Identification of Critical Residues in Gα₁₃ for Stimulation of p115RhoGEF Activity and the Structure of the Gα₁₃-p115RhoGEF Regulator of G Protein Signaling Homology (RH) Domain Complex

Abstract

RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein Gα₁₃ stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα₁₃ stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα₁₃ that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα₁₃ to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα₁₃ and not through its interaction with a secondary binding site. A crystal structure of Gα₁₃ bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα₁₃-Gα_i1 chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα₁₃.

Introduction

Heterotrimeric guanine nucleotide binding proteins (G proteins), composed of α, β, and γ subunits, act as molecular switches that cycle between an inactive, GDP-bound state and an active, GTP-bound state upon stimulation of G protein-coupled receptors (1). Once activated, the GTP-bound Gα subunit dissociates from the Gβγ dimer, both of which regulate the activity of multiple intracellular effectors to elicit cellular responses. The duration of signaling mediated by Gα is dictated by the lifetime of bound GTP. All Gα subunits have intrinsic GTPase activity that hydrolyzes GTP to GDP, and this rate of hydrolysis is enhanced by GAPs,² such as RGS proteins (2), which bind the switch regions of activated Gα subunits, stabilize the transition state for GTP hydrolysis, and thus accelerate the hydrolysis of GTP to GDP (3, 4). Once in the GDP-bound state, Gα subunits reassociate with Gβγ and are capable of initiating another round of signaling.

Gα₁₃ is one of two members of the G₁₂ family of G proteins (5) and has been implicated in regulating multiple cellular processes that depend on activation of the monomeric GTPase RhoA, including gene transcription, embryogenesis, and rearrangement of the actin cytoskeleton (6–8). A direct link between Gα₁₃ and RhoA was established with the discovery of p115RhoGEF, the founding member of a family of RhoA-specific GEFs containing an RH domain in their N termini (RH-RhoGEFs) (9). Together with p115RhoGEF, PDZ-RhoGEF and LARG constitute the RH-RhoGEF family in mammals (10, 11). The RH domain of p115RhoGEF has low sequence similarity to canonical RGS proteins but nevertheless functions as a GAP for Gα₁₃ (12). Although the structure of the RH domain of p115RhoGEF is similar to that of other RGS proteins (13), additional elements flanking the RGS box are required for GAP activity (14) and stability of the isolated domain (15). In addition to its RH domain, p115RhoGEF also contains the tandem DH/PH domains characteristic of Dbl family RhoGEFs. The nucleotide exchange activity of p115RhoGEF can be directly stimulated by Gα₁₃ in vitro (9), and Gα₁₃ acts synergistically with p115RhoGEF to activate Rho-dependent signaling in cells (16). Thus, p115RhoGEF functions as both an effector and a GAP for Gα₁₃. Although the relationship between p115RhoGEF and Gα₁₃ has been well established, the precise mechanism by which Gα₁₃ regulates the activity of p115RhoGEF remains to be fully elucidated.

The molecular mechanisms regulating p115RhoGEF activity are likely to be complex and may involve multiple intermolecular interfaces with Gα₁₃. It has been clearly demonstrated that Gα₁₃ binds directly to the RH domain of p115RhoGEF (12, 15). Furthermore, this domain is required for both basal and Gα₁₃-stimulated nucleotide exchange activity, suggesting that it plays a critical role in the activation mechanism (15). A structure of the RH domain of p115RhoGEF bound to an AlF₄⁻-activated Gα₁₃-Gα_i1 chimera has been solved by x-ray crystallography and revealed that the α subunit engages this domain through two distinct interfaces (17). In addition to interacting with switch regions I and II of Gα_13/i1 through an N-terminal extension of the RGS box, the RH domain also docks into the hydrophobic groove between the α₂ and α₃ helices of Gα_13/i1, a highly conserved effector interface among other Gα-effector pairs (18–21). However, many of the residues in the α₃ helix of this chimera are derived from Gα_i1, and its ability to activate p115RhoGEF has not been clearly demonstrated. Thus, the role of residues in the α₃ helix in regulating p115RhoGEF activity is unknown. Gα₁₃ has also been reported to bind to the isolated DH/PH domains of p115RhoGEF in vitro (15); however, it has no capacity to directly stimulate the GEF activity of this fragment. Therefore, the role of additional binding sites outside of the RH domain is also unclear.

Here, specific residues in Gα₁₃ that mediate activation of p115RhoGEF are identified. We demonstrate that mutation of these residues, which bind to the RH domain of p115RhoGEF, significantly impairs binding of Gα₁₃ to p115RhoGEF as well as stimulation of GEF activity in cells and in vitro. Our results suggest that the initial step in stimulation of exchange activity by Gα₁₃ involves allosteric regulation through interaction with the RH domain of p115RhoGEF. We also present a crystal structure of Gα₁₃ bound to the RH domain of p115RhoGEF, which differs in several respects from the previously crystallized complex using the Gα_13/i1 chimera (17) and reinforces the results of our biochemical analysis. This structure provides a more accurate picture of the Gα₁₃-p115RhoGEF RH interface, whereas the biochemical data provide new insight into the mechanism by which p115RhoGEF is activated by Gα₁₃.

EXPERIMENTAL PROCEDURES

Generation of Constructs

Gα₁₃ Mutants

Single point mutations were introduced into the mammalian expression vector pCMV5 harboring murine Gα₁₃ Q226L for use in SRE-luciferase assays or the baculovirus transfer vector pFastBac HTa (Invitrogen) encoding Gα_i/13 for protein expression using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The T274E/N278A double mutant was generated using either pCMV5-Gα₁₃ Q226L T274E or pFastBac HTa-Gα_i/13 T274E as the template. Sense and antisense primer pairs used to introduce each mutation were as follows: N270A, 5′-CGCCTTACAGAATCTCTGGCCATTTTTGAAACAATTG and 5′-CAATTGTTTCAAAAATGGCCAGAGATTCTGTAAGGCG; T274E, 5′-CTGAACATTTTTGAAGAGATTGTCAACAATCGGGTTTTCAGC and 5′-GCTGAAAACCCGATTGTTGACAATCTCTTCAAAAATGTTCAG; N278A, 5′-GAAACAATTGTCAACGCTCGGGTTTTCAGCAACG and 5′-CGTTGCTGAAAACCCGAGCGTTGACAATTGTTTC; T274E/N278A, 5′-GAAGAGATTGTCAACGCTCGGGTTTTCAGCAACG and 5′-CGTTGCTGAAAACCCGAGCGTTGACAATCTCTTC. Mutated bases are underlined. Introduction of the desired mutation was verified by automated dideoxy sequencing of each construct.

