Activation of p115-RhoGEF Requires Direct Association of Gα₁₃ and the Dbl Homology Domain

Background: p115-RhoGEF can be regulated by activated Gα₁₃.

Results: Both RGS and DH domains of p115-RhoGEF interact with Gα₁₃.

Conclusion: The binding of RGS to Gα₁₃ facilitates direct association of Gα₁₃ to DH to regulate its exchange activity.

Significance: RGS domains can act cooperatively with other domains to mediate effector regulation by G proteins.

Abstract

RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G₁₂ class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G₁₂ and G₁₃. Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα₁₃, the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα₁₃ in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα₁₃ docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα₁₃ and ablates the stimulation of p115 by Gα₁₃. Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα₁₃ also affect stimulation of p115 by Gα₁₃. Although the GAP activity of p115 is not required for stimulation by Gα₁₃, two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα₁₃ to the RH domain facilitates direct association of Gα₁₃ to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

Introduction

RGS-RhoGEFs³ are a homologous subfamily of RhoGEFs (guanine nucleotide exchange factors for Rho proteins) that contain regulator of G protein signaling (RGS) domains. There are three members of this subfamily: p115-RhoGEF (p115), PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). They represent potential regulatory links between G protein-coupled receptors that activate the G₁₂ class of heterotrimeric G proteins and RhoA-mediated pathways that lead to cytokinesis and transformation (1, 2). RGS-RhoGEFs catalyze the exchange of GDP for GTP on RhoA, a small GTPase of the Ras superfamily (3). Activated RhoA bound to GTP can then engage downstream effectors and influence cellular functions. Like all members of the large family of RhoGEFs (about 70 in the human genome), the GEF activity of RGS-RhoGEFs resides in their tandemly linked Dbl homology (DH) and pleckstrin homology (PH) domains (4, 5). The RGS homology (RH) domains are situated N-terminal to the DH/PH domains. The RH domains of p115 and LARG function as GTPase-activating proteins (GAPs) for Gα₁₃ and Gα₁₂ subunits, and binding of Gα subunits to their respective RhoGEFs stimulates their guanine nucleotide exchange activity toward RhoA (6–9). In addition to the RH domain, PRG and LARG also contain an N-terminal PDZ domain that has been shown to mediate interaction of the RGS-RhoGEFs with regulatory proteins (10–12).

The exact mechanism by which Gα₁₃ stimulates the exchange activity of p115 remains elusive (7–9). Studies on interactions between RGS-RhoGEFs and activated Gα₁₃ have focused primarily on the RH domain, which led to elucidation of crystal structures of the RH domain alone and complexes between RH and Gα₁₃ (13–16). The RH domains in RGS-RhoGEFs share low sequence similarity to the canonical RGS domain and require elements outside of the consensus RGS box for proper folding and binding to Gα₁₃. In p115, these include the ²³IIG and ²⁷EDEDF motifs located N-terminal to the consensus RGS box and the ¹⁶³MGM motif C-terminal to the RGS box (see Fig. 1A). When bound to Gα₁₃, RH occupies both the regulator binding and effector binding sites on the GTPase, mainly through direct interaction with its Ras-like domain. The presence of RH is crucial for stimulation of the GEF activity by Gα₁₃, as the activity of a p115 fragment missing the RH domain could not be regulated by Gα₁₃ (17). As expected, mutations within the ²⁷EDEDF motif resulted in the loss of GAP activity and a reduction in binding affinity of p115 toward Gα₁₃. The same mutations, however, had little impact on the stimulation of GEF activity by Gα₁₃ (18). This raises the question of whether direct association between RH and Gα₁₃ is actually required for the stimulation of GEF activity; it also suggests that regions outside of RH in p115 might interact with Gα₁₃ during the activation process. There is evidence that activated Gα₁₃ binds weakly to regions outside of RH; however, it has not been shown that the GEF activity of p115 is regulated by such interactions (9, 17).

FIGURE 1.

Two hydrophobic anchors in the RH domain of p115 are critical for stimulation of GEF activity by Gα₁₃. A, structural based sequence alignment of RH domains from p115 and PRG. The helices are represented with bars on top of the amino acid sequences. The sequence alignment was carried out using the program Clustal W (35). The shaded bars represent additional elements N- and C-terminal to the consensus RGS box (white bars). The ²³IIG motif and the ¹⁶³MGM motif, as well as the ²⁷EDEDF motif critical for the GAP activity of p115, are boxed. The open arrow labeled YFP indicates the position of the insertion of YFP between helices α9 and α10. B, nucleotide exchange assays with p115RH-L-DH/PH and RhoA. For each time course, 2 μm RhoA was mixed with 5 μm mant-GTP, and the exchange reaction was started at room temperature by the addition of buffer (basal, solid circles), 100 nm p115 (open circles), or 100 nm p115 with 100 nm Gα₁₃ bound to GDP·AlF₄⁻·Mg²⁺ (GDP·AMF, closed triangles). The subsequent increase in fluorescence (λ_ex = 356 nm, λ_em = 445 nm) was measured for 10 min. C, stimulation of the GEF activity of RH-L-DH/PH by activated Gα₁₃ bound to GDP·AMF or GTPγS. Binding of mant-GTP to RhoA was measured as shown in B. The initial rates were approximated by linear regression and plotted as the fold increase over basal exchange on RhoA for the indicated conditions: WT, wild-type p115; F31A, Phe-31 in ²⁷EDEDF mutated to alanine; ²³AAG, ²³IIG mutated to AAG; ¹⁶³AGA, ¹⁶³MGM mutated to AGA; and ²³AAG/¹⁶³AGA, combination of both mutations.

