Chimeric G (cid:1) i2 /G (cid:1) 13 Proteins Reveal the Structural Requirements for the Binding and Activation of the RGS-like (RGL)-containing Rho Guanine Nucleotide Exchange Factors (GEFs) by G (cid:1) 13 *

The (cid:1) -subunit of G proteins of the G 12/13 family stimu- late Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of G (cid:1) 12/13 to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between G (cid:1) 13 and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of G (cid:1) i proteins. Using G (cid:1) i2 , which does not bind RGL domains, as the backbone in which G (cid:1) 13 sequences were swapped or mutated, we observed that the switch region of G (cid:1) 13 is strictly necessary to bind

Rho GTPases, which include Rho, Rac, and Cdc42, play a central role in the regulation of a number of basic cellular events such as cell movement and changes in cell shape, as well as in the control of gene expression regulation and cell growth (1). These GTP-binding proteins act as molecular switches that are inactive in their GDP-bound form, and upon exchange of GDP for GTP, they adopt an active conformation in which they can interact with their specific effector molecules, thereby affecting their localization and/or activity (1)(2)(3). This nucleotide exchange is promoted by a large family of guanine nucleotide exchange factors (GEFs), 1 the vast majority of which are characterized by the presence of a dbl-homology (DH) and pleckstrin homology (PH) domain (2,4). These GEFs also exhibit a number of additional regulatory regions by which they are strictly controlled by a diverse array of upstream signaling pathways, including those initiated by cell adhesion molecules, tyrosine kinase growth factor receptors, as well as by G proteincoupled receptors (GPCRs) (2,5).
In particular for Rho, this GTPase participates in many physiological and pathological processes that involve the activation of GPCRs. For example, GPCRs such as those for thrombin and lysophosphatidic acid (LPA) promote cytoskeletal changes and expression from serum responsive element (SRE)regulated genes by activating Rho (6,7). Rho also participates in platelet aggregation (8) and in smooth muscle contraction when elicited by a large number of vasoactive hormones that act on GPCRs (9). The pathway by which these GPCRs stimulate Rho involves the activation of ␣-subunits of the G 12/13 and G q family of heterotrimeric G proteins. G␣ 12/13 in turn stimulate Rho through the direct interaction with a group of Rho GEFs characterized by the presence of a RGS-like (RGL) domain (10 -12), whereas G␣ q activates Rho through a still not fully understood mechanism (13,14).
The family of RGL-containing Rho GEFs comprises three members: PDZ-RhoGEF (PRG) and LARG, which contain an N-terminal PDZ domain, and p115-RhoGEF (p115), which lacks this N-terminal protein-protein interaction domain (10 -12). The PDZ domain of PRG and LARG mediates the interaction of these GEFs with membrane receptors including plexins of the B family and insulin-like growth factor receptor (15)(16)(17)(18). The RGL domain is followed by DH and PH homology domains, by which they promote the nucleotide exchange on Rho, and a long C-terminal domain that harbors regulatory properties (19 -21). The RGL domain is directly recognized by receptorstimulated G␣ 12/13 , thus providing a molecular bridge for the activation of Rho by G␣ 12/13 (19), and in the case of p115, this domain also acts as a GTPase-activating protein (GAP) for G␣ 13 (22). However, this GAP activity is not required to couple G 13 to Rho activation, as p115 mutants that possess a reduced GAP activity toward G␣ 13 and a decreased binding ability for this ␣-subunit, still exhibit a normal ability to stimulate Rho exchange when activated by G␣ 13 (23).
The G␣ subunit is composed by a ␣-helical domain and a GTPase domain, which includes a switch region that changes conformation upon nucleotide exchange, and is directly involved in binding and hydrolysis of GTP (24,25). In the inactive heterotrimer the G␣ subunit, with GDP bound to it, keeps stable interactions with the heterodimer G␤␥. Therefore, the effector motifs are covered in the heterotrimer, rendering both G␣ and G␤␥ unable to activate their respective effectors. Agonists acting on GPCRs induce a structural change that results in the nucleotide exchange and the dissociation of the heterotrimer, allowing the activation of G␣-and G␤␥-dependent effectors. Signaling is terminated by the hydrolysis of GTP to GDP by virtue of the intrinsic GTPase activity of G␣ subunits, which is further stimulated by the interaction with regulators of G protein signaling (RGS proteins).