Full-length Gα₁₃

Full-length Gα₁₃ (Gα₁₃ FL) was generated by first introducing a silent mutation into pCMV5-Gα₁₃ by QuikChange site-directed mutagenesis that eliminates an internal HindIII site using the primers 5′-GCCCGAGAGAAGCTCCATATTCCCTGGGG and 5′-CCCCAGGGAATATGGAGCTTCTCTCGGGC. Mutated bases are underlined. KpnI and HindIII sites were then introduced at the 5′- and 3′-ends, respectively, of Gα₁₃ by PCR using pCMV5-Gα₁₃ lacking the internal HindIII site as the template and the primers 5′-GGTACCGCGGACTTCCTGCCGTC and 5′-GACCAAGCTTTCACTGCAGCATGAGCTG. The PCR products were digested with KpnI and HindIII and ligated into the pFastBac HT(−) vector containing the N-terminal α₁ helix of Gα_i1 such that the construct contains, from the N terminus, a His₆ tag, residues 1–28 of Gα_i1, a TEV protease site, and residues 2–377 of Gα₁₃.

p115RhoGEFΔC

Tyr⁷⁶³ of p115RhoGEF was mutated to a stop codon using QuikChange site-directed mutagenesis using pFastBac HTc-p115RhoGEF as the template and the primers 5′-CACTGAGACTGCCGGATAACTGAAAGTCCCTGCCC and 5′-GGGCAGGGACTTTCAGTTATCCGGCAGTCTCAGTG. Mutated bases are underlined.

Protein Purification

His₆-Gα_i/13 (Wild Type and Mutant)

His₆-Gα_i/13 and His₆-Gα_i/13 harboring the N270A, T274E, N278A, or T274E/N278A mutation(s) were purified from the soluble fraction of Sf9 cells as described previously (22). The His₆ tag was removed for crystallography by incubating His₆-Gα_i/13 with 2% (w/w) His₆-TEV protease overnight at 4 °C. Uncut His₆-Gα_i/13 and His₆-TEV protease were removed using a HisTrap HP column (GE Healthcare Life Sciences). Gα_i/13 was subjected to gel filtration chromatography on a Superdex 200 10/300 size exclusion column (GE Healthcare) and eluted in buffer containing 20 mm HEPES, pH 8.0, 100 mm NaCl, 10 mm 2-mercaptoethanol, 1 mm MgCl₂, 10 μm GDP, and 10% glycerol. The protein was aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

Full-length Gα₁₃

His₆-Gα_i/13 FL was purified from Sf9 cells in the same fashion as His₆-Gα_i/13. After elution from nickel-NTA resin (Qiagen), fractions containing His₆-Gα_i/13 FL were pooled and incubated with 2% (w/w) His₆-TEV protease overnight at 4 °C to remove the His₆ tag and α₁ helix of Gα_i1. The protein was concentrated and buffer-exchanged to imidazole-free buffer using an Amicon Ultra 30k centrifugal filter device (Millipore Corp.). The protein was aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

p115RhoGEF

Sf9 cells expressing His₆-p115RhoGEF were resuspended in lysis buffer (20 mm HEPES, pH 8.0, 100 mm NaCl, 10 mm imidazole, 10 mm 2-mercaptoethanol, 16 μg/ml TPCK, TLCK, and PMSF, and 3.2 μg/ml leupeptin and lima bean trypsin inhibitor) and lysed by nitrogen cavitation. The lysate was clarified by centrifugation at 100,000 × g for 30 min at 4 °C. The soluble portion of the lysate was diluted 2-fold with lysis buffer and applied to a nickel-NTA column equilibrated with lysis buffer. The column was washed with 20 bed volumes of wash buffer (20 mm HEPES, pH 8.0, 400 mm NaCl, 20 mm imidazole, 10 mm 2-mercaptoethanol, 16 μg/ml TPCK, TLCK, and PMSF, and 3.2 μg/ml leupeptin and lima bean trypsin inhibitor), and bound protein was eluted from the column using elution buffer (20 mm HEPES, pH 8.0, 400 mm NaCl, 300 mm imidazole, 10 mm 2-mercaptoethanol, 16 μg/ml TPCK, TLCK, and PMSF, and 3.2 μg/ml leupeptin and lima bean trypsin inhibitor). Peak fractions containing His₆-p115RhoGEF were pooled and dialyzed overnight at 4 °C against dialysis buffer (20 mm HEPES, pH 8.0, 400 mm NaCl, 10 mm 2-mercaptoethanol) in the presence of 2% (w/w) His₆-TEV protease. The solution was supplemented with 10% glycerol and applied to a HisTrap HP column to remove remaining His₆-p115RhoGEF and His₆-TEV protease. Flow-through fractions were collected and applied to a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare) equilibrated in gel filtration buffer (20 mm HEPES, pH 8.0, 150 mm NaCl, 10 mm 2-mercaptoethanol, 10% glycerol). Fractions containing p115RhoGEF were pooled and subjected to further purification on a Superdex 200 HR 10/30 size exclusion column (GE Healthcare) equilibrated in gel filtration buffer. Fractions containing p115RhoGEF were pooled and concentrated using an Amicon Ultra 30k centrifugal filter device. The protein was aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

p115RhoGEFΔC

His₆-p115RhoGEFΔC(1–762) was purified from Sf9 cells in the same fashion as full-length p115RhoGEF except that the final round of size exclusion chromatography utilized a HiLoad Superdex 200 16/60 size exclusion column (GE Healthcare), and the protein was eluted in buffer containing 20 mm HEPES, pH 8.0, 100 mm NaCl, 1 mm EDTA, 10% glycerol, and 2 mm DTT. The protein was aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

GST-p115RhoGEF RH Domain

The GST-tagged RH domain of p115RhoGEF was purified from Escherichia coli as described previously (22).

RhoA

GST-RhoA lacking the C-terminal CAAX motif (RhoA(1–181)) was expressed from the plasmid pGEX-6P (GE Healthcare) in the E. coli strain BL21-CodonPlus (DE3)-RP (Stratagene). Cells were grown at 30 °C to an A₆₀₀ of 0.6–0.8, and protein expression was induced with 200 μm isopropyl β-d-1-thiogalactopyranoside for 6 h. Cells were resuspended in lysis buffer (50 mm HEPES, pH 7.5, 1 mm EDTA, 1 mm DTT, 200 mm NaCl, 5 mm MgCl₂, 10 μm GDP, 10% glycerol) and lysed with lysozyme and sonication. The lysate was clarified by centrifugation at 100,000 × g for 30 min at 4 °C. The soluble portion of the lysate was applied to a column of glutathione-Sepharose 4B beads (GE Healthcare) equilibrated with lysis buffer. The column was washed with 10 bed volumes of lysis buffer, followed by 20 bed volumes of cleavage buffer (50 mm Tris, pH 7.5, 1 mm EDTA, 1 mm DTT, 5 mm MgCl₂, 10 μm GDP). On-column cleavage of the GST tag was achieved using PreScission protease according to the manufacturer's protocol (GE Healthcare). Cleaved RhoA was eluted from the column using lysis buffer. Fractions containing RhoA were pooled and concentrated using an Amicon Ultra 15k centrifugal filter device. The protein was aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use. To form nucleotide-free RhoA for crystallography, the protein was incubated with an excess of EDTA on ice.

Biochemical Experiments

SRE-luciferase Assays

Assays were performed as described (23), with the following modification. Each well was co-transfected with 200 ng of pGL3-SRE.L, 100 ng of pCMV5-LacZ, and 10 ng of empty pCMV5 vector or the indicated Gα₁₃ construct using Lipofectamine2000 (Invitrogen). For immunoblotting of cell lysates, samples were harvested directly into SDS-PAGE sample buffer and boiled. Cell lysate was separated by SDS-PAGE, and Gα₁₃ was detected by Western blotting with the B860 antibody (24). GAPDH was detected using a monoclonal antibody (clone 6C5, Ambion).

