Characterization of GIV‐Gαi3, a Non‐Receptor GEF‐G protein complex involved in cell migration and metastasis

G‐protein coupled receptors (GPCRs) are the canonical guanine nucleotide exchange factors (GEFs) that activate trimeric G proteins. In contrast to GPCRs, little is known about the structure or function of non‐receptor GEFs. We previously reported that GIV/Girdin is a non‐receptor GEF for Gαi‐subunits that promotes cell migration and tumor cell metastasis (PNAS, 2009, 106: 3178–83). We discovered an evolutionarily conserved GEF motif in GIV that forms a unique interface with the Gα‐subunit. Mutation of a key residue (F1685) within this motif disrupted the Gαi‐GIV interaction and made GIV GEF‐deficient. We have now extensively characterized the molecular basis for the interaction between GIV and Gαi3 and its functional consequences. We found that GIV and Gαi3 can form a stable 1:1 complex in which the rate of nucleotide exchange, but not GTP hydrolysis, by the G protein is enhanced. Based on the preferential binding of GIV to Gαi versus Gαo, we have identified W258 in the Gαi‐subunit as a critical determinant for GIV binding. Mutation of W258 to the corresponding F in Gαo rendered Gαi3 GEF‐insensitive but did not perturb interaction with other Gα binding partners, i.e., Gβγ, GPCRs, GDIs or GAPs. When either the GEF‐deficient GIV mutant F1685A or GIV‐insensitive Gαi3 mutant W258F were expressed in HeLa cells they failed to undergo cell migration and to promote Akt signaling. These findings provide valuable information on the structural and biochemical basis for the biological functions of GIV as a Gαi activator which may be useful in the design of small molecules to disrupt the Gαi‐GIV interface for therapeutic purposes. Support: NIH DKI7780 and CA100768 (M.G.F). Susan G. Komen KG080079 (M.G‐M). NIH/T32 DK07202 and RSA from American Gastroenterology Association (P.G). McNair Student Research Scholarship (JE).