Here, a combination of molecular cloning, biochemical assays and small angle x-ray scattering (SAXS) was used to examine the interaction of activated Gα₁₃ with a C-terminally truncated p115 molecule that includes the tandemly linked DH and PH domains in addition to RH. We show that the ²³IIG and the ¹⁶³MGM hydrophobic motifs, respectively, located N- and C-terminal to the consensus RGS box rather than the ²⁷EDEDF motif are crucial for the stimulation of the GEF activity of p115 by activated Gα₁₃. Furthermore, activated Gα₁₃ interacts with the DH domain via a novel effector binding site located within its helical domain. Mutations of residues in the αB-αC loop of the helical domain negatively affect stimulation of p115 by Gα₁₃. The additional binding site in p115 for Gα₁₃ is located on the side of DH opposite to the binding site for the substrate RhoA. Mutations of residues in the predicted binding site for Gα₁₃ on DH abolished stimulation of GEF activity by Gα₁₃. These observations provide a comprehensive model for the molecular mechanism by which the intrinsic exchange activity of RGS-RhoGEFs is regulated by activated G proteins.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification

Coding regions of human p115-RhoGEF were subcloned into a pGEX-KG vector containing the protease recognition site for the tobacco etch virus (pGEX-KG-TEV) for proteolytic cleavage of the expressed domains from glutathione S-transferase as described previously (19, 20). His₆ tags were also inserted at the C termini of the p115 coding sequences. The proteins were expressed and purified from Escherichia coli strain BL21(DE3) as described (20). The expression and purification of a C-terminally truncated human RhoA (residues 1–181) was carried out as described previously (19). The N-terminally truncated Gα₁₃ (41–377) was expressed and purified from insect cells as described (13).

Nucleotide Exchange Assay

Fluorescence assays measuring the binding of N-methylanthraniloyl-GTP (mant-GTP; Invitrogen) were performed on a Fluorolog-3 spectrofluorometer at room temperature (λ_ex = 356 nm, λ_em = 445 nm, slits = 1/1 nm), as described previously (20). In each assay, 1–2 μm RhoA was incubated with 5 μm mant-GTP in reaction buffer (25 mm NaHEPES, pH 8.0, 50 mm NaCl, 1 mm DTT, and 5 mm MgCl₂) in a 200 μl cuvette. The exchange reaction was started by the addition of 100 nm p115, in the presence or absence of activated Gα₁₃.

Binding of GTPγS to Gα₁₃

Purified Gα₁₃ was exchanged into binding buffer (20 mm NaHEPES, pH 8, 1 mm EDTA, 1 mm DTT, 50 mm NaCl, and 10 μm GDP) and concentrated to 100–250 μm. The concentrate was adjusted to 0.5 mm MgSO₄ and 1 mm GTPγS and incubated at 25 °C for 48–72 h.

Size Exclusion Chromatography with p115 and Gα₁₃-GTPγS

Fragments of p115 were mixed with activated Gα₁₃ bound to GTPγS, in a buffer containing 25 mm NaHEPES, pH 8.0, 100 mm NaCl, 1 mm DTT, 1 mm EDTA, and 5 mm MgCl₂. The mixture was concentrated by Amicon-Ultra 4 (10 kDa) concentrators (Millipore) to a final volume of less than 1 ml and then loaded onto Superdex 200/75 columns (Amersham Biosciences) that had been pre-equilibrated with the same buffer.