In Vitro Trypsin Protection Assays

3 μg of Gα_i/13 protein (wild type or mutant) was diluted into reaction buffer (20 mm HEPES, pH 8.0, 5 mm MgCl₂, 100 μm GDP) and incubated on ice for 45 min in the presence or absence of AlF₄⁻ (10 mm NaF, 30 μm AlCl₃). Trypsin was added to a final concentration of 13.3% (w/w), and the samples were incubated at 30 °C for 15 min. The reaction was terminated by the addition of SDS-PAGE sample buffer and boiling.

GST Pull-down Assays

Assays were preformed as described previously (22), with the exception that bound proteins were released from the beads by the addition of SDS-PAGE sample buffer and boiling.

Single Turnover GAP Assays

GTPase assays were performed as described previously (12), except that reactions were incubated on ice. [γ-³²P]GTP (specific activity = 6,000 Ci/mmol) was obtained from PerkinElmer Life Sciences.

Gel Filtration of Full-length p115RhoGEF with His₆-Gα_i/13 or His₆-Gα_i/13 T274E/N278A

2.8 nmol of full-length p115RhoGEF were incubated with 5.6 nmol of His₆-Gα_i/13 (wild type or mutant) for 30 min on ice in gel filtration buffer (20 mm HEPES, pH 8.0, 1 mm EDTA, 2 mm DTT, 150 mm NaCl, 5 mm MgCl₂, 10 μm GDP) in the presence or absence of AlF₄⁻ (10 mm NaF, 20 μm AlCl₃). The total volume of each sample was 200 μl. The proteins were fractionated at 4 °C on a Superdex 200 HR 10/30 size exclusion column equilibrated in the same buffer as the sample.

In Vitro RhoGEF Assays

Assays were performed as described previously (25). [³⁵S]GTPγS (specific activity = 1,250 Ci/mmol) was obtained from PerkinElmer Life Sciences.

Crystallization and Data Collection

Crystallization

To form the Gα_i/13-p115ΔC-RhoA complex, Gα_i/13 was activated with 8× AlF₄⁻ (80 mm NaF, 160 μm AlCl₃) on ice for 15 min and mixed with an equimolar amount of p115RhoGEFΔC and nucleotide-free RhoA. The complex was subjected to size exclusion chromatography on a HiLoad Superdex 200 10/16 gel filtration column, and fractions containing the stoichiometric complex were pooled and concentrated using an Amicon4 10k centrifugal filter device. Screening of the Gα_i/13-p115RhoGEFΔC-RhoA complex was carried out using vapor diffusion at 20 °C. Diffraction quality crystals of the Gα_i/13-p115RhoGEF RH domain complex were grown against a reservoir solution containing 15% PEG 4000 and 0.1 m HEPES (pH 7.0). Single crystals were coated with the reservoir solution containing 20% PEG 400 as a cryoprotectant, mounted using a nylon loop, and flash-cooled in the cold stream of the goniometer.

Data Collection

Diffraction data were collected at 100 K with a wavelength of 1.0 Å at SPring-8 beam line BL41XU (Harima, Japan). Data were processed with the HKL2000 program (26). The structure of the Gα_i/13-p115RhoGEF RH domain complex was determined by molecular replacement with the program MOLREP (CCP4), using the RH domain of p115RhoGEF (Protein Data Bank entry 1IAP, chain A) and Gα_i/13 (Protein Data Bank entry 1ZCB) as search models. The model was corrected iteratively using O (27), and structure refinement was carried out using CNS (Crystallography and NMR System) (28). The final refinement statistics are shown in Table 1. The quality of the model was inspected by the program PROCHECK in the CCP4 suite (29). Structural similarities were calculated with DALI (30). Solvent-accessible surface area was calculated with the program AREALMOL in the CCP4 suite (29). Graphic figures were created with the program PyMOL (31).

TABLE 1.

Crystallography statistics

All numbers in parentheses refer to the highest resolution shell statistics.

Parameters	Values
Data collection
Space group	P1
Unit cell parameters	a = 50.5 Å, b = 70.6Å, c = 88.1 Å, α = 77.8°, β = 84.5°, γ = 80.1°
Wavelength (Å)	1.0
Resolution range (Å)	50–2.4
Redundancy	1.7
Unique reflections	37,777
Completeness (%)	81.8 (82.1)
I/σ(I)	9.0 (2.3)
Rsyma	0.074 (0.296)
Refinement
Resolution range (Å)	48.88–2.40 (2.49–2.40)
No. of reflections	37,316
R-Factor/Free R-factorb	0.205/0.280
No. of protein atoms	8,091
No. of magnesium ion atoms	2
No. of ligand atoms	66
No. of water molecules	87
Root mean square deviations
Bond lengths (Å)	0.009
Bond angles (degrees)	1.4
Average B-value (Å2)	51.6

^a R_sym = Σ|I_avg − Ii|/ΣIi, where Ii is the observed intensity and I_avg is the average intensity.

^b Free R-factor is calculated for 10% of randomly selected reflections excluded from refinement.

RESULTS

Previous studies have suggested that the region of Gα₁₃ responsible for stimulating the guanine nucleotide exchange activity of p115RhoGEF is located C-terminal to the three switch regions (23, 32). These results are supported by crystallographic data, which demonstrate that the α₃ helix and α₃-β₅ loop in Gα₁₃ form a putative effector binding site with the RH domain of p115RhoGEF (17). However, the capacity of residues in this region to stimulate GEF activity has not been directly demonstrated. We utilized these data along with the interaction between Gα_q and its effectors PLC-β and GRK2 as models in order to identify specific amino acid residues responsible for activating p115RhoGEF. Recent work has identified several residues that are critical for the interaction between Gα_q and these effectors, namely Ala²⁵³, Thr²⁵⁷, and Tyr²⁶¹ (20, 33).³ The Gα_q-PLC-β interaction was selected as a model because, like RH-RhoGEFs, PLC-β functions both as a GAP (34) and effector (35) for Gα_q. Based on these data, Asn²⁷⁰, Thr²⁷⁴, and Asn²⁷⁸ in Gα₁₃, which correspond to Ala²⁵³, Thr²⁵⁷, and Tyr²⁶¹ in Gα_q (Fig. 1A), were targeted for site-directed mutagenesis, and the effects of these mutations on the ability of Gα₁₃ to regulate p115RhoGEF activity were examined.

FIGURE 1.

