Functional consequences of G alpha 13 mutations that disrupt interaction with p115RhoGEF.
The G-protein alpha subunit, alpha(13), regulates cell growth and differentiation through the monomeric Rho GTPase. Alpha(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(13). The mechanism for the G alpha(13)/p115RhoGEF interaction is not well understood. Here, we determined specific alpha(13) residues important for its interaction with p115RhoGEF. GST-pulldowns and co-immunoprecipitation assays revealed that individually mutating alpha(13) residues Lys204, Glu229, or Arg232 to opposite charge residues disrupts the interaction of activated alpha(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(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(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(13)/p115RhoGEF interaction, there are also significant differences.
Grabocka
Elda
E
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Wedegaertner
Philip B
PB
D019943:Amino Acid Substitution; D001120:Arginine; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics* / Q000378:metabolism*; D018698:Glutamic Acid; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D014162:Transfection
D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D018698:Glutamic Acid; D001120:Arginine; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D008239:Lysine
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