Distinct regions of Gα13 participate in its regulatory interactions with RGS homology domain-containing RhoGEFs

Gα12 and Gα13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of Gα12 and Gα13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in Gα13 than in Gα12. At the same time, Gα13, but not Gα12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which Gα13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of Gα13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between Gα12 and Gα13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of Gα12 (residues 267–379) with that of Gα13 (residues 264–377) conferred gain-of-function to the resulting chimeric subunit, Gα12C13. The inverse chimera, Gα13C12, exhibited basal RhoA activation which was similar to Gα12. In contrast to GEF assays, GAP assays showed that Gα12C13 or Gα13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to Gα12 or Gα13, respectively. We conclude from these results that the carboxyl-terminal region of Gα13 (residues 264–377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal α helical and switch regions of Gα12 and Gα13 are responsible for their differential GAP responses to the RH domain.