Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

Background: G protein-coupled receptors mainly signal through heterotrimeric G-proteins.

Results: We have demonstrated that mutations in the extended Linker 2 impaired the activation of Gα_s by β-adrenergic receptors.

Conclusion: We have proposed a potential novel conduit from β-adrenergic receptors to the helical domain of Gα_s subunit via the extended Linker 2.

Significance: Our systematic mutagenesis studies provide insights into the activation mechanism of G-proteins by receptors.

Abstract

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gα_s mutant proteins (from a screen of 66 Gα_s mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gα_s. This extended Linker 2 is the target site of a natural product inhibitor of G_q. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β₂/β₃ loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.

Introduction

A structurally diverse repertoire of ligands, from photons to many hormones and neurotransmitters, activate G protein-coupled receptors (GPCRs)⁴ to elicit their physiological functions (1, 2). GPCRs comprise a large and diverse superfamily, and family members have been identified in organisms as evolutionarily distant as yeast and human. Heterotrimeric G-proteins (Gαβγ) directly relay the signals from GPCRs (3–5). These G-proteins are composed of α, β, and γ subunits. The β and γ subunits are tightly associated and can be regarded as one functional unit. G-proteins function as molecular binary switches with their biological activity determined by the bound nucleotide (3–5). Upon ligand-binding, GPCRs increase the exchange of GDP bound on the Gα subunit with GTP, in the presence of Gβγ subunits. This leads to the dissociation of the Gα subunit from the Gβγ dimer resulting in two functional subunits (Gα and Gβγ). Both Gα and Gβγ subunits signal to various cellular pathways.

Over the past 30 years, great progress has been made in understanding the mechanisms by which heterotrimeric G-proteins regulate their downstream targets (6, 7). Recently a series of crystal structures of GPCRs in the inactive and active states, bound with antagonists, inverse agonists, or agonists, have elucidated the structural basis for the modulation and activation of GPCRs by ligands (1, 8, 9). A crystal structure of the complex of β₂-adrenergic receptor and G_s has revealed the structural changes in β₂-adrenergic receptor and G_s, the interacting regions, and residues between a GPCR and a G-protein (10). However, the molecular mechanisms by which GPCRs activate G-proteins are still not completely understood (11, 12).

The structure of Gα subunits consists of two domains: a Ras-like GTPase domain and an α-helical domain (6) (Fig. 1, A and B). These two domains are linked by Linker 1 and Linker 2 (Fig. 1B). Between these two domains lies a deep cleft within which GDP or GTP is tightly bound (Fig. 1, A and B). The nucleotide is essentially occluded from the bulk solvent, leading to a proposal that the helical domain is the inhibitory barrier and provides the regulatory entry point by GPCRs or Gβγ subunits (13–16).

FIGURE 1.

Summary of mutated amino acid residues of Gα_s. A, crystal structure of a Gα subunit shows the Ras-like GTPase domain and the α-helical domain. The αF, Linker 2, and β2 are indicated. The bound GDP is colored in magenta. B, diagram of a Gα subunit shows the relative locations of elements discussed in the paper. C, all residues on Gα contacting Gβγ are labeled (in red) based on crystal structures. The two images are the same rotated 180°. D, summary of mutated residues of Gα_s (magenta and green letters). The secondary structure is assigned based on the crystal structure of Gα_s·GTPγS (PDB code 1AZT). The three switch regions are indicated by black boxes. The two linker regions are indicated by arrows. Green amino acid letters indicate the residues mutated in the 8 Gα_s mutants that are activated by β-ARs without Gβγ binding.

One of the major remaining problems in the biology of GPCR/G-protein signaling is to experimentally demonstrate whether GPCRs or Gβγ subunits play the catalytic exchange role in Gα protein activation (17). In theory, the question could be straightforwardly answered with purified proteins of GPCRs, Gα and Gβγ subunits. However, purified GPCRs had no guanine-nucleotide exchange effect on Gα in the absence of Gβγ subunits (18, 19). A GPCR could only activate Gα in the presence of Gβγ (18, 19), leading some to believe that GPCRs were not the real catalysts, although they were required to initiate the activation event from agonist binding. Yet, purified Gβγ subunits alone also could not accelerate the guanine nucleotide exchange on Gα subunits (3, 20). In fact, Gβγ subunits inhibit the basal nucleotide exchange activity of Gα subunits and behave as a guanine-nucleotide dissociation inhibitor (20). When the structure of Gβ was revealed to be similar to RCC1, a guanine-nucleotide exchange factor for the small GTPase Ran, Gβγ was proposed to be the real guanine-nucleotide exchange factor for Gα, whereas GPCRs served as a trigger (15, 21). Therefore, it remains a fundamental unsolved question: which one, a GPCR and/or Gβγ subunit, has the ability to catalyze the nucleotide exchange on Gα.

We reasoned that if GPCRs or Gβγ subunits are the nucleotide exchangers, we should be able to find mutants of GPCRs or Gβγ subunits that would accelerate the nucleotide exchange on Gα subunits in the absence of Gβγ or GPCRs, respectively. Alternatively, we might be able to find mutant Gα subunits that could be activated by GPCRs alone or by Gβγ alone. Here we describe our finding of a direct activation of some Gα_s mutant proteins by β-adrenergic receptors without Gβγ subunits. Although our data do not exclude a possible additional catalytic role for Gβγ, it clearly demonstrates that GPCRs by themselves have the catalytic ability to activate Gα subunits. Furthermore, from this systematic study, a pivotal role for the αF/Linker 2/β2 region (extended Linker 2) in Gα activation has been uncovered. Therefore, we propose that the opening and closing of the two domains (the Ras-like GTPase domain and the helical domain) with Linker 2 as a pivot provide one of the mechanisms for Gα activation by GPCRs.

EXPERIMENTAL PROCEDURES

G-protein Purification

Gα_s wild-type and mutant proteins were purified from Escherichia coli. The pGEX-6P-Gα_s plasmid was transformed into bacteria strain BL21(DE3). One liter of bacterial culture was grown at room temperature until the absorbance at 600 nm was ∼1. G-protein expression was induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for 18 h at room temperature. The bacterial pellet was resuspended in lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 0.1 mg/ml of lysozyme, and 0.2 mm PMSF) and incubated on ice for 30 min. After sonication, the lysate was spun down at 10,000 × g for 120 min at 4 °C. Glutathione-agarose resin (0.5 ml, from Sigma) was added to the supernatant after pre-equilibration of the resin with lysis buffer. The mixture was gently agitated at 4 °C for 3 h. After washing 3 times with 10 ml of washing buffer (50 mm Tris, pH 8.0, 100 mm NaCl, and 0.2 mm PMSF), GST-tagged Gα_s proteins were eluted with 0.5 ml of elution buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA, 1 mm DTT, and 10% glycerol). preScission protease (Amersham Biosciences) was used to cleave GST off at 4 °C overnight.

Gβγ Proteins Were Purified from Insect Hi5 Cells

One liter of Hi5 cell pellet was resuspended into 50 ml of lysis buffer (50 mm Tris, pH 8.0, 1 mm EDTA, and protease inhibitors: 10 μg/ml of leupeptin, 1 μg/ml of pepstatin A, 1 mm benzamidine, and 0.2 mm PMSF). After sonication, the lysate was spun down at 150,000 × g for 90 min at 4 °C. The membrane pellet was resuspended in 50 ml of lysis buffer. After homogenization, the lysate was centrifuged again. The final pellet was resuspended in 50 ml of extraction buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 2% dodecyl-β-d-maltoside (DM) and protease inhibitors). After centrifugation at 10,000 × g for 120 min at 4 °C, 1 ml of nickel-nitrilotriacetic acid-agarose beads pre-equilibrated with extraction buffer was added to the supernatant. The mixture was gently agitated overnight at 4 °C. After washing 3 times with 10 ml of washing buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 5 mm imidazole, and 0.2 mm PMSF), Gβγ was eluted with 10 ml of elution buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 10 mm EDTA, and 200 mm imidazole).

Gβγ Binding Assay (with Purified Gβ₁γ₂ Proteins)

Ten μg of Gα_s protein (with an N-terminal GST tag, attached to glutathione beads) were incubated in 100 μl of binding buffer (20 mm Hepes, pH 8.0, 2 mm GDP, 1 mm DTT, 150 mm NaCl, and 0.02% DM) with 100 nm Gβ₁γ₂ proteins overnight at 4 °C. After centrifugation, beads were washed three times with binding buffer and then eluted with binding buffer containing 10 mm reduced glutathione. After SDS-PAGE, Western blots were performed with anti-Gα_s and anti-G_β antibodies (Santa Cruz Biotechnology, Inc.).

Gβγ Binding Assay (with Membrane Preparations)

Membrane preparations were made from 293T cells. After solubilization, the supernatant (in 20 mm Hepes, pH 8.0, 2 mm GDP, 1 mm DTT, 150 mm NaCl, 1% DM, and protease inhibitors) was pre-cleared with glutathione beads. 500 μl of supernatant was mixed with 10 μg of Gα_s protein (with an N-terminal GST tag, attached to glutathione beads). The mixtures were rolled at 4 °C overnight. After centrifugation, beads were washed three times with binding buffer and then eluted with binding buffer containing 10 mm reduced glutathione. After SDS-PAGE, Western blots were performed with anti-Gα_s and anti-G_β antibodies.

Size Exclusion Chromatography

Size exclusion chromatography was used to examine the binding of Gα_s and its mutants to Gβ₁γ₂ in solution. Gα_s (1 μm) and Gβ₁(C68S)γ₂ (2 μm) were mixed and incubated on ice for 30 min. Samples were loaded on a Superdex 200 column (GE Healthcare Life Sciences) equilibrated with 20 mm Hepes, pH 8.0, 50 mm NaCl, and 1 mm EDTA at a flow rate of 0.5 ml/min. The elution was monitored at 280 nm, and 0.8-ml fractions were collected for subsequent SDS-PAGE analysis.

In Vivo cAMP Assay

Cells were plated on 6-well plates and treated with 1 mm isobutylmethylxanthine for 30 min at 37 °C. After washing twice with HEM buffer (20 mm Hepes, pH 7.4, 135 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO₄, 2.5 mm NaHCO₃, 0.1 mm Ro-20–1724, 0.5 unit/ml of adenosine deaminase, and 1 mm isobutylmethylxanthine), cells were treated with 0, 1 nm, 10 nm, 100 nm, 1 μm, 10 μm, and 100 μm isoproterenol in HEM buffer for 5 min at 37 °C. After two additional washes with HEM buffer, cells were harvested and lysed with 0.5% Triton X-100 containing 1 mm isobutylmethylxanthine. The amount of cAMP was measured with a Direct Cyclic AMP Enzyme Immunoassay kit (Assay Designs, Inc.).

