Functional consequences of G alpha sub(13) mutations that disrupt interaction with p115RhoGEF

• Published in: Oncogene Vol. 24; no. 13; pp. 2155 - 2165
• Main Authors: Grabocka, E, Wedegaertner, P B
• Format: Journal Article
• Language: English
• Published: 24.03.2005
• ISSN: 0950-9232
• DOI: 10.1038/sj.onc.1208414

The G-protein alpha subunit, alpha sub(13), regulates cell growth and differentiation through the monomeric Rho GTPase. alpha sub(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 G alpha sub(13). The mechanism for the G alpha sub(13)/p115RhoGEF interaction is not well understood. Here, we determined specific alpha sub(13) residues important for its interaction with p115RhoGEF. GST-pulldowns and co-immunoprecipitation assays revealed that individually mutating alpha sub(13) residues Lys204, Glu229, or Arg232 to opposite charge residues disrupts the interaction of activated alpha sub(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 alpha sub(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 alpha sub(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 alpha disrupt their interaction with regulator of G-protein signaling (RGS) proteins. These results demonstrate that, whereas several features of a typical alpha /RGS interaction are preserved in the alpha sub(13)/p115RhoGEF interaction, there are also significant differences.