In this regard, the crystal structure of the complex formed by RGS4-G␣ i (26), and that of the RGL domains of PRG and p115 (27,28), have provided a model for the likely mechanism by which the activity of G␣ subunits is terminated, as well as the process by which G␣ 13 and RGL-RhoGEFs might establish a functional interaction. Regarding the latter, in the proposed model the region including switch 1 and switch 2 within the GTPase domain of G␣ 12/13 , would be predicted to participate in the interactions with the RGL domain from RGL-RhoGEFs, whereas the N-terminal ␣-helical domain from G␣ 12/13 would not establish direct contact interactions with them. As no structural information is yet available for G␣ 12/13 members, we have addressed in this study the structural requirements for G␣ 13dependent stimulation of Rho by engineering chimeric G proteins using G␣ i2 , which does not activate RGL-RhoGEFs, as the backbone in which G␣ 13 sequences were swapped or mutated. The emerging results revealed that the entire switch region from G␣ 13 is necessary but insufficient to exert a Rho-stimulating activity when expressed in the context of G␣ i2 . In fact, G␣ 13 depends on most of its GTPase domain, excluding its C-terminal 36 amino acids, for a functional interaction with PRG. Within this region, the integrity of both switch 1 and switch 2 is strictly required for a maximal effect. Furthermore, membrane localization of G␣ 13 or the Rho-activating chimeric G␣ i2 subunits is also necessary for Rho activation, gene expression, and cell transformation. These findings indicate that specific structural features present in G␣ 13 together with additional molecular interactions occuring at the level of the plasma membrane are required for the effective coupling of G␣ 13 to the RGL-containing family of RhoGEFs, and ultimately to stimulate Rho.

MATERIALS AND METHODS
Bioinformatic Tools-The structure of G␣ i1 /RGS4 (26) was analyzed with the CN3D program (www.ncbi.nlm.nih.gov/Structure/CN3D/ cn3d.shtml) to identify amino acids in the G␣ i -RGS4-contact interface within 3 Å. Sequence alignment corresponding to the switch region from representative members of each G␣ protein family was performed using ClustalW (www.ch.embnet.org/software/ClustalW.html) and the figure prepared with Boxshade (www.ch.embnet.org/software/BOX_form.html).
DNA Constructs-The cDNAs for G␣ i2 SW␣ 13 QL and G␣ 13 SW␣ i2 QL chimeras were obtained by two consecutive PCR reactions, using human G␣ i2 -and human G␣ 13 GTPase-deficient mutants as templates, and cloned into the mammalian expression vector pCEFL2 by EcoRI and XbaI restriction sites that were introduced with the 5Ј-and 3Јprimers, respectively. The cDNAs corresponding to the N-terminal, switch, and C-terminal regions were first obtained by PCR in which the internal primers were designed to overlap with the sequence of the cDNA of the fragments to be fused with in the second PCR reaction. The corresponding chimeric cDNAs were obtained by a second PCR reaction in which the three initial fragments were mixed as templates. The second set of chimeras in which the content of G␣ 13 was extended from the switch region toward the extremes was also obtained by PCR, in this case the template for the second PCR was the mixture of two cDNA fragments corresponding to either N-or C-terminal domains, as indicated in the respective figures. From this second series, the chimera in which the content of G␣ 13 extended from the switch region toward the C-terminal end was used as template for further modifications, which included reduction of the contribution of G␣ 13 at the C-terminal end by substitution with the corresponding sequences from G␣ i2 ; and point mutations at the putative RGL-contact sites within the switch region and at the N-terminal myristylation signal. Point mutations were performed either by PCR to mutate the N-terminal myristylation signal by substituting Gly for Ala in the second codon, and by the QuikChange mutagenesis kit from Stratagene to substitute the putative RGL contact sites, following the manufacturer's instructions. All chimeric and mutant molecules were sequenced at the NIDCR DNA sequencing facility, and their expression was confirmed by Western blot using antibodies detecting the corresponding N-terminal domain. The sequence for the different primers will be made available upon request.