In Vitro Adenylyl Cyclase Activation Assay

Adenylyl cyclase activation assay of Sf9 membrane preparations was performed as previously described (22). Briefly, adenylyl cyclase isoform V (dog) was recombinantly expressed in Sf9 cells, and the adenylyl cyclase-containing membranes were prepared. Gα_s protein was activated by adding a buffer consisting of 10 mm NaF, 10 mm MgCl₂, 30 μm AlCl₃ and incubated at 30 °C for 1 h. Activated Gα_s proteins with membrane preparations of adenylyl cyclase V were incubated in buffer of 50 mm Tris, pH 7.6, 2 mm isobutylmethylxanthine, 1 mm ATP, 10 mm MgCl₂, 20 mm creative phosphate, 100 units/ml of creative phosphokinase at 30 °C for 10 min. The samples were boiled for 3 min to stop the reaction. After spinning for 3 min at 16,000 × g, the supernatant was used for cAMP measurement with Direct Cyclic AMP Enzyme Immunoassay kit.

Purification of Turkey β₁-Adrenergic Receptor Proteins

Turkey β₁-adrenergic receptor protein was purified as described previously (23). Turkey β₁-adrenergic receptor (residues 34–424 with a mutation C116L) cDNA was subcloned into the pVL1393 vector. Hi5 cells were infected with the recombinant baculovirus carrying the turkey β₁-adrenergic receptor at a density of 2 × 10⁶/ml. Sixty-hours later, cells were harvested and resuspended in lysis buffer (50 mm Tris-HCl, pH 8, 1 mm EDTA, 10 μg/ml of leupeptin, 9 mm benzamidine, 5 μg/ml of pepstatin A, and 2 mm PMSF). After homogenization, the cell lysate was centrifuged at 2,000 × g for 10 min and the supernatant was centrifuged again at 150,000 × g at 4 °C for 1.5 h. The membrane pellet was resuspended in lysis buffer and homogenized again. After spinning down at 150,000 × g at 4 °C for 1.5 h, the final pellet was then resuspended in membrane extraction buffer (50 mm Tris-HCl, pH 8, 350 mm NaCl, 3 mm imidazole, 2% DM, 10 μg/ml of leupeptin, 9 mm benzamidine, 5 μg/ml of pepstatin A, and 1 mm PMSF) and rolled at 4 °C for 3 h. After centrifugation at 150,000 × g at 4 °C for 1 h, solubilized membrane proteins were incubated with nickel-nitrilotriacetic acid-agarose (Qiagen) overnight. After washing with buffer containing 50 mm Tris-HCl, pH 8, 350 mm NaCl, 3 mm imidazole, 0.1% DM, 10 μg/ml of leupeptin, 9 mm benzamidine, 5 μg/ml of pepstatin A, and 1 mm PMSF, the receptor protein was eluted down by an imidazole gradient.

GTPγS Loading Assay

Agonist-stimulated GTPγS loading to G-proteins was performed as previously described (19). Gα_s (with or without 1 μm Gβγ) and turkey β₁-adrenergic receptor (20 nm) together with 10 μm alprenolol or isoproterenol in 200 μl of loading buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 10 mm MgCl₂, 1 mm EDTA, 0.02% DM, and 5 μm GDP) were incubated on ice for 20 min. After incubation at 30 °C (for wild-type and Gα_s219) or at room temperature (for Gα_s263 and Gα_s195) for 5 min, 100 nm GTPγS was added. At various times, 40-μl aliquots were removed and added to 1 ml of termination buffer (20 mm Tris, pH 8.0, 100 mm NaCl, and 25 mm MgCl₂, ice cold) and loaded onto nitrocellulose membrane (Schleicher & Schuell BioScience). After 3 washes with 1 ml of termination buffer, 4 ml of scintillation liquid were added to the membrane and ³⁵S was counted to measure GTPγS loading. 5 mm GDP was used to determine nonspecific binding.

Gα_s and Receptor Binding Assay

Different concentrations of Gα_s proteins (with an N-terminal GST tag, attached to glutathione beads) were incubated in 100 μl of binding buffer with 80 nm β-AR proteins (with a FLAG tag) at 4 °C for 1 h. GST (600 nm) was used as control. After centrifugation, beads were washed three times with washing buffer. After SDS-PAGE, Western blots were performed with anti-Gα_s and anti-FLAG M2 antibodies.

RESULTS AND DISCUSSION

Identification of Gα_s Mutants Defective in Gβγ Binding

To identify Gα subunits that could be activated by GPCRs in the absence of Gβγ subunits, we first characterized Gα_s mutants that were defective in Gβγ binding. The crystal structures of Gα and Gαβγ have been solved (24, 25). The conformational differences between the free and Gβγ-bound forms of Gα-GDP mainly involve residues that directly interact with Gβ (24, 25). Based on the crystal structures of Gα_tGβ₁γ₁ and Gα_i1Gβ₁γ₂ complexes, residues in the N-terminal, Switch I (Arg-185 to Ile-193, encompassing Linker 2 region), and Switch II region of Gα subunits are involved in contacting or binding of Gβγ (24, 25) (Fig. 1C, red residues). Furthermore, from the crystal structures of the Ras superfamily GTPases and their guanine nucleotide-exchange factors, such as Ras and Sos1, Arf1 and Sec7, Rac1 and Tiam1, EF-Tu and EF-Ts, as well as Ran and RCC1 (26–30), guanine nucleotide-exchange factors interact extensively with and remodel the Switch I and II regions of GTPases. Therefore, we have performed alanine scanning mutagenesis of every residue in these regions as well as some residues in Switch III and its adjacent regions (Table 1 and Fig. 1D).

