Chimeric G alpha(i2)/G alpha(13) proteins reveal the structural requirements for the binding and activation of the RGS-like (RGL)-containing Rho guanine nucleotide exchange factors (GEFs) by G alpha(13)

The alpha-subunit of G proteins of the G(12/13) family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of Galpha(12/13) to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between Galpha(13) and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of Galpha(i) proteins. Using Galpha(i2), which does not bind RGL domains, as the backbone in which Galpha(13) sequences were swapped or mutated, we observed that the switch region of Galpha(13) is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of Galpha(13) was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of Galpha(13) and chimeric Galpha(i2) proteins was also necessary for Rho activation. These findings revealed the structural features by which Galpha(13) interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs.