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 G␣ 13 stimulates the guanine nucleotide exchange factor (GEF) activity of RHRhoGEFs, leading to activation of RhoA. The mechanism by which G␣ 13 stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in G␣ 13 that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of G␣ 13 to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by G␣ 13 and not through its interaction with a secondary binding site. A crystal structure of G␣ 13 bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a G␣ 13 -G␣ i1 chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by G␣ 13 .Heterotrimeric guanine nucleotide binding proteins (G proteins), composed of ␣, ␤, and ␥ 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 G␣ subunit dissociates from the G␤␥ dimer, both of which regulate the activity of multiple intracellular effectors to elicit cellular responses. The duration of signaling mediated by G␣ is dictated by the lifetime of bound GTP. All G␣ 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 G␣ 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, G␣ subunits reassociate with G␤␥ and are capable of initiating another round of signaling.G␣ 13 is one of two members of the G 12 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 G␣ 13 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 (RHRhoGEFs) (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 G␣ 13 (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 nucleoti...

Phosphorylation-mediated regulation of GEFs for RhoA

“…Structural and biochemical experiments have established that GTP-bound Gα 12/13 subunits make direct contacts with the RH-domain, and that this interaction positively regulates RhoGEF activity. [34][35][36][37][38] It has also been shown that the RH domain of p115 and LARG also can function as negative regulator of Gα 12/13 by increasing its intrinsic hydrolysis rate of GTP to GDP. 33,37,39 While interactions of RH-RhoGEFs with Gα 12/13 is believed to be the primary and the most well-characterized mode of regulation, several reports also indicate that additional control could be provided by phosphorylation of tyrosine residues.…”

Plasma Membrane Association of p63 Rho Guanine Nucleotide Exchange Factor (p63RhoGEF) Is Mediated by Palmitoylation and Is Required for Basal Activity in Cells

“…In resting cells, RH-RhoGEFs are distributed throughout the cytoplasm of unstimulated cells and translocate to the PM upon activation of G␣ 12 or G␣ 13 subunits (19 -21). Direct activation of these RhoGEFs has been difficult to demonstrate in vitro with the exception of p115RhoGEF (22), suggesting that the dominant mechanism by which G␣ 12/13 subunits activate RHRhoGEFs is via membrane recruitment.…”

Gastrin-stimulated Gα13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells

“…6). Alignment is based upon the crystal structure of the RH domain of p115GEF (41,42). Despite low 32% sequence identities shared between the RH domains of p115RhoGEF and PDZRhoGEF, these domains share nearly identical three-dimensional structures, a root mean square deviation of 0.608 Å on C␣ values (41,42).…”

“…Next, we aligned the C-terminal portion of Rgnef (entries P97433, Q8N1W1, and P0C6P5) to the RH domains of the p115RhoGEF family. Relying on this alignment, we constructed a computational model of the Rgnef RH-like domain bound to G␣ 13 using Modeler (40) based on the homology to the RH-like domain of ArhGEF11, for which the structure in complex with G␣ 13 (Protein Data Bank code 3CX8) is available (41,42). The molecular complex was refined using energy minimization with AMBER 12.0 employing the Amberff99SB-ILDN force field (43).…”