Cell Lines and Transfections-Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum. For transient transfections, tissue culture plates were treated for 10 min with phosphate-buffered saline containing 5 g/ml poly-D-lysine before seeding the cells to prevent them from detaching from the plates during the transfection procedure and thereafter. Transient transfections in HEK 293T cells were performed using the Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. NIH 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) calf serum, and were used to monitor the transforming potential of the different chimeras as described (7).
Western Blots and Protein-Protein Interactions-Transfected cells were lysed at 4°C in a buffer containing 50 mM Tris, pH 7.4, 0.15 M NaCl, 1% Triton X-100, 20 mM ␤-glycerophosphate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 10 g/ml leupeptin, and insoluble material was removed by centrifugation. Lysates containing ϳ50 g of total cellular protein or affinity isolated proteins (see below) were analyzed by Western blotting after SDSpolyacrylamide gel electrophoresis and visualized by enhanced chemiluminescence detection (Amersham Biosciences) using rabbit anti-G␣ 13 (SC-410, Santa Cruz Biotechnology) or rabbit anti-G␣ i2 (SC-7276, Santa Cruz Biotechnology) depending on whether the N-terminal domain of the transfected G protein chimeras was from G␣ i2 or G␣ 13 and goat anti-rabbit (Cappel) IgGs coupled to horseradish peroxidase as a secondary antibody. To test the ability of the different chimeras to interact with RGL domain from PRG, lysates from HEK 293T transfected cells were incubated with bacterially expressed six histidine-tagged recombinant RGL isolated with talon beads (Clontech) as previously described (27).
Luciferase Assays-Cells in 24-well plates were transfected with different expression plasmids together with 0.1 g of pSRE luciferase reporter plasmid, pNull Renilla, and pcDNAIII-␤-gal (a plasmid expressing ␤-galactosidase) to normalize for transfection efficiency. Firefly and Renilla luciferase activities present in cell lysates were assayed using a dual-luciferase reporter system (Promega), and light emission was quantitated using a Monolight 2010 luminometer (Analytical Luminescence Laboratory) as specified by the manufacturer (39).
In Vivo Rho Activation Assay-HEK 293T cells were transfected using the Lipofectamine Plus TM reagent. The day after transfection, cells were cultured for 24 h in serum-free Dulbecco's modified Eagle's medium and assayed for Rho activity using the Rho-binding domain (RBD) of rhotekin bound to glutathione-Sepharose beads to isolate the GTP-bound forms of Rho, as previously described (14). Briefly, serumstarved HEK-293T cells transfected with the indicated plasmids were lysed at 4°C in a buffer containing 20 mM HEPES, pH 7.4, 0.1 M NaCl, 1% Triton X-100, 10 mM EGTA, 40 mM ␤-glycerophosphate, 20 mM MgCl 2 , 1 mM Na 3 VO 4 , 1 mM dithiothreitol, 10 g/ml aprotinin, 10 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were incubated with GST-rhotekin-RBD previously bound to glutathione-Sepharose beads and washed four times with lysis buffer, and associated GTP-bound forms of Rho were released with protein loading buffer and revealed by Western blot using a monoclonal antibody against RhoA (26C4, Santa Cruz Biotechnology). The content of Rho in total cell lysates was determined as a reference.