Collection of SAXS Data

The purified RH-L-DH/PH fragment of p115 was dialyzed overnight in 25 mm NaHEPES, pH 8.0, 200 mm NaCl, 5 mm β-mercaptoethanol, 1 mm EDTA, 10 mm DTT, and 5% glycerol to ensure a precise buffer match for background subtraction of the scattering arising with the buffer alone from that of the protein sample. The complex of p115 RH-L-DH/PH and activated Gα₁₃ bound to GDP·AlF₄⁻·Mg²⁺ was dialyzed overnight in 25 mm NaHEPES, pH 8.0, 200 mm NaCl, 5 mm β-mercaptoethanol, 1 mm EDTA, 10 mm DTT, 0.05 mm AlCl₃, 5 mm MgCl₂, 5 mm NaF, and 5% glycerol. Triton X-100 was added to the sample to a final concentration of 0.5% before the measurement to prevent aggregation. The addition of Triton X-100 had no effect on the stimulation of GEF activity by activated Gα₁₃ (supplemental Fig. S1). The samples for SAXS were at a concentration between 1 and 4 mg/ml. Measurements were taken at 10 °C using the SAXS instrument at the BioCAT beamline of the Advanced Photon Source at the Argonne National Laboratory as described previously (20). The samples were analyzed by size exclusion chromatography before and after the SAXS measurements to determine concentration and oligomerization state. Scattering profiles (intensity I versus scattering vector Q) were reduced, and the SAXS data were merged using IGOR Pro software (WaveMetrics, Inc.) with macros written by the BioCAT staff. Structural parameters and the distance distribution functions, P(r), were calculated with GNOM (21) using data up to a Q of 0.30 and 0.31 Å⁻¹, for p115 alone and the p115-Gα₁₃ complex, respectively.

Ab Initio Modeling

Low resolution molecular shape reconstructions from the experimental scattering data were performed with GASBOR (22). GASBOR searches a chain-compatible spatial distribution of an exact number of dummy residues, corresponding to the Cα atoms of protein amino acids. The Cα chain is folded to minimize the discrepancy between the scattering curve calculated from the folded model and the experimental scattering curve. More than 80 GASBOR calculations were performed, and 20 calculations with the smallest standard deviation relative to the experimental data were selected for averaging by DAMAVER (23) to generate the final model; this represents the most probable conformation reconstruction for the protein. The molecular envelopes of RH-L-DH/PH, alone or in a complex with activated Gα₁₃, were calculated based on SAXS data using the program Situs (24), and the crystal structures of DH/PH (20) and the Gα₁₃-RH complex (14) were then fit into the envelope using the program Chimera (25).

RESULTS

Two Conserved Hydrophobic Motifs outside of the Canonical RGS Box in p115-RhoGEF Are Critical for Stimulation of GEF Activity by Gα₁₃

The GEF activity of RGS-RhoGEFs can be stimulated by activated G₁₂ class Gα subunits in vivo, and some of the RGS-RhoGEFs can be stimulated in vitro by activated Gα₁₃ (1). In the case of p115, Gα₁₃ bound to GDP·AlF₄⁻·Mg²⁺ (GDP·AMF), which mimics the transition state of GTP hydrolysis, is more efficient at stimulating the GEF activity of p115 than Gα₁₃ bound to GTPγS, a nonhydrolyzable analog of GTP (Fig. 1C). This is anticipated because p115 is a GAP for Gα₁₃, and it has a much higher affinity toward Gα₁₃ bound to GDP·AMF than toward Gα₁₃ bound to GTP. The GAP activity of RGS-RhoGEFs is conveyed by the RH domain (14), particularly by a series of acidic residues followed by a phenylalanine located N-terminal to the RGS box (the ²⁷EDEDF motif; Fig. 1A). A recent study of the interfaces observed in various complexes of RH-Gα₁₃ revealed two conserved hydrophobic anchors in RH (13): the ²³IIG motif at the very N terminus of RH and the ¹⁶³MGM motif just C-terminal to the RGS box (Fig. 1A). Mutation of either one of the two hydrophobic anchors greatly diminished binding between RH and activated Gα₁₃ (data not shown) and greatly reduced stimulation of p115 by Gα₁₃ (Fig. 1C). However, the reduction can be at least partially recovered by using higher concentrations of activated Gα₁₃ (Fig. 1C). Gα₁₃ could not activate p115 containing mutations of both isoleucines of ²³IIG and the two methionines of ¹⁶³MGM, even in the presence of higher concentrations of activated Gα₁₃ (Fig. 1C). In contrast, the F31A mutation had much less impact on Gα₁₃-stimulated GEF activity of p115 (Fig. 1C). This is consistent with an earlier study demonstrating that mutations within the ²⁷EDEDF motif led to a loss of GAP activity and a reduction in binding affinity between RH and Gα₁₃, but the same mutations did not diminish Gα₁₃-stimulated GEF activity in the background of full-length p115 (18). In fact, the GAP activity is not required at all for the stimulation of GEF activity by Gα₁₃. The replacement of RH in p115 with that from PRG, which is not a GAP, had little effect on Gα₁₃-stimulated GEF activity (supplemental Fig. S2) (17). Taken together, these data show that the physical association of RH and Gα₁₃, mediated by the two conserved hydrophobic anchors outside of the RGS box, is critical for stimulation of GEF activity by activated Gα₁₃.