Characterization of Gα_i/13 mutants. A, a primary sequence alignment of murine Gα₁₂, Gα₁₃, and Gα_q was generated using the program T-Coffee. Residues in Gα₁₃ analyzed by site-directed mutagenesis in this study are underlined. Secondary structure was assigned based on the crystal structure of the Gα_13/i1-p115 RH domain complex (Protein Data Bank entry 1SHZ) (17). B, the T274E and N278A mutations in Gα₁₃ impair Rho activation in cells. HeLa cells were transiently transfected with empty vector or the indicated Gα₁₃ QL construct. The luciferase activity of cell lysates was determined as described under “Experimental Procedures.” Total cell lysate was immunoblotted for either Gα₁₃ or GAPDH. Data are presented as the mean ± S.E. (error bars) of triplicate determinations from a single experiment, representative of three independent experiments with similar results. Data were analyzed by one-way analysis of variance followed by Dunnett's post-test. Statistically significant difference from Gα₁₃ QL is shown as follows. ns, not significant; **, p < 0.01. C, Gα_i/13 mutants can undergo activation-dependent conformational changes. Gα_i/13 (wild type or mutant) was subjected to limited trypsin digestion in the presence or absence of AlF₄⁻. After proteolysis, proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The primary protected species is indicated with an arrow. The positions of molecular mass standards, in kDa, are shown on the left.

The T274E and N278A Mutations Impair Rho Activation in Cells

A constitutively active mutant of Gα₁₃, Gα₁₃ Q226L (hereafter referred to as Gα₁₃ QL), has been shown to stimulate transcription from the SRE in a Rho-dependent manner when overexpressed in cells (6). Therefore, to test the ability of the N270A, T274E, N278A, and T274E/N278A mutants to stimulate Rho activation, HeLa cells, which endogenously express RH-RhoGEFs and RhoA, were transiently co-transfected with plasmids encoding each mutant in the background of Gα₁₃ QL and a construct encoding the luciferase reporter gene under the control of the SRE. The activation status of Rho in cells was indirectly measured as luciferase activity of cell lysates. In this system, the N270A mutation in Gα₁₃ QL did not affect Rho activation compared with Gα₁₃ QL (Fig. 1B). However, both the T274E and N278A mutations decreased Rho activation by ∼50%, and Rho activity could be reduced to nearly basal levels by the T274E/N278A double mutation. Immunoblotting of cell lysates demonstrated equal expression levels of the constructs. These data suggested that residues Thr²⁷⁴ and Asn²⁷⁸, but not Asn²⁷⁰, are important for the ability of Gα₁₃ to stimulate Rho activation in cells.

Gα_i/13 N270A, T274E, N278A, and T274E/N278A Are Functional Gα Subunits

To analyze these mutants in more detail, each mutation was introduced into a construct harboring amino acid residues 1–28 of Gα_i1 and 47–377 of Gα₁₃ (hereafter referred to as Gα_i/13). The resulting protein was expressed and purified from Sf9 cells for biochemical reconstitution assays. The use of this Gα_i/13 chimera facilitates the production of recombinant protein by increasing the yield from Sf9 cells, and it retains all of the biochemical properties characteristic of native Gα₁₃, including the ability to regulate RH-RhoGEFs (22). In order to confirm that the mutants were functional Gα subunits, limited trypsin digestion assays were performed. In their inactive, or GDP-bound, form, Gα subunits adopt a conformation that renders them susceptible to digestion by the serine protease trypsin (36). However, in the active form, which can be induced by binding to the reversible activator AlF₄⁻, the conformation changes such that the Gα subunit is protected from digestion, largely due to ordering of the α₂ helix in switch II, although trypsin still cleaves the N terminus of the Gα subunit. Thus, the protected species runs at a smaller molecular weight than the native protein. Trypsin digestion assays can therefore be used to determine if a Gα subunit is capable of adopting an activated conformation. As shown in Fig. 1C, each Gα_i/13 mutant was protected from tryptic digestion in the presence of AlF₄⁻, as evidenced by the existence of the lower molecular weight species, indicating that these mutants were capable of undergoing the activation-dependent conformational changes indicative of a functional Gα subunit.

The T274E and N278A Mutations Disrupt Binding to the RH Domain of p115RhoGEF and Impair GAP Activity

Because these mutations were known to be in the Gα₁₃-p115RhoGEF RH domain interface (17), the ability of these mutants to interact with the RH domain was assessed using GST pull-down assays, and their capacity to respond to the GAP activity of this domain was determined using single turnover GTPase assays. The activation-dependent interaction between each mutant and the RH domain of p115RhoGEF is shown in Fig. 2. Although the N270A mutation had no apparent effect on the ability of Gα_i/13 to interact with GST-p115 RH, the Gα_i/13 T274E mutant had significantly reduced binding, whereas the N278A mutation produced an intermediate effect. The Gα_i/13 T274E/N278A double mutant had no detectable binding to the RH domain under these conditions.

FIGURE 2.

The T274E and N278A mutations impair binding of Gα_i/13 to the RH domain of p115RhoGEF. Gα_i/13 (wild type or mutant) was incubated with a 50-fold molar excess of GST-p115 RH in the presence or absence of AlF₄⁻. GST-p115 RH was pulled down (PD) with glutathione-Sepharose beads. Bound proteins were released by boiling and separated by SDS-PAGE. Gα_i/13 was detected by immunoblotting (IB). GST-p115 RH was stained with Coomassie Brilliant Blue. Data presented are from one experiment, representative of three independent experiments with similar results.

Previous work has established that the isolated RH domain (amino acids 1–252) of p115RhoGEF is as effective a GAP for Gα₁₃ as the full-length protein (15). Therefore, GST-p115 RH was also used to examine the effects of these mutations on p115 RH-stimulated GAP activity in vitro (Fig. 3). Wild type Gα_i/13 has a measurable rate of basal GTP hydrolysis, which can be markedly increased by the presence of GST-p115 RH (Fig. 3A). At 0 °C, 10 nm GST-p115 RH increased the initial velocity of the hydrolysis reaction (V_o) from ∼0.08 to ∼1.8 fmol/s, or ∼22-fold (Fig. 3F). Gα_i/13 N270A displayed a very similar rate of RH domain-stimulated hydrolysis (Fig. 3F). Consistent with the fact that Gα_i/13 T274E binds the RH domain of p115RhoGEF poorly (Fig. 2), its response to the GAP activity of the RH domain was also significantly impaired. In the presence of the RH domain, GTP hydrolysis by Gα_i/13 T274E was reduced to nearly basal levels (Fig. 3, C and F). In the case of Gα_i/13 N278A, the reduction in GAP activity in response to the RH domain was ∼50% (Fig. 3, D and F). Like Gα_i/13 T274E, the double mutant, Gα_i/13 T274E/N278A, had a rate of RH-stimulated hydrolysis that was reduced to essentially basal levels (Fig. 3, E and F). Therefore, the T274E mutation strongly affects the ability of Gα_i/13 to bind the RH domain of p115RhoGEF and respond to its GAP activity, whereas the effect of the N278A mutation was also clear but less pronounced. The N270A mutation had no detectable effect on RH domain binding or GAP activity under the assay conditions used in Figs. 2 and 3.

FIGURE 3.

The T274E and N278A mutations in Gα_i/13 impair the GAP response to the RH domain of p115RhoGEF. A–E, Gα_i/13 or the indicated mutant was preloaded with [γ-³²P]GTP (final Gα concentration 8–10 nm), and hydrolysis of bound GTP was initiated by mixing with buffer containing 8 mm MgSO₄ and 1 mm GTP in the presence (●) or absence (■) of 10 nm GST-p115 RH. Reactions were incubated on ice, and aliquots were removed and quenched in activated charcoal slurry (pH 3) at the indicated times. F, the apparent initial rate of GTP hydrolysis by Gα_i/13 or the indicated mutant is shown in the presence (black bar) or absence (gray bar) of 10 nm GST-p115 RH. Data presented are from one experiment representative of three independent experiments with similar results.