Cell Fractionation-HEK 293T cells in 10-cm dishes were transfected with the indicated chimeras and grown for 48 h. Cells were washed once with phosphate-buffered saline and then dislodged from the plate by washing and pelleted at low speed. The cell pellet was suspended in 0.5 ml of lysis buffer (50 mM Tris-HCl, pH 8, 2.5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin), and cells were lysed by 10 passages through a 27-gauge needle. Lysed cells were centrifuged at 2500 rpm for 5 min at 4°C to remove nuclei and intact cells. The supernatant was centrifuged at 14,000 rpm for 60 min at 4°C, and the pellet (particulate fraction) was suspended in an equal volume of lysis buffer. The supernatant was further centrifuged at 100,000 ϫ g for 5 min in a Beckman airfuge to obtain the soluble fraction and mixed with Laemmli sample buffer. Fractions were analyzed by Western blotting using anti-G␣ i2 antibody as indicated before.
Focus Forming Assays-NIH 3T3 cells were transfected by the calcium phosphate precipitation technique with different expression plasmids together with 1 g of pcDNAIII-␤-gal, a plasmid expressing the enzyme ␤-galactosidase, adjusting the total amount of plasmid DNA with empty vector. The day after transfection, the cells were washed in medium supplemented with 5% calf serum and then maintained in the same medium until foci were scored, 2-3 weeks later. Duplicate plates were fixed with phosphate-buffered saline containing 2% (v/v) formaldehyde and 0.2% (v/v) glutaraldehyde and stained at 37°C for ␤-galactosidase activity with a phosphate-buffered saline solution containing 2 mM MgCl 2 , 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , and 0.1% 5-bromo-4chloro-3-indolyl-␤-D-galactopyranoside (X-gal) to evaluate the transfection efficiency.

RESULTS
The Switch Region of G␣ 13 Is Required but Not Sufficient for Rho Activation-The structure of the RGL domain from two of the three known members of G␣ 12/13 -responsive RhoGEFs has recently been solved (27,28). The similarity of the structures of these RGL domains with the known RGS structures raised the possibility that the functional interaction between G␣ 12/13 and RGL-RhoGEFs might be similar to that of the complex formed by G␣ i and RGS4 shown in Fig. 1A (26). Residues in G␣ i that provide surface areas in direct contact with RGS4 are indicated. In particular, this region can be subdivided into 3 switch areas, which are highly conserved among each G protein ␣-subunit family. Of interest, all 6 residues involved in the interaction between G␣ i and RGS4 are also conserved in G␣ s and G␣ q , but 3 of them are quite distinct in G␣ 12 and G␣ 13 , as depicted in Fig. 1B. Nonetheless, as no information regarding the threedimensional characteristics of G␣ 13 family members is available, the molecular basis of their interaction with RGL-Rho-GEFs and the consequent activation of Rho remain to be determined. To begin addressing this issue, we first evaluated the possibility that the switch region from G␣ 13 contains all the structural elements required to elicit Rho activation. For this purpose, we engineered G protein ␣-subunits in which the switch region from active G␣ 13 , G␣ 13 QL, replaced the corresponding region from active G␣ i2 , G␣ i2 QL (Fig. 1B). The resulting chimera, named G␣ i2 SW␣ 13 QL, contains the first 173 amino acids (Met 1 -Gln 173 ) from G␣ i2 followed by a central part (Asp 174 -Ile 266 ), including de switch region from G␣ 13 QL, and the C-terminal (Leu 267 -Phe 355 ) from G␣ i2 , as indicated. The reciprocal chimera, G␣ 13  and the C terminus (Leu 288 -Gln 377 ) from G␣ 13 . The activity of these chimeras was determined by their ability to stimulate a mutant SRE that responds to Rho (7), and by their ability to interact with the RGL domain of PRG and to stimulate Rho. The different chimeric G proteins were detected by antibodies recognizing the N-terminal domain of either G␣ i2 or G␣ 13 (Fig.  1C). As expected, a GTPase-deficient G␣ 13 strongly stimulated Rho-dependent pathways, while active G␣ i2 was unable to do so. However, surprisingly, a chimeric G␣ i2 containing what was expected to be the critical region for the interaction with RGL-RhoGEFs, did not gain the ability to stimulate Rho or to interact with PRG (G␣ i2 SW␣ 13 QL, Fig. 1C). G␣ 13 , on the other hand, lost these properties when its switch region was replaced by that of G␣ i2 (G␣ 13 SW␣ i2 QL, Fig. 1C). Direct evaluation of Rho-GTP content in transfected HEK-293T cells indicated that swapping the switches from G␣ 13 into G␣ i2 provided to the resulting chimera only a very limited ability to stimulate Rho, while the reciprocal chimera was unable to do so (Fig. 1C, lower panel).
