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

Nicole Hajicek,Mutsuko Kukimoto-Niino,Chiemi Mishima-Tsumagari,Christina R Chow,Mikako Shirouzu,Takaho Terada,Maulik Patel,Shigeyuki Yokoyama,Tohru Kozasa

doi:10.1074/jbc.m110.201392

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 Gα(13) stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα(13) stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα(13) that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα(13) to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα(13) and not through its interaction with a secondary binding site. A crystal structure of Gα(13) bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα(13)-Gα(i1) chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα(13).

Highlights

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-cou
Previous studies have suggested that the region of G␣13 responsible for stimulating the guanine nucleotide exchange activity of p115RhoGEF is located C-terminal to the three switch regions [23, 32]
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

Summary

Generation of Constructs

G␣13 Mutants—Single point mutations were introduced into the mammalian expression vector pCMV5 harboring murine G␣13 Q226L for use in SRE-luciferase assays or the baculovirus transfer vector pFastBac HTa (Invitrogen) encoding G␣i/13 for protein expression using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s protocol. Full-length G␣13—Full-length G␣13 (G␣13 FL) was generated by first introducing a silent mutation into pCMV5-G␣13 by QuikChange site-directed mutagenesis that eliminates an internal HindIII site using the primers 5Ј-GCCCGAGAGAAGCTCCATATTCCCTGGGG and 5Ј-CCCCAGGG-. KpnI and HindIII sites were introduced at the 5Ј- and 3Ј-ends, respectively, of G␣13 by PCR using pCMV5-G␣13 lacking the internal HindIII site as the template and the primers 5Ј-GGTACCGCGGACTTCCTGCCGTC and 5Ј-GACCA-. The PCR products were digested with KpnI and HindIII and ligated into the pFastBac HT(Ϫ) vector containing the N-terminal ␣1 helix of G␣i1 such that the construct contains, from the N terminus, a His tag, residues 1–28 of G␣i1, a TEV protease site, and residues 2–377 of G␣13. P115RhoGEF⌬C—Tyr763 of p115RhoGEF was mutated to a stop codon using QuikChange site-directed mutagenesis using pFastBac HTc-p115RhoGEF as the template and the primers 5Ј-CACTGAGACTGCCGGATAACTGAAAGTCCCTGCCC and 5Ј-GGGCAGGGACTTTCAGTTATCCGGCAGTCTC-.

Protein Purification

Biochemical Experiments

Crystallization and Data Collection

RESULTS

Data collection Space group Unit cell parameters

DISCUSSION