Received for publication May 3, 2004.
Revised July 12, 2004.
Accepted for publication July 13, 2004.

Critical Role of Lysine 204 in Switch I Region of G₁₃ for Regulation of p115RhoGEF and LARG

Abstract

Heterotrimeric G proteins of the G12 family regulate the Rho GTPase through RhoGEFs which contain an amino-terminal RGS domain (RGS-RhoGEFs). Direct regulation of the activity of RGS-RhoGEFs p115 or LARG by G₁₃ has previously been demonstrated. However, the precise biochemical mechanism by which G₁₃ stimulates the RhoGEF activity of these proteins has not yet been well-understood. Based on the crystal structure of G _i1 in complex with RGS4, we mutated the G ₁₃ residue lysine 204 to alanine (G ₁₃ K204A) and characterized the effect of this mutation in its regulation of RGS-RhoGEFs p115 or LARG. Compared to wild-type G₁₃, G₁₃ K204A induced much less serum response factor (SRF) activation when expressed in HeLa cells. Recombinant G₁₃ K204A exhibits normal function in terms of nucleotide binding, basal GTP hydrolysis, and formation of heterotrimer with . We found that lysine 204 of G₁₃ is important for interaction with the RGS domain of p115 or LARG and for the GAP activity of these proteins. In addition, the K204A mutation of G₁₃ impaired its regulation of the RhoGEF activity of p115 or LARG. We conclude that lysine 204 of G₁₃ is important for interaction with RGS-RhoGEFs and is critically involved in the regulation of their activity.

Key words: G12,13;other G's, RGS proteins, Cdc42, rho, rac, other small G proteins, Mutagenesis/Chimeric approaches