TABLE 1.

Summary of the characterization of 66 Gα_S mutants

GTPγS binding and in vitro activation of adenylyl cyclase (AC) were shown as percentage of the values obtained with wild-type Gα_S. The increase of cAMP in cells is shown as the fold increase from each individual experiment. ΔN31 and ΔN38 are deletion mutants of the N-terminal 31 or 38 amino acids, respectively.

First we identified Gα_s mutants that could not bind Gβγ. We purified recombinant proteins of wild-type Gα_s and 66 mutant Gα_s proteins from E. coli (Table 1 and some examples shown in Fig. 2A). (Here we used the short spliced variant of bovine Gα_s. The difference between the short and long splice forms of Gα_s is an insertion of 14 amino acids after residue Gly-70.) We also purified recombinant Gβ₁γ₂ proteins from insect Hi5 cells (Fig. 2A). To test the functionality of these purified Gα_s mutant proteins, we performed in vitro GTPγS binding assays and adenylyl cyclase activation assays for all Gα_s mutants (Table 1). Other than eight mutants (Table 1), all Gα_s mutant proteins were able to bind to GTPγS, implying these purified proteins were stable and functional. Furthermore, using membrane preparations from Sf9 insect cells infected with recombinant baculoviruses carrying adenylyl cyclase type V, we performed in vitro adenylyl cyclase activation assays (Table 1, and some examples shown in Fig. 2, B–E). In addition to the 8 mutants that could not bind GTPγS in vitro, 4 more mutants could not activate adenylyl cyclase in vitro (Table 1). Three (Asn-265, Arg-266, and Trp-267) of these 4 residues are in the α3/β5 loop, which directly contacts the catalytic domain of adenylyl cyclase as revealed by the crystal structure of the complex of Gα_s and the catalytic domains of adenylyl cyclase (31). Thus, most of the purified mutant Gα_s proteins were stable and functional.

FIGURE 2.

Functional characterization of Gα_s and its mutants. A, Coomassie Blue staining shows the purified Gα_s proteins (some representatives), GST and Gβ₁γ₂. B–E, in vitro activation assays of adenylyyl cyclase by Gα_s and its mutants (some representatives). F and G, in vitro binding of Gα_s and representative mutants to Gβ₁γ₂. H, size exclusion chromatography of heterotrimer formation. Upper panels: elution profiles are shown for wild-type Gα_s alone, Gα_sK219A alone, Gβ₁γ₂ alone, the mixture of wild-type Gα_s/Gβ₁γ₂, and the mixture of Gα_sK219A/Gβ₁γ₂. Lower panels, SDS-PAGE analysis of the elution fractions. One representative experiment from three independent experiments is shown for each case. WB, Western blot.

We next tested the interaction of these Gα_s mutants with Gβγ. For the in vitro binding experiments, we incubated wild-type Gα_s and mutant Gα_s proteins with purified Gβ₁γ₂ proteins. Glutathione beads were used to pulldown Gα_s proteins. Co-precipitation of Gβ₁γ₂ was detected with anti-Gβ antibody. As shown in Table 1 (and some examples in Fig. 2F), wild-type Gα_s and 48 mutant Gα_s proteins pulled down Gβ₁γ₂, 18 other mutant Gα_s proteins (including Gα_sE195A, Gα_sK219A, or Gα_sW263A) did not bind Gβ₁γ₂. To confirm these binding data and to show that the 18 mutant Gα_s proteins could not interact with other Gβ subunits in addition to Gβ₁, we used membrane preparations of 293T cells as the source of Gβγ subunits (293T cells at least express Gβ₁, Gβ₂, and Gβ₄). The results were the same as the binding experiments with purified Gβ₁γ₂ (Table 1 and some examples shown in Fig. 2G). As a third approach, we used size exclusion chromatography to verify the inability of some Gα_s mutants binding to Gβγ. As shown in Fig. 2H, wild-type Gα_s, Gα_sK219A, and Gβ₁γ₂ eluted as single major peaks from the size exclusion column, demonstrating the homogeneity of the subunit preparations. When combined with excess Gβ₁γ₂, wild-type Gα_s showed a two-peaks elution profile: one was the free Gβ₁γ₂ and the other was the complex of Gα_s-Gβ₁γ₂ with a shorter retention time as compared with the isolated wild-type Gα_s (Fig. 2H). On the other hand, Gα_sK219A showed a two-peak elution prolife with the same retention times as free Gβ₁γ₂ and free Gα_sK219A, indicating that Gβ₁γ₂ had little effect on the retention time of Gα_sK219A and that Gα_sK219A was unable to form a complex with Gβ₁γ₂ (Fig. 2H). Similar results were observed with Gα_sE195A and Gα_sW263A, which could not form trimers with Gβ₁γ₂. Together, these data demonstrate that 18 Gα_s mutants (including Gα_sE195A, Gα_sK219A, and Gα_sW263A) are unable to interact with Gβγ subunits, and the locations of these 18 Gα_s mutants are displaced in Fig. 3 (red and green residues).