The Structural Requirements for Rho Activation by G␣ 13 Extend toward the C-terminal Domain of G␣ 13 -As swapping the switch region from G␣ 13 QL into G␣ i2 QL was not sufficient to confer to G␣ i2 the ability to stimulate Rho, we predicted that additional structural elements from G␣ 13 were required to support this effect. In order to test this possibility, we engineered additional chimeras where the contribution from G␣ 13 QL into the G␣ i2 backbone was extended from the switch region toward their N or C termini (Fig. 2, upper panel). Chimeras in which only the N-or C-terminal domains were swapped were also tested as controls. The expression of each chimera was confirmed by Western blotting (Fig. 2, lower  panel). By this approach, we found that a chimeric G protein (G␣ i2 SW-C␣ 13 QL), that includes the switch region and the C-terminal end, was able to stimulate Rho-dependent luciferase reporter gene comparable to that of the GTPase-deficient G␣ 13 QL (Fig. 2, lower panel).
The Active G␣ i2 SW-C␣ 13 QL Chimera Requires the Integrity of the Switch Region and an Adjacent C-terminal Extension-To narrow down the minimal structural requirements for G␣ 13 QL to stimulate Rho, the active chimera (G␣ i2 SW-C␣ 13 QL) was further modified by replacing additional sequences for those from G␣ i2 corresponding to each of the switch subregions, or the C-terminal domain, as indicated in Fig. 3, upper panel. The expression of this set of chimeras was demonstrated by Western blot (Fig. 3, lower panel). When either switch 1 or switch 1 and 2 in G␣ i2 SW-C␣ 13 QL were substituted by those from G␣ i2 , the resulting chimeras, G␣ i2 SW2,3-C␣ 13 QL and G␣ i2 QLSW3-C␣ 13 , respectively, were unable to stimulate Rhodependent pathways (Fig. 3, lower panel). On the other hand, the contribution from G␣ 13 at the C-terminal end of G␣ i2 SW-C␣ 13 QL was narrowed down by substituting it for sequences from G␣ i2 as indicated in Fig. 3, upper panel. Based on this approach, we observed that the last 36 amino acids in G␣ i2 SW-C␣ 13 QL (Pro 320 -Gln 356 ) could be replaced with those from G␣ i2 with no reduction in the Rho-stimulating activity of the resulting chimera, G␣ i2 SW-320␣ 13 QL, whereas a further reduction, replacing the last 61 amino-acids (Pro 295 -Gln 356 ), resulted in a chimeric G protein, G␣ i2 SW-295␣ 13 QL, that was unable to activate Rho. G␣ i2 SW-320␣ 13 QL was therefore referred to herein as the "minimal active chimera" (Fig. 3, lower panel).