Gα_i/13 T274E/N278A Cannot Bind to Full-length p115RhoGEF

Next, we assessed the ability of the T274E/N278A double mutant to bind to full-length p115RhoGEF by size exclusion chromatography. As shown in Fig. 4, upon activation with AlF₄⁻, Gα_i/13 forms a stable complex with p115RhoGEF, as evidenced by its elution in earlier, p115RhoGEF-containing fractions. In contrast, binding of the double mutant to full-length p115RhoGEF in the presence of AlF₄⁻ was not detectable at the level of Coomassie Brilliant Blue staining. Given that micromolar concentrations of p115RhoGEF and Gα_i/13 T274E/N278A were insufficient to form a stable complex during the course of gel filtration, the affinity of Gα_i/13 T274E/N278A for full-length p115RhoGEF is likely to be quite low, suggesting that the primary binding site for Gα₁₃ is in the RH domain.

FIGURE 4.

The T274E/N278A double mutation in Gα_i/13 impairs binding to full-length p115RhoGEF. p115RhoGEF was incubated with Gα_i/13 and GDP (A), Gα_i/13 and AlF₄⁻ (B), Gα_i/13 T274E/N278A and GDP (C), or Gα_i/13 T274E/N278A and AlF₄⁻ (D). The molar ratio of Gα_i/13 (wild type or mutant) to p115RhoGEF was 2:1. Proteins were fractionated on a Superdex 200 size exclusion column, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Fraction numbers are indicated. The positions of molecular mass standards, in kDa, are shown on the left.

Gα₁₃ T274E/N278A Fails to Activate p115RhoGEF in Vitro

We next examined whether Gα_i/13 T274E/N278A could regulate p115RhoGEF activity directly by measuring GTPγS binding to RhoA in vitro. Although Gα_i/13 was able to stimulate RhoA activation through p115RhoGEF (Fig. 5A), Gα_i/13 T274E/N278A failed to increase GTPγS binding to RhoA over the p115RhoGEF only condition. These results demonstrate that the T274E/N278A double mutation inhibits the ability to Gα_i/13 to stimulate the nucleotide exchange activity of p115RhoGEF.

FIGURE 5.

Analysis of RhoA activation in vitro. GTPγS binding to RhoA (final concentration 500 nm) was measured in the presence of buffer, 5 nm p115RhoGEF, or 5 nm p115RhoGEF and 100 nm of the indicated AlF₄⁻-activated Gα subunit. Samples were incubated at 30 °C and quenched in ice-cold buffer containing 10 mm MgSO₄ at the indicated time. A, Gα_i/13 T274E/N278A fails to stimulate p115RhoGEF activity in vitro. Samples are as follows: RhoA only (●), p115RhoGEF (■), p115RhoGEF plus Gα_i/13 T274E/N278A (▴), and p115RhoGEF plus Gα_i/13 (♦). Data are presented as the mean of single determinations pooled from two independent experiments. B, full-length Gα₁₃ is a more efficacious activator of p115RhoGEF in vitro than Gα_i/13. GTPγS binding to RhoA was determined after a 20-min incubation as described above. Data are presented as the mean ± S.E. (error bars) of single determinations pooled from three independent experiments. Data were analyzed by one-way analysis of variance, followed by Dunnett's post-test. Statistically significant difference from the p115RhoGEF condition is shown as follows. ns, not significant; **, p < 0.01. C, the T274E/N278A double mutation significantly reduces p115RhoGEF activation by full-length Gα₁₃. Samples are as follows: RhoA only (●), p115RhoGEF (■), p115 RhoGEF plus Gα₁₃ T274E/N278A (▾), p115RhoGEF plus Gα₁₃ (▴), and Gα₁₃ alone (□). Data are presented as the mean ± S.E. of single determinations pooled from three independent experiments.

Recently, we successfully generated recombinant, full-length Gα₁₃ comprising amino acid residues 2–377. In the course of analyzing this protein, we found that it is a more efficient activator of RhoA in vitro than a functionally equivalent amount of Gα_i/13 (Fig. 5B). This suggests that the N-terminal region of Gα₁₃ may play a role in efficient stimulation of RhoGEF activity. Due to the higher activity of Gα₁₃ in the in vitro RhoGEF assay, we chose to confirm the effect of the T274E/N278A mutations in the background of this full-length protein and examined its ability to stimulate RhoGEF activity. 100 nm full-length Gα₁₃ was able to significantly increase the amount of GTPγS bound to RhoA compared with the p115RhoGEF only condition (Fig. 5, B and C). In contrast, RhoA activation stimulated by a functionally equivalent amount of Gα₁₃ T274E/N278A was significantly reduced compared with wild type Gα₁₃ (Fig. 5C). The effects of the T274E and N278A mutations on the ability of Gα₁₃ to stimulate p115RhoGEF activity in vitro are shown in supplemental Fig. 1. We note that in contrast to the SRE assay, in which the activity of both Gα₁₃ QL T274E and Gα₁₃ QL N278A was reduced ∼50% (Fig. 1B), Gα₁₃ T274E failed to stimulate RhoA activation in vitro, whereas Gα₁₃ N278A had no apparent defect. However, in vitro, the functional concentration of Gα₁₃ and its mutants was quantitated by [³⁵S]GTPγS binding, which allows for a comparison between functionally equivalent amounts of protein. In contrast, the SRE assay is a cell-based overexpression system that precludes the tight control over the concentration of Gα₁₃ afforded by our reconstitution assay. Thus, it is difficult to make a direct comparison between the behavior of the mutants in vitro and in cell-based experiments. Taken together, the results using both Gα_i/13 and Gα₁₃ are consistent and in agreement with the results of the cell-based assays, thus providing strong evidence that residues in the α₃ helix of Gα₁₃ regulate the GEF activity of p115RhoGEF.

X-ray Crystallography of the Gα_i/13-p115RhoGEF RH Domain Complex

To gain more insight into the interaction between Gα_i/13 and p115RhoGEF, we purified the ternary complex of Gα_i/13, a deletion mutant of p115RhoGEF lacking the region C-terminal to the PH domain (p115RhoGEFΔC), and RhoA by size exclusion chromatography in the presence of AlF₄⁻ and subjected the complex to crystallization screening. Platelike crystals formed at 20 °C after ∼6 weeks and contained a complex of Gα_i/13 and the RH domain of p115RhoGEF. However, the region C-terminal to the RH domain, including the DH and PH domains and RhoA, was not present in the crystal. The x-ray crystal structure of the p115RhoGEF RH domain in complex with Gα_i/13, GDP, Mg²⁺, and AlF₄⁻ was determined at 2.4 Å resolution (Table 1). The structure contained two pairs of the Gα_i/13-p115RhoGEF RH domain complex in the asymmetric unit (A-B and C-D; molecules A and C designate Gα_i/13, and molecules C and D designate the p115RhoGEF RH domain (see supplemental Tables 1 and 2)). The final model contains residues 47–336 and 341–372 of molecule A; residues 22–34, 46–85, 93–121, and 134–233 of molecule B; residues 47–336 and 341–368 of molecule C; residues 22–32, 46–85, 92–115, 138–176, and 183–233 of molecule D; 2 GDP; 2 Mg²⁺; 2 AlF₄⁻; and 87 water molecules. For the purpose of discussing the crystal structure, we hereafter refer to the complex as Gα₁₃-p115 RH because the N terminus of Gα_i/13 containing the residues from Gα_i1 was disordered, and thus, only residues native to Gα₁₃ were present.