FIGURE 3.

Spatial locations of 18 Gα_s mutants that are defective in Gβγ binding. The structure displayed is Gα_i1β₁γ₂ (PDB code 1GP2). Gα, yellow; Gβ, gray; Gγ, gold. Corresponding residues for the 18 Gα_s mutants are highlighted (red and green). Green labeled residues are the 8 Gα_s mutants that are defective in Gβγ binding but can still be activated by β-ARs. Red labeled residues are the 10 Gα_s mutants that are defective in Gβγ binding and could not be activated by β-ARs. A and B are the same image rotated 180°.

Activation of Gα_s Mutants by β-Adrenergic Receptors in Cells

Because our goal was to find Gα_s mutants that could be potentially activated by GPCRs in the absence of Gβγ, we next investigated the activation of all 66 Gα_s mutants by GPCRs in Gα_s-deficient cells. Gα_s-deficient mouse embryonic fibroblast (MEF) cells were derived from Gα_s knock-out mouse embryos (32, 33). Because exon 2 of the Gα_s gene was deleted, none of the two alternative spliced variants of Gα_s were present in these Gα_s^−/− MEF cells (32, 33). We have made stable cell lines with these Gα_s^−/− MEF cells expressing Gα_s and all 66 Gα_s mutants (in pcDNA3.1/hygromycin vector). Stimulation of these cells with isoproterenol, which activates the endogenous G_s-coupled β-adrenergic receptors, increased cellular cAMP accumulation in cells expressing wild-type and 37 mutant Gα_s proteins (Table 1, and some examples shown in Fig. 4). Among the 18 Gα_s mutants that could not interact with Gβγ, 8 mutants (Gα_sD182A, Gα_sL184A, Gα_sR185A, Gα_sE195A, Gα_sF198A, Gα_sK219A, Gα_sW263A, and Gα_sN23A/I26A/E27A/L30A/D33A) could still mediate receptor activation of adenylyl cyclases leading to cAMP increase (Fig. 4, B-E, and green in Fig. 1D). As control, Gα_s^−/− MEF cells did not show any cAMP increase upon isoproterenol stimulation (Fig. 4A). The functionality of these Gα_s mutant proteins was also verified by cholera toxin, a direct activator of Gα_s (Fig. 4). These cellular studies demonstrate that 8 Gα_s mutants are able to functionally couple to β-adrenergic receptors, are activated by the receptors, and stimulate the downstream effector adenylyl cyclases, leading to the cellular accumulation of cAMP, even though they could not bind to Gβγ.

FIGURE 4.

In vivo activation of adenylyl cyclase by Gα_s and representative mutants. A, isoproterenol (ISO) or cholera toxin (CTX) failed to increase the cellular cAMP levels in Gα_s^−/− MEF cells. B–E, Both ISO and CTX increased the cellular cAMP levels in Gα_s^−/− cells stably re-expressing wild-type Gα_s (B), Gα_sE195A (C), Gα_sK219A (D), or Gα_sW263A (E). Data shown are mean ± S.D. of three independent experiments.

Activation of Purified Gα_s Mutants by Purified β-Adrenergic Receptors in Vitro in the Absence of Gβγ

To directly demonstrate that a GPCR has the capability to accelerate the guanine-nucleotide exchange on Gα without Gβγ, we performed biochemical studies with purified recombinant proteins of a GPCR and Gα_s mutants in vitro. We purified recombinant turkey β₁-adrenergic receptors from insect Hi5 cells (23) (Fig. 5A). Gα_s proteins were initially purified as GST fusion proteins and the GST tag was removed by protease cleavage (Fig. 5B). Among the 8 Gα_s mutants identified above (able to be activated by β-AR in cells but unable to bind Gβγ), 5 (D182A, L184A, R185A, F198A, and N23A/I26A/E27A/L30A/D33A) were unstable after removal of the GST tag. Therefore, we examined the activation of the remaining 3 Gα_s mutants (E195A, K219A, and W263A) by β-adrenergic receptor. The activation was monitored by the initial rate of GTPγS loading onto Gα_s subunits. As reported previously, purified β-adrenergic receptors had no effect on the rate of GTPγS loading (∼90 fmol/min) onto wild-type Gα_s proteins in the absence of Gβγ subunits (19, 34) (Fig. 5C). The rate of GTPγS loading on Gα_s was the same in the presence of isoproterenol (an agonist for β-adrenergic receptor) or alprenolol (an antagonist). The initial rate of GTPγS loading to Gα_s alone was similar to that of Gα_s with β-adrenergic receptor in the presence of the antagonist alprenolol. In contrast, in the presence of purified Gβγ proteins, isoproterenol increased the initial rate of GTPγS loading onto Gα_s (∼450 fmol/min) by 4-fold compared with that in the presence of alprenolol (∼115 fmol/min) (Fig. 5D). The fold-increase is similar to that reported in previous reconstituted systems, reflecting a relatively high basal nucleotide exchange rate of Gα_s (19, 34).

FIGURE 5.