Point Mutations on the Putative RGL Contact Sites within Switch 1 and 2 of the Minimal Active Chimera G␣ i2 SW-320␣ 13 QL Reduce Its Activity-Based on the structure shown in Fig. 1A (26), amino acids from G␣ i2 switch 1 and switch 2 region that were predicted to make contact with RGS4 were re-introduced by site-directed mutagenesis into the equivalent positions in G␣ i2 SW-320␣ 13 QL (Fig. 4, upper panel) (SW1 and SW2 mutants). The expression of these mutants was demonstrated by Western blot (Fig. 4, lower panel). Furthermore, as shown in Fig. 4, lower panel, G␣ i2 SW-320␣ 13 QL harboring mutations in the putative RGL contact sites in switch 1 or switch 2 exhibited a reduced ability to stimulate the SRE, which was nearly half of that of G␣ i2 SW-320␣ 13 QL, while the double mutant showed no activity. The ability of these chimeras to interact with PRG and to stimulate the increase in GTP-Rho was aligned with their activity detected in the luciferase reporter assay as shown in Fig. 4

, lower panel. An N-terminal Lipid Modification in the G␣ i2 SW-320␣ 13 QL Chimera Is Not Required for Binding to PRG, but Is Necessary for a Functional Interaction Leading to the Activation of Rho-
The minimal G␣ i2 SW-320␣ 13 QL active chimera possesses a G␣ i2 N-terminal region that exhibits a myristylation signal instead of the palmitylation signal known to be present in G␣ 13 (29). Thus, we tested if this myristylation signal had any influence on the ability of this chimera to interact with PRG. A G␣ i2 SW-320␣ 13 QL mutant chimera (G␣ i2 G2A-SW-320␣ 13 QL), in which the myristylation signal was mutated, was well expressed (Fig. 5B, lower panel) but exhibited a strongly reduced ability to interact with the plasma membrane as judged by membrane fractionation (Fig. 5A, lower panel). Interestingly, this mutant was still able to interact with PRG, supporting that a stable interaction between G␣ 13 and PRG does not require additional, membrane-derived elements. However, neither this mutant G␣ i2 chimera nor a mutant G␣ 13 lacking palmitylation sites stimulated the accumulation of GTP-Rho (Fig. 5B, lower panel and data not shown). Thus, the interaction between G␣ 13 and PRG is required but not sufficient for the activation of Rho. Indeed, membrane association or the subsequent interaction with membrane-associated molecules might be also required to induce the GEF activity of the G␣ 13 -RGL-containing GEF complex.
G␣ i2 SW-320␣ 13 QL Induces Cell Transformation-In order to examine whether the structural features providing the ability to activate Rho were related to those determining the transforming potential of G␣ 13 we tested the focus-forming activity of the chimeras and point mutants derived from the minimal active chimera to induce focus formation. As shown in Fig. 6, the efficiency of G␣ i2 SW-320␣ 13 QL and its mutants to induce transformation of NIH 3T3 fibroblasts correlated nicely with its ability to stimulate Rho (see above). On the other hand, as expected, the myristylation-deficient G␣ i2 G2A-SW-320␣ 13 QL chimera was not transforming (results not shown). DISCUSSION In this report we have investigated the nature of the structural elements required for the functional interaction between G␣ 13 and RGL-containing RhoGEFs. We took advantage of the fact that members of the G␣ i family of heterotrimeric G protein subunits do not bind to PRG and cannot stimulate Rho and its downstream pathways to use the primary sequence of G␣ i2 as the backbone in which G␣ 13 sequences were swapped. Our findings indicate that the switch region within the GTPase domain of G␣ 13 is necessary for the activation of Rho, and within this  Fig. 1B were substituted in G␣ i2 -SW-320␣ 13 QL chimera by introduction of the amino acids from G␣ i2 present at the equivalent position. SW1 mutant corresponds to G␣ i2 -SW-320␣ 13 QL in which PTK was replaced for KTT at the corresponding positions; the amino acids in bold correspond to those numbered as 1 and 2 in the alignment in Fig. 1B. SW2 mutant corresponds to G␣ i2 -SW-320␣ 13 QL in which RWFE in switch 2 was replaced for KWFH. In this case the amino acids in bold correspond to those identified with numbers 4 and 5 in Fig. 1B. SW1ϩSW2 chimera corresponds to G␣ i2 -SW-320␣ 13 QL with all the point mutations indicated for SW1 and SW2. The graph in the middle panel shows the luciferase reporter for Rhodependent SRE activity of HEK 293T cells transfected with the indicated chimeras. The expression and ability of different GTPase-deficient G␣ i2 , G␣ 13 and chimeric G proteins to interact with the RGL domain from PRG was determined by Western blot analysis in total cell extracts (TCE) or after affinity purification with the RGL domain of PRG (AP RGL), using antibodies against the N-terminal domain of either G␣ 13 or G␣ i2 , as indicated on the left. Below, their ability to promote the activation of endogenous RhoA (AP GST) is shown. As a reference, the content of RhoA in total cell extracts (TCE) is shown at the bottom panel. Control cells were transfected with empty vector. region, two residues in switch 1 and switch 2 were identified that are strictly required for this function. However, neither these residues, which act in an additive fashion, nor the entire switch region are alone sufficient to exert a Rho-stimulating activity when introduced into G␣ i2 . Indeed, additional structural elements located toward the C terminus of G␣ 13 are required to bind PRG, whereas lipid modification, and hence membrane localization of G␣ 13 or chimeric G␣ i2 subunits, is necessary for Rhoactivation upon binding to RGL-containing GEFs.