Overall Structure

In general, the structure of the Gα₁₃-p115 RH complex is similar to the previously reported crystal structure of the Gα_13/i1 chimera-p115RhoGEF RH domain complex (17). Both Gα_13/i1 and Gα₁₃ interact with the RH domain of p115RhoGEF through two distinct surfaces (Fig. 6). The N-terminal extension of the RH domain, the βN-αN hairpin, makes extensive contacts with the helical domain and switch regions I and II of Gα_13/i1. This interface is also present in the current structure; however, the βN strand is disordered (Fig. 6). Additionally, the RGS box binds to an effector-like interaction surface consisting of the α₂ and α₃ helices and α₃-β₅ loop of both Gα_13/i1 and Gα_i/13 (Fig. 6). However, unlike the Gα_i/13 used in the current study, Gα_13/i1 contains several residues from Gα_i1 in its effector interface, and its ability to stimulate RhoGEF activity has not been clearly demonstrated. The result of these substitutions is a significant difference in the effector interfaces between the two structures at the atomic level.

FIGURE 6.

Structure of the Gα₁₃-p115 RH complex. Top, the Gα₁₃-p115RhoGEF RH domain complex. The complex is shown as a ribbon diagram, with Gα₁₃ colored in green and the RH domain of p115RhoGEF in yellow. GDP, Mg²⁺, and AlF₄⁻ are shown as space-filling spheres. Carbon, oxygen, nitrogen, phosphate, magnesium, aluminum, and fluoride atoms are colored white, red, dark blue, orange, purple, light gray, and light blue, respectively. The disordered region in p115RhoGEF between the GAP interface and the RGS box is shown as a dashed yellow line. Bottom, a detailed view of the Gα₁₃-p115 RH domain effector interface. The Gα₁₃-p115 RH complex is shown on the left, and for comparison, the Gα_13/i1-p115 RH complex (Protein Data Bank entry 1SHZ) is shown on the right (17). The former is colored as in the top, whereas in the latter structure, Gα_13/i1 is colored blue, and the p115 RH domain is purple. Residues that contribute to the Gα-p115 RH interface are labeled. In Gα_13/i1, residues that are not native to Gα₁₃ are indicated in red. Hydrogen bonds are depicted as dashed lines. Oxygen and nitrogen atoms are colored red and blue, respectively.

The Effector Interface

Compared with Gα_13/i1, Gα₁₃ is shifted 3 Å closer to the RH domain of p115RhoGEF. This is probably a reflection of the higher affinity of this domain for Gα₁₃ versus Gα_13/i1. Additionally, the bulky side chain of Trp²⁸⁰ in Gα_13/i1, which corresponds to Val²⁸⁰ in Gα₁₃, may sterically inhibit the formation of a tight complex. Gα₁₃ forms more hydrogen bonds with p115 RH as compared with Gα_13/i1 (9 versus 3 possible hydrogen bonds) (Fig. 6). In Gα₁₃, Glu²⁷³ forms an ion pair with Lys¹⁶⁰ in the RH domain of p115RhoGEF. In contrast, Glu²⁷³ in Gα_13/i1 makes no contact with the RH domain, whereas Lys¹⁶⁰ participates in a van der Waals interaction with Ser²⁷⁴ of Gα_13/i1. The backbone of Thr²⁷⁴ in Gα₁₃ (Ser²⁷⁴ in Gα_13/i1) engages the side chain of Gln⁶⁹ of the RH domain through hydrogen bonding, whereas Gα_13/i1 interacts with Gln⁶⁹ via a hydrogen bond with the side chain of Asn²⁷⁸. Given that this is one of the three possible hydrogen bonds between Gα_13/i1 and the RH domain of p115RhoGEF, perturbing this interaction might be expected to result in a large decrease in binding. However, the results of our experiments with the N278A mutant of Gα₁₃ suggest that this residue is not as critical for the interaction with the RH domain of p115RhoGEF as Thr²⁷⁴. Consistent with this notion, in Gα₁₃, Asn²⁷⁸ does not participate in hydrogen bonding to p115 RH but rather is in hydrophobic contact with Leu⁶⁸. Arg²⁷⁹ of Gα₁₃ makes extensive contacts with p115 RH, including a hydrogen bond to the backbone carboxyl group of Phe⁷⁰, a residue that has previously been implicated in binding to Gα₁₃ (14). In contrast, Phe⁷⁰ makes no contact with Gα_13/i1. Additionally, Arg²⁷⁹ hydrogen-bonds to two additional residues in the RH domain, Ala⁶⁷ and Leu⁶⁸, through a main chain-side chain bond and a main chain-main chain bond, respectively. As a result of these amino acid differences, the calculated surface area buried in the effector interface of the Gα₁₃-p115 RH complex (1,600 Ų) is significantly larger than that of the Gα_13/i1-p115 RH complex (1,200 Ų).

The GAP Interface

Although there are significant differences in the effector interface between Gα₁₃ and Gα_13/i1, both interact with the GAP surface of the RH domain in an essentially identical manner (supplemental Fig. 2), which is consistent with the fact that the α helical domain and switch regions of Gα_13/i1 are largely derived from Gα₁₃. The interaction between the catalytic residue Arg²⁰⁰ of Gα₁₃ and Glu²⁷ of p115 RH is found in both structures. Arg²⁶⁰ in Gα_13/i1, which hydrogen-bonds with the main chain carbonyl of Ile²³ and forms an ion pair with Asp²⁸ in p115 RH, participates in the same bonding interactions in the Gα₁₃-p115 RH structure. Likewise, the interaction between Met²⁵⁷ from Gα₁₃ and Phe³¹ of p115 RH, which in the Gα_13/i1-p115 RH domain structure is a van der Waals contact, is also present in the current structure (supplemental Table 1).