In vitro activation of Gα_s by the β-adrenergic receptor. A, Coomassie Blue staining shows the purified turkey β-adrenergic receptor. B, Coomassie Blue staining shows some examples of purified G proteins (after GST cleavage). C, activation of Gα_s by the β-adrenergic receptor in the presence of alprenolol (■) or isoproterenol (●). D, activation of Gα_s + Gβ₁γ₂ by the β-adrenergic receptor in the presence of alprenolol (■) or isoproterenol (●). E, activation of Gα_sE195 by the β-adrenergic receptor in the presence of alprenolol (■) or isoproterenol (●). F, activation of Gα_sE195 + Gβ₁γ₂ by the β-adrenergic receptor in the presence of alprenolol (■) or isoproterenol (●). G, in vitro binding of Gα_s and representative mutants to β-AR. Different concentrations (150, 300, and 500 nm) of Gα_s and mutant proteins were used. H, left two panels, measurement of the initial rate of GTPγS loading onto wild-type Gα_s (top panel) or Gα_sK219A mutant (bottom panel) in the presence of different concentrations of Gβ₁γ₂. Right two panels, measurement of the initial rate of GTPγS loading onto wild-type Gα_s in the presence of Gβ₁γ₂ (top panel) or the Gα_sK219A mutant (bottom panel) in the absence of Gβ₁γ₂. One representative experiment from three independent experiments is shown for A-G. In H, data are shown as mean ± S.E.

With purified Gα_sE195A, we found that, without Gβγ subunits, isoproterenol increased the rate of GTPγS loading by about 2.4-fold compared to that with alprenolol (from ∼39 fmol/min with alprenolol to ∼95 fmol/min with isoproterenol) (Fig. 5E). Addition of Gβγ subunits did not significantly change the rates with either isoproterenol (∼105 fmol/min) or alprenolol (∼40 fmol/min) (Fig. 5F). Similar results were obtained with Gα_sK219A and Gα_sW263A mutants: without Gβγ subunits, isoproterenol increased the rate of GTPγS loading by 2∼3-fold on Gα_sK219A and Gα_sW263A mutants, compared to that with alprenolol. Furthermore, the mutations did not seem to alter the interaction between Gα_s and β-AR (Fig. 5G). Moreover, increasing the concentrations of Gβγ enhanced the initial rate of GTPγS loading onto wild-type Gα_s, but not Gα_sK219A (Fig. 5H). Additionally, increasing the concentrations of β-AR elevated the initial rate of GTPγS loading onto both wild-type Gα_s (in the presence of Gβ₁γ₂) and Gα_sK219A (in the absence of Gβ₁γ₂) (Fig. 5H). These biochemical experiments clearly demonstrate that purified β₁-adrenergic receptors can accelerate the guanine nucleotide exchange on Gα_s in the absence of Gβγ subunits. We should note that previous studies with rhodopsin and transducin had indicated that high concentrations of rhodopsin might activate transducin in the absence of Gβγ, and that Gβ₁ or Gγ₁ knock-out mice still had some light response (35–40). Thus, GPCRs possess the ability to catalyze the nucleotide exchange on Gα subunits.

Role of Extended Linker 2 in Gα Activation

How do the 8 Gα_s mutations (Gα_sD182A, Gα_sL184A, Gα_sR185A, Gα_sE195A, Gα_sF198A, Gα_sK219A, Gα_sW263A, and Gα_sN23A/I26A/E27A/L30A/D33A) alleviate the requirement of Gβγ for the activation of Gα_s by β-adrenergic receptors? When mapped onto the crystal structure of the complex of β₂-AR and G_s, 3 of these 8 residues (Asp-182, Leu-184, and Arg-185) are in αF helix (Fig. 6 A and B). Two of the 8 residues (E195 and F198) are in β2 sheet (Fig. 6B). αF and β2 flank Linker 2, which connects the Ras-like GTPases domain and the α-helical domain of Gα_s (Figs. 6, A and B, and 1D). Also, within Linker 2, mutations of Arg-187, Leu-189/Thr-190, Ile-193, and Thr-196 all blocked β-AR induced cAMP increases in cells even though these mutants could bind to Gβγ, GTPγS, and activate adenylyl cyclase in vitro (Table 1). Hence, almost all residues in the extended Linker 2 are critical for Gα activation. Although some mutations (such as R185A and E195A) enable Gα activation by GPCR in the absence of Gβγ, other mutations (such as S191A and I193A) block Gα activation by GPCR in cells. When comparing the structures of inactive Gα_i-GDP and active Gα_i-GTPγS, or the structures of inactive Gα_t-GDP and active Gα_t-GTPγS, Linker 2 is moved toward the γ-phosphate, bringing the side chain oxygen of Thr-190 into the coordination sphere of the Mg²⁺ ion where it replaces one of the water molecules observed in the structure of the GDP form (24, 25). Furthermore, it has been shown that Linker 2 changes the conformation upon GPCR activation (41).

FIGURE 6.