The crystal structure of the complexes formed by RGS4 with G␣ i1 (26) and RGS9 with G␣ t (30) revealed that switch 1 and switch 2 within the G␣-GTPase domain harbors critical interacting residues at the interface between the G␣ subunit and the RGS protein. Considering the structural similarity between the recently reported structures of the RGL domains from PRG and p115 and the RGS domain of RGS4 and RGS9, it would be expected that G␣ 13 might use similar elements to bind RGL-containing GEFs as those employed by G␣ i1 or G␣ t for their respective complexes with these RGS proteins (27,28). If so, the replacement of the three switches in G␣ 13 QL for those of G␣ i2 QL would be expected to result in a G protein ␣-subunit incapable of activating Rho. Conversely, the presence of the switch region from G␣ 13 QL would be expected to confer G␣ i2 QL the ability to stimulate Rho. Our results indicate that the first premise is correct while the second is not, as additional elements toward the C-terminal end of the GTPase domain of G␣ 13 are required to activate Rho and its downstream pathways.
Regarding the contribution of the G␣ 13 switch region, the structure of RGS4 bound to G␣ i1 revealed that threonine 182, present in switch 1 of G␣ i1 , exhibits the strongest interaction with this RGS (26). This residue is conserved among all G protein ␣-subunits but in G␣ 12 and G␣ 13 , which exhibit a lysine in this particular position. Similarly, histidine 213 in switch 2 of G␣ i1 is part of the contact surface with RGS4, and this residue is also conserved among all G protein ␣-subunits with the exemption of G␣ 12/13 . To analyze whether these residues in G␣ 13 participate in binding to RGL-RhoGEFs, we mutated them together with adjacent non-conserved residues for those corresponding to G␣ i2 in the minimal fully functional G␣ i2 -G␣ 13 chimera. When these putative contact sites in either switch 1 or switch 2 were mutated, a reduced ability to stimulate Rho was observed that was abolished when mutations in both switch regions were introduced simultaneously. These findings indicated that these residues in switch 1 and switch 2 are strictly required to interact with RGL-containing GEFs, and are likely to contribute to the contact interface between G␣ 12/13 and the RGL-like domain in an additive fashion. This observation may help explain why ␣-subunits of the G q , G i , and G s G protein family fail to bind PRG and other RGL-containing GEFs (11,12), and, conversely, why G␣ 12 and G␣ 13 may not bind to and interact with RGS4 and other related RGSs (26, 31). Thus, these particular residues within switch 1 and 2 may determine the choice of effector molecules and GAPs available to each G protein family, a key event for achieving signal specificity upon GPCR activation.
On the other hand, the need of sequences in addition to the switch region from G␣ 13 to stimulate Rho, suggests that either the overall structure of the GTPase domain of G␣ 13 may differ from that of G␣ i , and thus its C-terminal region may be required for the correct spatial positioning of the residues present in the switch region, or that the overall structures of G␣ i and G␣ 13 might be indeed similar, but that additional interactions may exist beyond those involving the switch region of G␣ 13 that may be required for the functional coupling of this G␣ subunit with RGL-RhoGEFs. Ongoing attempts to crystallize these chimeric G proteins bound to the RGL domain may ultimately help elucidate the nature of the molecular determinants of the active G␣ 13 conformation.