Structural Aspects of the N270A, T274E, and N278A Mutations

In the Gα₁₃-p115 RH domain complex, Asn²⁷⁰ of Gα₁₃ interacts with p115 RH primarily via hydrophobic contact with Met¹⁶³. However, this interaction is well outside of the effector interface, supporting the results of cell-based experiments demonstrating that the N270A mutation does not impact GEF activity. In contrast, the T274E mutation resulted in reduced Rho activation in cells and severely disrupted binding to p115 RH and the GAP response. These observations are consistent with the fact that Thr²⁷⁴ hydrogen-bonds, via its backbone carbonyl, to Gln⁶⁹ in the p115 RH domain, a residue known to be important for Gα₁₃ binding (14). It is likely that the introduction of a longer, charged side chain in place of Thr²⁷⁴, as is the case with the T274E mutation, disrupts the interaction with Gln⁶⁹ and that this reduced binding results in impaired GAP and GEF activity. Additionally, in molecules C and D, Thr²⁷⁴ in Gα₁₃ makes additional contacts with p115 RH via hydrogen bonding of its side chain to the side chain of Lys¹⁶⁰ and hydrophobic interaction with Met¹⁶⁵. Thus, the effects of the T274E mutation may also be manifest in the form of steric clashing and perturbation of hydrophobic contacts. To probe the role of the side chain of Thr²⁷⁴ in the interaction with the RH domain of p115RhoGEF, we made several additional point mutations at this position in Gα₁₃ and assessed the activity of these mutants in the SRE assay. As shown in Fig. 7A, neither the T274A nor the T274S mutation affected Rho activation by Gα₁₃ QL in this system. Although the T274V mutant had slightly reduced activity, the effect was not nearly as severe as the T274E mutation. Thus, these data strongly suggest that Thr²⁷⁴ interacts with the RH domain primarily through its backbone. Asn²⁷⁸ interacts with p115 RH solely through a hydrophobic interaction with residue Leu⁶⁸. Thus, the modest decreases in binding to the RH domain and GAP activity caused by the N278A mutation are consistent with the fact that Asn²⁷⁸ does not contribute substantially to the interface with p115 RH. In addition, other residues in Gα₁₃ that we did not initially target for site-directed mutagenesis also contribute to the interface with the RH domain of p115RhoGEF, namely Glu²⁷³ and Arg²⁷⁹. In order to determine if these residues contribute to the ability of Gα₁₃ to stimulate GEF activity, the E273K and R279E mutants were generated and analyzed by an SRE assay. As shown in Fig. 7B, mutation of these residues impairs the ability of Gα₁₃ to stimulate Rho activation in cells, consistent with the notion that the Gα₁₃-p115 RH interface has the capacity to regulate effector activity. Thus, the results of the current biochemical analysis as well as previously published mutational analysis of p115RhoGEF (14) are more consistent with the Gα₁₃-p115 RH interface presented here than with the interface between the RH domain and Gα_13/i1.

FIGURE 7.

Mutational analysis of the effector interface between Gα₁₃ and p115 RH. A, the T274A and T274S mutations in Gα₁₃ fail to impair Rho activation in cells. HeLa cells were transiently transfected with empty vector or the indicated Gα₁₃ QL construct. The luciferase activity of cell lysates was determined as described under “Experimental Procedures.” Total cell lysate was immunoblotted for either Gα₁₃ or GAPDH. Data are presented as the mean ± S.E. (error bars) of triplicate determinations from a single experiment, representative of three independent experiments with similar results. Data were analyzed by one-way analysis of variance followed by Dunnett's post-test. Statistically significant difference from Gα₁₃ QL is shown as follows. **, p < 0.01; ***, p < 0.001. B, the E273K and R279E mutations in Gα₁₃ impair Rho activation in cells. Assays were performed as in A. Statistically significant difference from Gα₁₃ QL is shown as follows. **, p < 0.01.

Comparison with PDZ-RhoGEF

Interestingly, the effector interface between Gα₁₃ and the RH domain of p115RhoGEF is largely conserved in PDZ-RhoGEF (Fig. 8 and supplemental Table 3). Although Asn²⁷⁰ of Gα₁₃ participates in hydrophobic interaction with p115 RH, mutation of this residue has no apparent effect on binding, GAP, or GEF activity in cells. There is no interaction between this residue and PDZ-RhoGEF. In contrast, residue Thr²⁷⁴ of Gα₁₃, due to rotation of the side chain, makes more extensive contact with PDZ-RhoGEF RH than with p115RhoGEF RH. In addition to the interaction with Gln³⁵¹, which is analogous to Gln⁶⁹ in p115RhoGEF, via its backbone carbonyl group, Thr²⁷⁴ is also involved in hydrophobic interactions with Lys⁴³⁹ and Leu⁴⁴⁴. Residue Asn²⁷⁸ in Gα₁₃ also interacts with Gln³⁵¹ of PDZ-RhoGEF via hydrophobic contact. Arg²⁷⁹ of Gα₁₃, which has extensive interaction with the RH domain of p115RhoGEF, makes fewer contacts with PDZ-RhoGEF. However, the main chain-main chain interaction between Arg²⁷⁹ and Leu⁶⁸ of p115RhoGEF is conserved in the analogous residue of PDZ-RhoGEF, Ser³⁵⁰. Thus, the prominent features of the effector interface between Gα₁₃ and p115RhoGEF RH are conserved in the Gα₁₃-PDZ-RhoGEF RH crystal structure as well, suggesting that this interface may also play a role in regulating the GEF activity of PDZ-RhoGEF.

FIGURE 8.

Comparison of the interaction between Gα₁₃ and the RH domains of p115RhoGEF and PDZ-RhoGEF. Top, the effector interface formed by Gα₁₃ and the RH domain of p115RhoGEF. The interface is depicted as a ribbon diagram, with Gα₁₃ colored in green and the RH domain in yellow. Oxygen and nitrogen atoms are colored red and blue, respectively. Hydrogen bonds are depicted as dashed lines. Residues that contribute to the GAP interface are labeled in gray, whereas hydrophobic residues that contribute to the effector interface by the RH domain are labeled in light blue. Residues in Gα₁₃ that affect its capacity to stimulate the GEF activity of p115RhoGEF are labeled in red, and their bonding partners in the RH domain are labeled in purple. The conformationally flexible residue Phe²³⁴ in Gα₁₃ is labeled in black. Bottom, the effector interface formed by Gα₁₃ and the RH domain of PDZ-RhoGEF (Protein Data Bank entry 3CX7) (45). The interface is depicted as a ribbon diagram, with Gα₁₃ colored in olive green and the RH domain in light blue. Atoms and amino acid residues are labeled as in the top.

DISCUSSION

The RH domains found in RH-RhoGEFs, such as p115RhoGEF, are characterized by low sequence identity to classical RGS domain-containing proteins, such as RGS2 and RGS4 (12, 37). Additionally, unlike these RGS proteins, the RGS box of p115RhoGEF does not contain the residues necessary to accelerate GTP hydrolysis by Gα₁₃; these catalytic residues are found in the acidic βN-αN hairpin N-terminal to the RGS box (14, 17). The RH domain of p115RhoGEF has also been implicated in the regulation of nucleotide exchange activity because deletion of the first 288 amino acids of p115RhoGEF impairs both basal and Gα₁₃-stimulated GEF activity (15). Given that removal of the first 42 amino acids of p115RhoGEF, which are required for GAP activity, has no effect on the ability of Gα₁₃ to stimulate GEF activity (15), the GAP and GEF functions are clearly distinct properties of the RH domain. However, specific residues in Gα₁₃ responsible for stimulating p115RhoGEF activity have not been identified. Although x-ray crystallography has demonstrated that a Gα_13/i1 chimera forms a putative effector-like interface with the RH domain of p115RhoGEF (17), the capacity of residues in this interface, many of which are derived from Gα_i1, to regulate GEF activity is unknown.