Diagrams of Gα activation by GPCRs. A, ribbon presentation of 8 Gα_s mutants that are defective in Gβγ binding, but are still activated by β-adrenergic receptors in cells. The structure displayed is the complex of β₂-AR·G_s (PDB code 3SN6). β₂-AR, dark blue; Gα_s, yellow; Gβ, gray; Gγ, gold. Residues in the 8 Gα_s mutants (that are defective in Gβγ binding, but are still activated by β-adrenergic receptors in cells) are in green. B, detailed view of the 5 residues in the αF/Linker 2/β2 region. C, detailed view of the residues from the other 3 Gα_s mutants. These figures were drawn with MolScript and Raster three-dimensional programs. D, diagram of the clam-shell model of Gα activation. The helical domain opens away from the Ras-like GTPase domain, allowing GDP released. E, diagram of the rolling-top model of Gα activation. The helical domain (the top) slides and rotates away from the Ras-like GTPase domain, leading to GDP release. Dashed lines are unmodeled residues in the β₂-AR/G_s structure. F, zoomed view of the link from ICL2 of β₂-AR to the αF/Linker 2/β2 region of Gα_s from the structure of the β₂-AR·G_s complex. Some parts of Gα_s were removed for clarity. ICL2, intracellular loop 2 of β₂-AR. G, diagram of Gα_q with its inhibitor of YM-254890.

A critical role for the extended Linker 2 in Gα activation is consistent with the observation from the crystal structure of the complex of β₂-AR and G_s, which shows a large rotation (∼127°) of the α-helical domain relative to the Ras-like GTPase domain upon G-protein activation (Fig. 6, A and E) (10). αF/Linker 2, through the β₂-strand, is connected to the β₂/β₃ loop, which interacts with intracellular loop 2 of β₂-AR (Fig. 6F). Furthermore, experiments using various biophysical measurements suggest a “clam-shell” like opening model for Gα activation by GPCRs, in which the helical domain opens away from the Ras-like GTPase domain (42–45) (Fig. 6D). Here the clustering of several Gα_s mutants critical for GPCR activation of Gα_s around the extended Linker 2 suggests a possibility that Linker 2 may serve as the “hinge” in this clam-shell model (Fig. 6D). Similarly, in the “rolling-top” model (in which the α-helical domain slides and rotates away along the sideways from the Ras-like GTPase domain) observed in the β₂-AR·G_s complex structure, this hinge is stretched (some residues in the linkers were disordered and thus were unmodeled in the structure) (10)(Fig. 6E). Mutations of residues in this hinge could make it easy to open the interface between the two domains of Gα or to enhance the interdomain motions. Preventing this interdomain opening by cross-linking inhibits GPCR-catalyzed G-protein activation (42). We should note that, in addition to Linker 2, Linker 1 also connects the Ras-like GTPase domain and the α-helical domain. Indeed a point mutation (G56P) in Linker 1 of transducin increased the basal exchange rate and exhibited some degrees of activation at high levels of rhodopsin in the absence of β₁γ₁ (35). The role of Linker 1 in Gα-protein activation by GPCRs requires future systematic investigation. Here we propose that a GPCR via its intracellular loop 2 directly interacts with the β₂/β₃ loop of Gα to communicate to Linker 2 and αF, resulting in the rotation of the helical domain and the release of GDP (Fig. 6, E and F).

Linker 2 as the Target Site of a Natural Product Inhibitor for G_q

The crystal structure of G_q and a small molecule natural product inhibitor shows the inhibitor binds to a hydrophobic cleft and directly contacts Linker 2 (Fig. 6G) (46). This inhibitor, YM-254890, inhibits the guanine-nucleotide exchange reaction by preventing the GDP release (46). Also, this inhibitor blocks the AlF₄⁻-induced conformational change in Gα_q (46). It is proposed that this inhibitor stabilizes an inactive GDP-bound form (46). These structural data suggest that Linker 2 might be critical for G-protein activation, and that the Linker 2 region is a potential therapeutic targeting site.

Role of αN, α3, and Switch Regions in Gα Activation in Cells

In addition to the extended Linker 2, our data have uncovered some other residues that are essential for Gα_s activation in cells by β-ARs. The N-terminal residues (Asn-23, Ile-26, Glu-27, Leu-30, and Asp-33) and Trp-263 are located at or close to the receptor-interacting surface (10, 15, 47, 48)(Fig. 6, A and C). Trp-263 is close to the α₃/β₅ loop. This region is analogous to GEF contact sites in other GTPases such as Ras and EF-Tu (26, 27). The crystal structure of β₂-AR/G_s has revealed that β₂-AR interacts with the αN/β₁ hinge; this may explain the role of the 5 N-terminal residues (Asn-23, Ile-26, Glu-27, Leu-30, and Asp-33) in the activation of Gα by GPCR.

Lys-219 is located within Switch II (Figs. 1D and 6C). Lys-206 of Gα_t (corresponding to Lys-219 in Gα_s) had been identified to be important for Gα_t activation by rhodopsin (49). Several residues near Lys-219 of Gα_s such as Gly-212 and Gln-213 are critical for triggering conformational changes in Switch II through an interaction with the γ-phosphate in GTP and for stabilizing the transition state for GTP hydrolysis, respectively (13, 14, 50, 51). Indeed, most Gα_s mutations in the Switch I and II regions (such as Ile-193, Thr-196, Asp-209, Gly-212, Arg-214, Asp-215, Arg-217, Arg-218, Trp-220, Ile-221, and Gln-222) could not be activated by β-adrenergic receptors in cells, despite their ability to bind to Gβγ subunits, bind to GTPγS, and activate adenylyl cyclases in vitro (Table 1). Arg-187 and Thr-190 (in Switch I) as well as Gly-212 (in Switch II) contact the γ-phosphate of GTP (6). Together, these data demonstrate that αN, α3, Switch I and Switch II are critical for Gα activation by GPCRs.

