Mapping the Gα₁₃ binding interface of the rgRGS domain of p115RhoGEF

Structural requirements for function of the Rho GEF (guanine nucleotide exchange factor) regulator of G protein signaling (rgRGS) domains of p115RhoGEF and homologous exchange factors differ from those of the classical RGS domains. An extensive mutagenesis analysis of the p115RhoGEF rgRGS domain was undertaken to determine its functional interface with the Gα₁₃ subunit. Results indicate that there is global resemblance between the interaction surface of the rgRGS domain with Gα₁₃ and the interactions of RGS4 and RGS9 with their Gα substrates. However, there are distinct differences in the distribution of functionally critical residues between these structurally similar surfaces and an additional essential requirement for a cluster of negatively charged residues at the N terminus of rgRGS. Lack of sequence conservation within the N terminus may also explain the lack of GTPase-activating protein (GAP) activity in a subset of the rgRGS domains. For all mutations, loss of functional GAP activity is paralleled by decreases in binding to Gα₁₃. The same mutations, when placed in the context of the p115RhoGEF molecule, produce deficiencies in GAP activity as observed with the rgRGS domain alone but show no attenuation of the regulation of Rho exchange activity by Gα₁₃. This suggests that the rgRGS domain may serve a structural or allosteric role in the regulation of the nucleotide exchange activity of p115RhoGEF on Rho by Gα₁₃.