Our results demonstrate that Gα₁₃ regulates p115RhoGEF activity via the classical effector interface it forms with the RH domain. Specifically, mutation of Thr²⁷⁴ and Asn²⁷⁸ in the α₃ helix of Gα₁₃ impairs the ability of Gα₁₃ to stimulate Rho activation in cells, impairs binding to p115RhoGEF, and reduces the capacity of Gα₁₃ to stimulate the guanine nucleotide exchange activity of p115RhoGEF in vitro. We also determined the structure of the Gα₁₃-p115RhoGEF RH complex and found that this effector interface differs in several important respects from the interface formed with Gα_13/i1. Importantly, these new structural data are strongly supported by the results of our biochemical experiments.

In the context of previously reported data, our results are consistent with earlier studies that broadly defined the RhoGEF-activating surface of Gα₁₃ as the region C-terminal to the Ras-like domain (32), more specifically the last 100 amino acid residues (23). However, given that we have demonstrated that the interaction between the RH domain and Gα₁₃ also regulates the effector activity of p115RhoGEF, this suggests that the previously described interface between Gα₁₃ and the DH/PH domains of p115RhoGEF may not contribute significantly to stimulation of GEF activity (15). If the RH domain represents the sole binding site for Gα₁₃, how might this interaction regulate the activity of the DH/PH domains? One study using PDZ-RhoGEF has suggested that the RH domain may work in concert with elements found in the linker region between the RH and DH domains to regulate GEF activity (38). Specifically, an acidic cluster of residues located in the linker may interact directly with residues in the DH domain to autoinhibit PDZ-RhoGEF activity. Mutation of these residues enhances GEF activity in vitro but only in the absence of the RH domain. Recently, the linker between the RH and DH domains of p115RhoGEF has also been implicated in regulating basal GEF activity, although the mechanism of autoinhibition appears to be distinct from that employed by PDZ-RhoGEF (39). A stretch of 40 amino acids located in the linker region of p115RhoGEF has been proposed to disrupt the interaction between the DH domain and residues in the “GEF switch” immediately N-terminal to it, an interaction that is critical for basal GEF activity. Thus, one consequence of Gα₁₃ binding to the RH domain may be to reorient elements within the linker region of these RH-RhoGEFs, allowing for enhanced exchange activity.

A comparison of the structures of Gα₁₃ bound to the RH domains of p115RhoGEF and PDZ-RhoGEF reveals that it binds this domain in a nearly identical manner. Additionally, the T274E and N278A mutations in Gα₁₃ also impair the interaction with the RH domain of LARG in vitro (data not shown). Thus, it is likely that Gα₁₃ engages the RH domains of all three RH-RhoGEFs in the same manner and stimulates RhoGEF activity through a similar mechanism. Although LARG is clearly regulated directly by Gα₁₃ in vitro (40), stimulation of the RhoGEF activity of PDZ-RhoGEF by Gα₁₃ in vitro has not been detectable under the conditions tested (15). However, PDZ-RhoGEF has been linked to Gα₁₃-mediated Rho activation in cells (10). Thus, although the effector interface formed between Gα₁₃ and the RH domain of p115RhoGEF bears striking resemblance to the Gα₁₃-PDZ-RhoGEF RH interface, in the latter case, this interaction is not sufficient to regulate RhoGEF activity in vitro. Post-translational modification, such as phosphorylation, or interaction with a co-activating protein may be required to render PDZ-RhoGEF susceptible to regulation by Gα₁₃.

It is also possible that activation of p115RhoGEF by Gα₁₃ requires multiple intermolecular interfaces. Binding of Gα₁₃ to the RH domain may result in a conformational change that brings additional regions of p115RhoGEF, perhaps the catalytic DH/PH domains, into contact with Gα₁₃, allowing for efficient activation of exchange activity. Although activated Gα₁₃ clearly fails to stimulate the GEF activity of p115RhoGEF fragments lacking the RH domain, it has been reported to bind to the isolated DH/PH domains of p115RhoGEF in vitro (15). Additionally, the N terminus of Gα₁₃, which is replaced by the αN helix of Gα_i1 in Gα_i/13 and is disordered in the present crystal structure, may contribute to the binding interface with p115RhoGEF. This hypothesis is supported by the fact that Gα₁₃ exhibits more robust activity in the RhoGEF assay compared with Gα_i/13 in vitro (Fig. 5B). The results of our gel filtration analysis suggest that Gα_i/13 T274E/N278A cannot bind to p115RhoGEF and are consistent with the in vitro RhoGEF assays demonstrating that Gα_i/13 T274E/N278A fails to stimulate RhoA activation over the p115RhoGEF condition. These data argue in favor of a model in which Gα₁₃ binds exclusively to the RH domain; however, it is difficult to rule out the possibility of additional low affinity binding sites outside of the RH domain. Given that the RH domain occupies the classical effector interface on Gα₁₃, any additional interface would probably be a novel surface that has not been observed in other Gα-effector pairs. Aside from the residues reported here, however, screening of additional Gα₁₃ mutants has so far failed to identify other residues that affect its capacity to stimulate Rho activation in cells.⁴

Little is known about the mechanism by which the other G₁₂ family member, Gα₁₂, stimulates RH-RhoGEF activity. p115RhoGEF also functions as a GAP for Gα₁₂ and serves as an effector in cells; however, Gα₁₂ is incapable of activating p115RhoGEF in vitro (9). Because Gα₁₂ and Gα₁₃ have a high degree of identity at the primary sequence level (∼69%) and have nearly identical α₃ helices and α₃-β₅ loops, it is worthwhile to consider biochemically whether Gα₁₂ and Gα₁₃ bind to the RH domain of p115RhoGEF using the same interface. When mutations corresponding to the Gα₁₃ mutants described here were introduced into Gα₁₂, the pattern of Rho activation in SRE assays and binding to the RH domain of p115RhoGEF was similar to that observed with the Gα₁₃ mutants (supplemental Fig. 3). This suggests that the binding site for Gα₁₂ overlaps with that of Gα₁₃ in the RH domain of p115RhoGEF and probably explains the observation that activated Gα₁₂ can inhibit Gα₁₃-mediated activation of p115RhoGEF despite the fact that Gα₁₂ cannot directly activate p115RhoGEF in vitro (9). However, these data imply that the inability of Gα₁₂ to stimulate p115RhoGEF is not due to a difference in primary sequence, as had previously been suggested (23). Thus, the reasons behind this lack of activity remain unknown. However, as another RH-RhoGEF, LARG, needs to be tyrosine-phosphorylated in order to be responsive to Gα₁₂ in vitro (40), this may be the case with p115RhoGEF as well.

Although the precise activation mechanism of p115RhoGEF remains to be fully elucidated, we have identified residues in Gα₁₃ that play a role in regulating the nucleotide exchange activity of p115RhoGEF. However, given that p115RhoGEF activity is also reported to be regulated by subcellular localization (41–43) and post-translational modification (44), the activation mechanism in cells is likely to be complex. Ongoing efforts to crystallize Gα₁₃ and Gα₁₂ with p115RhoGEF constructs containing the RH domain and DH/PH domains will aid in directly addressing the presence of additional binding sites and will help to clarify the molecular mechanism by which heterotrimeric G proteins stimulate activation of this RhoGEF.