A Binding Site for Activated Gα₁₃ outside of the RH Domain

The interaction between RH domains and activated Gα₁₃ has been well characterized (13, 14). The binding affinities between RH and Gα₁₃ range between 10 nm to 1 μm, depending on the specific RH domain and the state of the GTPase.⁴ Binding of Gα₁₃ to regions outside of the RH domain has been suggested (9, 17), but the evidence is limited. The affinity between truncated p115 missing RH and activated Gα₁₃ is low, such that no physical association between the two could be observed by size exclusion chromatography (Fig. 2). In contrast, activated Gα₁₃ and RH form a stable complex that is dependent on two interfaces (13, 14). The N-terminal 41 amino acids in RH include one of these interfaces, the ²³IIG motif and the ²⁷EDEDF motif, which are located N-terminal to the consensus RGS box. Truncation of this N-terminal segment (^ΔNRH; Fig. 2A) results in a sharp decrease in the affinity of the RH domain itself toward activated Gα₁₃. Although the intact RH domain readily forms a stable complex with activated Gα₁₃, ^ΔNRH failed to form a complex with Gα₁₃-GTPγS that can be detected by size exclusion chromatography (Fig. 2B). A fragment of p115 consisting of L-DH/PH also failed to bind Gα₁₃-GTPγS (Fig. 2B). However, the fragment consisting of both ^ΔNRH and L-DH/PH can form a weak but readily observable complex with the activated α subunit (Fig. 2B). This strongly suggests the existence of a weak binding site for activated Gα₁₃ within the L-DH/PH region in addition to the known site in ^ΔNRH.

FIGURE 2.

An additional binding site for activated Gα₁₃ outside of RH. A, schematic representation of truncated forms of p115 used; the included amino acids are listed in parentheses. B, left panels, chromatograms from size exclusion chromatography with p115 and Gα₁₃-GTPγS are aligned based on elution volumes; peaks corresponding to free (unbound) Gα₁₃ with elution volumes around 26 ml are outlined in a gray box. Right panels, SDS-PAGE gels showing components of eluted peaks of protein. Lanes on gels are aligned based on volumes of elution. Molecular masses of protein standard makers (first lanes on the left) are labeled. Fractions corresponding to free (unbound) Gα₁₃ are outlined in a gray box. The same amount of Gα₁₃, which was premixed with equal molar of the p115 fragment, was used in all four experiments. The scale of absorption (Abs.) in the last panel (at bottom) was changed because of variations in detector efficiency.

Solution Structure of the C-terminally Truncated p115

Dimerization of p115 in solution is dependent on the domain C-terminal to PH (1). Thus, the C-terminally truncated proteins used in Figs. 1 and 2 are monomeric. We determined the solution structure of the C-terminally truncated p115 (RH-L-DH/PH) at 20 Å resolution using SAXS. The molecular envelope of RH-L-DH/PH was obtained by ab initio shape reconstruction from experimental SAXS data (22). The experimental scattering profile is shown in Fig. 3A. Low angle scattering intensity calibration with cytochrome c indicated that RH-L-DH/PH exists as a monomer in solution, with a distance distribution function, P(r), that is characteristic of an elongated molecule (Fig. 3A, inset). The calculated molecular envelope of RH-L-DH/PH confirms this, with the longest dimension of the molecule at ∼260 Å. The crystal structure of p115 DH/PH (20) fits well into the calculated molecular envelope (Fig. 3B), occupying half of the envelope. The shape and volume of this region closely resembles those calculated for solution envelopes of the DH/PH domains in the absence of RH (20) or without the linker (data not shown). The linker region and RH putatively occupy the volume of the molecular envelope below the DH domain.

FIGURE 3.

Solution structures of p115 RH-L-DH/PH. A, solution x-ray scattering profile for p115 RH-L-DH/PH. The distance distribution function (inset), P(r), of RH-L-DH/PH was computed from the x-ray scattering using the program GNOM (21). B, solution structure of RH-L-DH/PH of p115. The solution structure (molecular envelope) is depicted as a mesh and superimposed onto the crystal structure of the p115 DH/PH domains (blue ribbon). The large unoccupied region in the molecular envelope beneath the DH domain presumably contains the linker region and RH. The schematic representation of RH-L-DH/PH of p115 is depicted underneath the molecular envelope.

The spatial relationship between DH/PH and RH is not evident. The linker region is disordered in the crystal structure of L-DH/PH and partially disordered in solution (20). Crystal structures of RH domains are available; however, the exact location of RH within the volume below DH (Fig. 3B) could not be unambiguously determined. To identify the location of RH, we inserted YFP between helices α9 and α10 of RH (Fig. 1A) and determined the molecular envelope of the ^YFPRH-L-DH/PH fusion protein by SAXS (supplemental Fig. S3). The YFP fusion protein is elongated, with the longest dimension similar to that of the native p115. The portion of the ^YFPRH-L-DH/PH envelope containing the DH/PH domains can be overlapped relatively well with that of the native protein. There is almost no change in shape or volume at the distal end (supplemental Fig. S3) of the envelope, suggesting that YFP, and hence the RH domain, is not located in this area. The major difference between the two envelopes is evident in the center region underneath the DH domain. Insertion of YFP in RH caused a 20° bend of the molecule in the middle, a strong indication that RH (and YFP) is located in this region, near the bottom of the DH domain. The close proximity of RH to DH is also supported by the calculated molecular envelope of RH-L-DH/PH bound to activated Gα₁₃.