The critical role of Switch I and II regions in Gα activation is similar to the activation of Ras-superfamily GTPases by their GEFs. GEFs for small GTPases have different structures (26–30). They contact GTPases at the same as well as different amino acid residues and induce different conformational changes on GTPases to drive out the GDP. However, they all utilize a two-sided attack to release positive charges (the Mg²⁺ ion and the invariant lysine residue in the P loop (phosphate-binding loop)) from their interaction with the phosphates of the nucleotide (52). These GEFs interact extensively with and remodel Switch I and II regions of GTPases, which form part of the binding pocket for Mg²⁺ and the γ-phosphate of GTP. Thus, although GPCRs are unusual GEFs because they do not contact these switch regions directly, they still require/remodel these switch regions for the activation of Gα.

Possible Role of Gβγ in Gα Activation

Our mutagenesis studies unexpectedly reveal a role for residues Trp-263 (in α3) and Asn-265 (in the α3-β5 loop) in Gβγ binding (Table 1). From the structure of the Gα_i·GDP/Gβγ complex, this region is not directly involved in contacting with Gβγ. However, this region is right next to Switch III and forms an elaborate inter-dependent network of polar interactions with the Switch II region, which is critical for interacting with Gβγ (24). Although we did not address the role of Gβγ in the guanine-nucleotide exchange on Gα, we should mention that there were two models that proposed a catalytic role for Gβγ (15, 16, 21). Gβγ subunits contact the switch regions of Gα subunits (24, 25), and have a structure similar to RCC1 (30). Hence, Gβγ subunits would be more like GEFs if only Gβγ subunits could be proven to possess GEF activity without GPCRs. The role of Gβγ in Gα activation could be complex. Gβγ stabilizes GDP binding on Gα, thus serving as a guanine-nucleotide dissociation inhibitor. GPCR activation could release the inhibition of Gβγ. However, the nucleotide exchange rate of Gα, in the absence of Gβγ, is still slower than that in the presence of GPCRs. This would indicate that, in addition to releasing Gβγ, GPCRs act on the nucleotide exchange on Gα. Gβγ subunits could serve an additional catalytic role to augment the complex formation between GPCRs and Gα subunits, similar to the role of ELMO in facilitating nucleotide exchange on Rac by Dock180 (53). Indeed, our data (Table 1, Fig. 5, E and F) show that, whereas Gα_sE195A could be activated by β-ARs in the absence of Gβγ, the extent of activation was suboptimal relative to wild-type Gα_s in the presence of Gβγ. This implies that Gβγ somehow contributes to the activation process.

Previously two models were proposed for a catalytic role for Gβγ subunits in activating Gα subunits. In the “lever” model (15, 21), GPCRs cause a tilt (or a rotation) of Gβγ relative to Gα. The membrane proximal part (interacting with the Gα N-terminal helix) of Gβγ moves closer to Gα. The opposite end of Gβγ moves away from Gα and, at the same time, pulls along the interacting parts of Gα. These interacting residues from Switch I and II of Gα are part of the lip of the nucleotide binding pocket. Therefore, GDP can exit permitting exchange by GTP. In the second “gear shift” model (16), GPCRs also cause a tilt of Gβγ. However, the rotating direction is opposite of that in the lever model. Gβγ moves closer to Gα. The N terminus of Gγ (the membrane distal end of Gβγ) moves to contact and displace the helical domain of Gα, creating an exit route for GDP. The exact role of Gβγ in the exchange reaction needs further investigation.

Conclusions

Upon ligand-binding, GPCRs initiate the exchange of GDP bound on Gα subunits of G-proteins with GTP. However, the detailed molecular mechanisms by which GPCRs activate G-proteins are not well understood. Here we reveal some new insights on this process. We demonstrate that a GPCR could activate some Gα mutants in the absence of Gβγ, and that the extended Linker 2 in Gα is critical for Gα activation by GPCRs. These data will advance our understanding of this critical cellular signaling process.

The closed and open states of the cleft (the GDP/GTP binding site) are determined by relative pivoting of the Ras-GTPase domain and the α-helical domain at Linker 2. The closed state of the cleft is represented in all crystal structures of Gα subunits. One of the open states of the cleft is captured in the β₂-AR/G_s crystal structure. This model of activation of Gα subunits suggests that factors binding at the interface of the Ras-GTPase domain and the helical domain might facilitate cleft closure, thus serving as an inhibitor of Gα activation (Fig. 6G). Given this pivot model, the two domains of Gα could be either clam-shell opening or rolling-top expansion including a relative rotation of the GTPase domain and the helical domain around an axis through the linkers (Fig. 6, D and E). Results from several biophysical studies are consistent with the clam-shell opening, and that the crystal structure of the β₂-AR·G_s complex is in line with the rolling-top movement. Therefore, in addition to the established role for α5-helix of Gα connecting GPCRs to the guanine-nucleotide binding pocket, we show here that the extended Linker 2 connects GPCRs to the nucleotide binding pocket as well as the α-helical domain of Gα. In conclusion, our data demonstrate that GPCRs possess the capability to catalyze the nucleotide exchange on Gα subunits, and that the extended Linker 2 is critical for Gα activation by GPCRs.