¹Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th Street, 839 BLSB, Philadelphia, PA 19107, USA

Correspondence: P Wedegaertner, E-mail: P_Wedegaertner@mail.jci.tju.edu

Received 20 August 2004; Revised 23 November 2004; Accepted 23 November 2004; Published online 14 February 2005.

Abstract

The G-protein subunit, ₁₃, regulates cell growth and differentiation through the monomeric Rho GTPase. ₁₃ activates Rho through direct stimulation of the guanine nucleotide exchange factor p115RhoGEF, which contains a regulator of G-protein signaling homology domain (RH) in its N-terminus. Through its RH domain, p115RhoGEF also functions as a GAP for G₁₃. The mechanism for the G₁₃/p115RhoGEF interaction is not well understood. Here, we determined specific ₁₃ residues important for its interaction with p115RhoGEF. GST-pulldowns and co-immunoprecipitation assays revealed that individually mutating ₁₃ residues Lys204, Glu229, or Arg232 to opposite charge residues disrupts the interaction of activated ₁₃ with the RH domain of p115RhoGEF or full-length p115RhoGEF. We further demonstrate that mutation of Glu229, and to a lesser extent Lys204 or Arg232, disrupts the ability of activated ₁₃ to induce the recruitment of p115RhoGEF to the plasma membrane (PM) and to activate Rho-mediated serum response element-luciferase gene transcription. Interestingly, an ₁₃ mutant where a conserved Gly was mutated to a Ser (G205S) retained its ability to bind to p115RhoGEF, induce p115RhoGEF recruitment to the PM, and activate Rho-dependent signaling, even though identical Gly to Ser mutations in other disrupt their interaction with regulator of G-protein signaling (RGS) proteins. These results demonstrate that, whereas several features of a typical /RGS interaction are preserved in the ₁₃/p115RhoGEF interaction, there are also significant differences.