Solution Structure of a p115-Gα₁₃ Complex

The solution structure of RH-L-DH/PH from p115 (Fig. 3B) in a complex with activated Gα₁₃ bound to GDP·AMF was determined by SAXS at a resolution of 20 Å. The experimental scattering profile is shown in Fig. 4A. Low angle scattering intensity calibration with cytochrome c indicated that the complex is monomeric in solution. Comparison of the P(r) function of p115 alone and that of the complex indicates that the overall shapes of the two are similar, with the longest dimension being 260 Å in both cases (Fig. 4B). The molecular envelope of the RH-L-DH/PH-Gα₁₃ complex was obtained by ab initio shape reconstruction from experimental SAXS data. Although the overall shape of the complex resembles that of the RH-L-DH/PH alone, a large increase in volume (∼65,000 ų) was observed in the midsection of the molecular envelope, near the bottom of the modeled DH domain. This additional volume can accommodate a Gα₁₃ molecule (calculated at ∼50,000 ų). As discussed above, binding of the RH domain to activated Gα₁₃ is critical for both the activation of p115 and the formation of a stable complex between p115 and Gα₁₃ in solution (Fig. 2B). It is therefore reasonable to assume that the RH domain remains bound to Gα₁₃ in this complex. Hence, the RH domain is located near the DH domain where the increase in mass is observed, as concluded from inspection of the molecular envelope of the YFP fusion protein (supplemental Fig. S3). The crystal structure of the p115-RH-Gα₁₃ complex (14) can be fit into the calculated solution envelope together with the DH/PH domains (Fig. 4C). In this model, activated Gα₁₃ contacts the DH domain, opposite to the site at which RhoA binds (Fig. 4D). Importantly, the binding site for Gα₁₃ on the DH domain does not overlap with the binding site for RhoA. The DH domain appears to form an interface with the helical domain of Gα₁₃ rather than the Ras-like domain, which is engaged with the RH domain as observed in the structures of RH-Gα₁₃ complexes (13, 14). Thus, Gα₁₃ utilizes both the helical and the Ras-like domains for interaction with p115. We do note that the positioning of RhoA in this model (Fig. 4D) is based exclusively on crystal structures of DH/PH-RhoA complexes (26, 27). A solution structure of the ternary complex may provide further insight into the orientation of RhoA in the presence of Gα₁₃.

FIGURE 4.

Solution structures of the complex of p115 RH-L-DH/PH bound to Gα₁₃-GDP·AMF. A, solution x-ray scattering profile for the complex, with the distance distribution function (inset), P(r). B, comparison of the distance distribution functions of p115 alone and its complex with Gα₁₃. The overall shapes of the two are similar, with the longest dimensions being identical at ∼260 Å. C, solution structure of RH-L-DH/PH of p115 bound to Gα₁₃-GDP·AMF. The solution structure (molecular envelope) is depicted as a mesh and superimposed onto the crystal structures of the p115 DH/PH domains (blue ribbon) and the complex of RH (green ribbon) bound to Gα₁₃-GDP·AMF (red ribbon). The helical domain and the Ras-like domain of Gα₁₃ are outlined with brackets and labeled with H and R, respectively. The unoccupied region in the molecular envelope beneath the RH domain is likely to be the location for most of the linker region. D, a model for the ternary complex of RH-L-DH/PH bound to activated Gα₁₃ and nucleotide-free RhoA. RhoA (orange ribbon) was modeled into the binding site on DH/PH of p115 based on crystal structures of PRG-DH/PH or LARG-DH/PH bound to nucleotide-free RhoA (26, 27). The location of RhoA was not experimentally determined by SAXS in this study.

Mutations in a Predicted Binding Site for Gα₁₃ on DH Abolish Gα₁₃-stimulated GEF Activity

The model of the RH-L-DH/PH-Gα₁₃ complex derived from interpretation of SAXS data features an interface between the DH domain of p115 and the helical domain of Gα₁₃ (Fig. 4C). Structures of DH domains from RGS-RhoGEFs (20, 26, 27) consist of six major α-helices (Fig. 5A). Unlike p115, the GEF activity of PRG, a member of the RGS-RhoGEF family, cannot be stimulated by activated Gα₁₃ in vitro, even though its RH domain binds tightly to the α subunit (13). Thus, differences in the amino acid sequences and tertiary structures of the DH domains of p115 and PRG provide clues to the location of the binding site at which Gα₁₃ exerts GEF stimulatory activity toward p115. Two DH segments that are poorly conserved between p115 and PRG were identified at the putative interface between DH and Gα₁₃ in the SAXS model of the RH-L-DH/PH-Gα₁₃ complex (Fig. 5, A and B). The first segment is located in the α2b helix, consisting of amino acids 475–483 (α2b). The second segment is located within the α3b helix, including residues 503–507 (α3b). None of these residues in α2b or α3b are involved in binding to RhoA (Fig. 5A). Replacement of residues in α2b or α3b in p115 with those from PRG had little effect on the basal exchange activity of p115 toward RhoA, indicating that these substitutions do not affect the structure of the DH recognition site for RhoA (Fig. 5C and supplemental Fig. S4). Although changes in α2b had no effect on GEF activity stimulated by Gα₁₃, swapping of residues from PRG into α3b in p115 abolished stimulation by Gα₁₃ in vitro (Fig. 5C).

