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Critical Role of Lysine 204 in Switch I Region of G₁₃ for Regulation of p115RhoGEF and Leukemia-Associated RhoGEF

Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois

Heterotrimeric G proteins of the G12 family regulate the Rho GTPase through RhoGEFs that contain an amino-terminal regulator of G protein signaling (RGS) domain (RGS-RhoGEFs). Direct regulation of the activity of RGS-RhoGEFs p115 or leukemia-associated RhoGEF (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 G₁₃ residue lysine 204 to alanine (G₁₃K204A) and characterized the effect of this mutation in its regulation of RGS-RhoGEFs p115 or LARG. Compared with wild-type G₁₃, G₁₃K204A induced much less serum-response factor activation when expressed in HeLa cells. Recombinant G₁₃K204A 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 GTPase-activating protein 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.

Received for publication May 3, 2004.

Accepted for publication July 13, 2004.

Address correspondence to: Dr. Tohru Kozasa, 835 South Wolcott Ave., M/C 868, Chicago, IL 60612. E-mail: tkozas{at}uic.edu

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