Ultimately, how binding to G␣ 13 results in the activation of RGL-containing GEFs and the subsequent Rho stimulation is at the present unknown. In prior studies, we have not observed any inter-or intramolecular interactions between the RGL domain and the DH-PH cassette or any other region in PRG, LARG, and p115 (20). Furthermore, removal of the RGL domain has only a limited stimulatory effect (12), suggesting that the RGL domain is unlikely to act as a negative regulatory region whose inhibitory activity is relieved upon binding to G␣ 12/13 . Instead, the observation that membrane localization of G␣ 13 (29) and the minimal active G␣ i2 -G␣ 13 chimera is required to stimulate Rho activation, suggest that the binding of G␣ 12/13 to the RGL-domain is not sufficient to activate RGL-containing GEFs, but that the recruitment of this GEF family to the membrane is a major determinant of the ability of G␣ 12/13 to stimulate Rho. Indeed, whereas the N-terminal ␣-helical domain does not provide structural elements for the activation of Rho, the lipid modification is needed to bring the chimeric G␣ i2 -G␣ 13 G protein to the membrane in order to be active. In this regard, the fact that it does not appear to exist a preferential lipid modification on the G␣ protein, as myristylation of G␣ i2 (32,33) can functionally replace the palmitylation of G␣ 12/13 to induce cell transformation (34) and to stimulate Rho, and each target preferentially a distinct membrane compartment (35,36), this process is unlikely to involve the interaction of RGL-containing GEFs with additional molecules exhibiting restricted membrane subdomain distribution. Instead, membrane recruitment through the RGL-domain may enhance the local concentration of these RhoGEFs at the inner face of the plasma membrane. These GEFs may then interact through other regions with membrane components, such as membrane phospholipids through their PH domain, thus either causing a conformational change in the DH domain that then becomes activated, or adopting an orientation with respect to the lipid bilayer that facilitates the interaction of the DH domain with membrane-bound Rho. This membrane interaction might be also required for the regulatory properties of tyrosine or serine/threonine protein kinases that modulate RGL-RhoGEFs, such as focal adhesion kinase and PAK4 (21,37), respectively. These, as well as other possibilities are under current investigation.
In conclusion, our results indicate that the structural requirements for the functional interaction between G␣ 13 and RGL-RhoGEFs involve a large fraction of the GTPase domain of G␣ 13 that includes the switch region, and in particular two conserved residues within switch 1 and 2 that act in an additive fashion, a C-terminal extension, and the N-terminal domain of G␣ 13 , which does not participate in binding to these GEFs but is strictly required to stimulate Rho and cell transformation FIG. 6. The minimal active G␣ i2 -SW-320␣ 13 QL chimera requires the integrity of the putative RGL-contact sites for G␣ 13 -dependent full transforming activity. A, representative experiment showing the transforming activity of GTPase-deficient G␣ i2 , G␣ 13 , RhoA, G␣ i2 -SW-295␣ 13 QL, G␣ i2 -SW-320␣ 13 QL,and different variants of the last one with point mutations at the putative RGL contact sites (schematic representation of these chimeras is shown in Figs. 3 and 4), which was determined by transfecting them into NIH3T3 fibroblasts and monitoring the foci formation after several weeks in culture as described under "Materials and Methods." The control included cells transfected with a plasmid expressing the enzyme ␤-galactosidase. B, graph represents the transforming activity of GTPase-deficient RhoA, G␣ i2 , G␣ 13 , and the indicated chimeric G proteins, reported as the mean Ϯ S.E. of three independent experiments. due to its membrane targeting lipid modification. As increasing interest on the structural characteristics of RGS-G protein interactions has emerged from the potential use of small molecule inhibitors that may block or regulate such interactions (38), we can envision that the present findings defining the structural determinants required for the coupling of G␣ 13 to RGL-RhoGEFs and Rho activation may facilitate the identification of novel therapeutic approaches for the treatment of the numerous human diseases that are dependent on the dysfunction of GPCRs that act on G␣ 12/13 to stimulate Rho.