FIGURE 5.

Mutations at the predicted Gα₁₃-binding site on DH abolish the Gα₁₃-stimulated GEF activity of p115. A, sequence alignment of DH domains from p115 and PRG. Residues involved in contacts with nucleotide-free RhoA are marked with dots over the alignment. Helices are represented with bars on top of the amino acid sequences. The sequence alignment was carried out using the program Clustal W (35). The two potential binding sites for Gα₁₃ targeted for mutagenesis are colored red. B, ribbon diagrams with superimposed structures of DH domains from p115 (wheat) and PRG (gray). Side chains in α3b are depicted as sticks. Oxygen and nitrogen atoms are colored red and blue, respectively. C, stimulation of the GEF activity of RH-L-DH/PH by activated Gα₁₃. Binding of mant-GTP to RhoA was measured as shown in Fig. 1B. Initial rates were approximated by linear regression, and the increases of exchange rate over basal RhoA were plotted for the following proteins: WT, wild-type p115; α2b, mutant p115 where residues 475–483 were substituted with corresponding residues from PRG; and α3b, mutant p115 where residues 503–507 were substituted with corresponding residues from PRG. D, effects of single point mutations in the α3b region on stimulation by Gα₁₃. E, effects of single point mutations of Trp⁵⁰⁷ on stimulation by Gα₁₃.

Three of the five amino acids in α3b are exposed to solvent in the crystal structures of p115 and PRG DH domains and are not conserved in the amino acid sequences of the domains (Fig. 5B). These include Glu⁵⁰⁴ (Ala⁸²¹ in PRG), Ser⁵⁰⁶ (Glu⁸²³ in PRG), and Trp⁵⁰⁷ (Glu⁸²⁴ in PRG). Single point mutations at any of these three positions in p115 had varied effects on stimulation of GEF activity by Gα₁₃ (Fig. 5D). Mutation of Ser⁵⁰⁶ to glutamate in p115 had no effect on this regulation. The change of glutamate 504 to alanine decreased the response of p115 to activated Gα₁₃. Mutation of the exposed tryptophan 507 to glutamate, the corresponding residue in PRG, completely abolished Gα₁₃-stimlated GEF activity of p115. None of these point mutations affected the basal exchange activity of p115 in the absence of Gα₁₃. The p115 fragment, ^ΔNRH-L-DH/PH, bearing the W507E mutation also failed to bind activated Gα₁₃ as assayed by size exclusion chromatography (Fig. 2). Mutations of Trp⁵⁰⁷ to either a tyrosine or an alanine also greatly reduced Gα₁₃-stimulated GEF activity of p115 (Fig. 5E).

The αB-αC Loop in the Helical Domain of Gα₁₃ Is a Novel Effector Binding Motif

The α3b helix in the DH domain of p115, which includes Trp⁵⁰⁷, forms a protruding bulge on the surface of DH that is predicted to interact with the helical domain of Gα₁₃ in our model (Fig. 6A). Q-SiteFinder, a ligand-binding site prediction program (28), identifies a hydrophobic pocket between the αB and αC helices of Gα₁₃ in the helical domain (Fig. 6, B–D) as a possible site for protein-protein interaction. The shape of this pocket appears roughly complementary to the protruding bulge formed by Trp⁵⁰⁷ from DH. These surfaces are in close proximity in the low resolution model of the p115-Gα₁₃ complex derived from SAXS (Fig. 4C). Part of the α3b helix from DH, including residues 502–510, can be docked into this hydrophobic pocket in the αB-αC loop on Gα₁₃ using the program PatchDock (29). In this model, Trp⁵⁰⁷ from DH docks into the hydrophobic pocket on Gα₁₃ formed by the side chains of Phe¹²⁵, Ala¹²⁹, Pro¹³⁰, Met¹³¹, Ala¹³², Val¹³⁷, Val¹⁴¹, and Tyr¹⁴⁵ (Fig. 6D). Glu⁵⁰⁴ from DH also makes van der Waals contacts with residues from this region. Single point mutations of these hydrophobic residues in the αB-αC region had varied effects on the stimulation of GEF activity of p115 by Gα₁₃ in vitro (Fig. 6E). Mutation of Ala¹²⁹ to lysine or Ala¹³² to leucine resulted in a 40% reduction in stimulation of GEF activity of p115 by Gα₁₃. Similar reduction of stimulation of p115 was observed with a double mutation of Phe¹²⁵ and Tyr¹⁴⁵ to alanines. Such a reduction in stimulation of p115 correlated with a lowered efficacy of the mutant when compared with the wild-type Gα₁₃ (Fig. 6F). These mutations had no effect on the intrinsic GTPase activity of Gα₁₃ or its ability to bind guanine nucleotide (supplemental Fig. S5).

