with 10 μM GTP[γS], 10 μM GDP, or 10 μM GDP plus 20 μM AlCl3 and 10 mM NaF at 22°C for 60 min. Samples were further incubated in the absence or presence of trypsin (0.1 mg/ml) on ice for 60 min and trypsin-resistant fragments of αs (arrows) were visualized by SDS/PAGE followed by Coomassie blue staining.
Figure 5
Figure 5
The arginine-glutamate hasp of Gα. Switch (Swi) regions 1–3 in the crystal structure of αt⋅GTP[γS] (3) are shown in relation to bound GTP (green; γ-phosphate colored red) and Mg2+ ion (magenta). Except for side chains of key amino acids, only main chains are depicted. The γ-phosphate of GTP (red) is linked to two amino acids (yellow, not labeled) in switch 1; these include R174 (on the left side of the picture) and T177. R174 corresponds to R201 of αs, the target of ADP ribosylation by cholera toxin. T177 (T 204 in αs) is linked to the γ-phosphate via bound Mg2+ (magenta). The γ-phosphate is linked to switch 2 by the main chain amide of G199 in αt (red, not labeled; corresponding to G226 in αs). The intramolecular hasp is the salt bridge between R204 (red; cognate to R231 in αs) in switch 2 (the α2 helix) and E241 (blue; E268 of αs) in α3. The hasp helps to maintain both the tight binding of GTP and the GTP-induced active conformation by fastening switch 2 and α3, thus reinforcing linkage of the glycine residue in switch 2 to the γ-phosphate of GTP.
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