FIGURE 6.

A binding pocket for DH within the helical domain of Gα₁₃. A, ribbon diagram of the DH domain of p115. The molecule is positioned such that the proposed Gα₁₃-binding site on DH (yellow) is facing the reader. The side chain of Trp⁵⁰⁷ is shown as sticks. The surface of DH is shown and colored gray. B, ribbon diagram of Gα₁₃. The molecule is positioned such that the proposed DH binding site (green) is facing the reader. The side chain of Phe¹²⁵ is shown as sticks. The surface of Gα₁₃ is shown and colored gray. C, structural based sequence alignment of Gα₁₃ and Gα₁₂ helical domains. Helices are represented with bars on top of the amino acid sequences. The sequence alignment is carried out by the program Clustal W (35). Residues targeted for mutagenesis from the hydrophobic pocket are colored red. D, ribbon diagram depicting a modeled interface between α3b and the hydrophobic pocket in the helical domain of Gα₁₃ using the same color scheme as in A and B. Side chains of residues involved are depicted as stick models. Residues from the helical domain of Gα₁₃ that impact the stimulation of p115 when mutated are marked with asterisks. E, stimulation of the GEF activity of full-length p115 by activated Gα₁₃. Reactions contained 2 μm RhoA, 30 nm p115, and 300 nm Gα₁₃ (wild type or mutant). The effect of mutations in Gα₁₃ on the initial rate of the GEF activity of p115 is compared against wild-type Gα₁₃ (100%). Each experiment was repeated at least twice. F, stimulation of p115 with different concentrations of Gα₁₃. The initial rate of the GEF activity of p115 (approximated by linear regression with the first 4 min of a 6-min reaction) was assessed in the presence of increasing concentrations of activated Gα₁₃ (wild type or mutant). The reactions contained 2 μm RhoA and 30 nm p115: WT (closed circles), F125A (closed triangles), F125A/Y145A (open circles).

DISCUSSION

Earlier studies on interactions between RGS-RhoGEFs and activated Gα₁₃ have been primarily focused on RH and its interaction with Gα₁₃. The residues in the α3b helix of DH described here are the first outside of the RH domain to be implicated as direct mediators for activation by G proteins. The binding affinity between DH and activated Gα₁₃ is low; hence DH alone may not be sufficient to associate with the α subunit. An important function of the RH domain, as suggested by data presented in this report, might be to hold activated Gα₁₃ in a position for optimized interaction with the α3b helix in the DH domain of p115.

The DH and RH domains of RGS-RhoGEFs are separated by long linker regions. Interpretation of the size exclusion chromatography data (Fig. 2) and the low resolution molecular envelopes of Gα₁₃ with p115 (Fig. 4) is most consistent with a model in which Gα₁₃ interacts simultaneously with the RH and the DH domains of p115. Because the 150-residue linker region in p115 intervenes between RH and DH, it must therefore adopt a hairpin conformation, in which the N and C termini of the linker are nearly juxtaposed. The linker region from p115 would occupy the portion of the molecular envelope distal to the PH domain and beneath the RH domain (Fig. 4). The linker region in p115 appears to be rigid, because molecular envelopes of p115 alone and its complex with Gα₁₃ are of the same length. Our results differ from a recent study of PRG by SAXS (30). Like that of p115, the RH-L-DH/PH fragment of PRG is also an elongated structure. However, the RH and DH modules of PRG are well separated in the SAXS model by the linker region. The linker connecting RH and DH in PRG appears to be very flexible and changes both in length and in overall shape in the presence of the N-terminal PDZ domain (not present in p115) or when bound to RhoA. It is not clear how Gα₁₃ would affect the overall shape of PRG in solution, because PRG cannot be stimulated by Gα₁₃ in vitro.

The hairpin conformation of the p115 linker in our model allows the C terminus of the linker to contact the RH domain as the polypeptide chain returns from its excursion to the hairpin back toward the DH domain. This is supported by the observation that C-terminally extended RH constructs (RH-L) are not stably expressed unless the full-length linker is included in the construct (data not shown). RH-L constructs missing the C-terminal 40 residues in the linker region did not express well as soluble protein. Furthermore, removal of the N-terminal ∼100 residues of the linker region (RH-^ΔN2l-DH/PH; Fig. 7A) did not affect stimulation by Gα₁₃ (Fig. 7B) or the apparent interaction between DH and Gα₁₃ (Fig. 7C). Complete removal of the linker region, however, led to a significant increase in the basal exchange activity of p115 and diminished stimulation by Gα₁₃ (Fig. 7B). This is consistent with recent reports in which the linker region in RGS-RhoGEFs has been shown to play an autoinhibitory role (20, 31). The presence of the 40 amino acids in the linker immediately preceding the start of DH lowers the basal exchange activity of these RhoGEFs. A recent study suggests that this segment from the linker perturbs the folding of the GEF switch at the N terminus of DH in p115 (20). The same segment in the linker would interact with RH in our model. A possible scenario for Gα₁₃-stimulated GEF activity is that the binding of Gα₁₃ to RH and DH leads to conformational changes at the RH-L interface, which leads to formation of a productive interface between the GEF switch and RhoA that is required for enhanced GEF activity. Thus, binding of activated Gα₁₃ to RH, and to a less extent to DH, relieves the autoinhibition imposed by the linker region of p115.

FIGURE 7.

Removal of part of the linker region does not affect stimulation of p115 by Gα₁₃. A, schematic representation of the truncated forms of p115 used; the included amino acids are listed in parentheses. B, stimulation of the GEF activity of RH-ΔL-DH/PH by activated Gα₁₃. Binding of mant-GTP to RhoA was measured as shown in Fig. 1. Removal of the first 100 amino acids in the linker region had no effect on stimulation by Gα₁₃. The last 40 residues within the linker region immediately preceding DH is important for activation by Gα₁₃. C, stimulation of the GEF activity of mutated RH-^ΔN2l-DH/PH by activated Gα₁₃. The constructs are: WT, wild-type p115-^ΔN2LΔC; α2b, mutant p115-^ΔN2LΔC where residues 475–483 were substituted with corresponding residues from PRG; α3b, mutant p115-^ΔN2LΔC where residues 503–507 were substituted with corresponding residues from PRG; and α2b+α3b, mutant p115-^ΔN2LΔC where both α2b and α3b regions were substituted with corresponding regions from PRG.

Interaction between the DH domain and the α subunit of a G protein has been reported before. p63-RhoGEF, a GEF for the small GTPase Rho, can be activated by Gα_q. In the crystal structure of the Gα_q-p63-RhoGEF complex, parts of the α2 and α3 helices from the DH domain also make contact with the Gα subunit (32), but in this case, the segments in Gα_q that interact with DH come from the Ras-like domain of Gα_q, rather than its helical domain. Nevertheless, interactions between the DH domains of RhoGEFs and their regulatory proteins might be a common mechanism for regulation.

The proposed binding of the p115 DH domain to the helical domain of Gα₁₃ defines a new effector interface for heterotrimeric G proteins and the potential to define the contribution of direct activation of the GEF activity of p115 in physiological function. All heterotrimeric G protein α subunits possess two domains: a Ras-like domain that makes direct contacts with regulators and effectors and a less well functionally characterized helical domain. Thus far, the only protein-protein interactions that have been ascribed to the helical domain are between Gα_i and the GoLoco motif of RGS14 (33) and between Gα₁₃ and the RH domain of RGS-RhoGEFs (13, 14). The α3b helix in the DH domain of p115, which includes Trp⁵⁰⁷, forms a protruding bulge on the surface of DH that is predicted to interact with the helical domain of Gα₁₃ in our models (Fig. 4). One of the potential binding sites for the DH domain of p115 within the helical domain of Gα₁₃ includes the αB-αC helices, because these surfaces are in close proximity in the low resolution model of the p115-Gα₁₃ complex derived from SAXS (Figs. 4C and 6). The hydrophobic pocket in the helical domain is unique to Gα₁₃. As described in previous studies (14), a notable feature of the helical domain of Gα₁₃ that is not present in other known Gα structures is the “helical insert” (αB1) between the αB and αC helices, which forms part of this hydrophobic pocket (Fig. 6, B–D). In Gα₁₂, the other G protein that interacts with the RGS-RhoGEFs, the loop connecting the αB and αC helices is shortened by four amino acids (Fig. 6C). Comparison of the structures of Gα₁₃ and Gα₁₂ (34) reveals that αB1 is not present in Gα₁₂. The GEF activity of p115 can be stimulated by activated Gα₁₃, but not Gα₁₂, in vitro (7). The lack of the αB1 helix or an intact hydrophobic pocket in the helical domain of Gα₁₂ might explain this observation. The GEF activity of p115 can be stimulated by activation of Gα₁₂ in vivo. However, this activation is likely due to the localization of p115 to the plasma membrane where free RhoA is located. The conformation of the helical domain is not significantly different between the GDP- and the GTP-bound forms of G protein α subunits. Therefore it is anticipated that any specific regulation of effectors by this region would require coincident interactions of effector molecules with the conformationally flexible switch regions within the Ras-like domain as observed here. This could represent a common mechanism by which RGS domains in effector proteins could both regulate turnover of the G proteins and provide assistance to other sites that regulate effector activities.