Identification of potential mechanisms for regulation of p115 RhoGEF through analysis of endogenous and mutant forms of the exchange factor

Rho GTPases play a fundamental role in numerous cellular processes that are initiated by extracellular stimuli including agonists that work through G protein-coupled receptors. A direct pathway for such regulation was elucida ted by the identification of p115 RhoGEF, an exchange factor for RhoA that is activated through its RGS domain by G alpha (13). Endogenous p115 RhoGEF was found mainly in the cytosol of serum-starved cells but partially local ized to membranes in cells stimulated with lysophosphatidic acid. Overexpre ssed p115 RhoGEF was equally distributed between membranes and cytosol; eit her the RGS or pleckstrin homology domain was sufficient for this partial t argeting to membranes. Removal of the pleckstrin homology domain dramatical ly reduced the in vitro rate of p115 RhoGEF exchange activity. Deletion of amino acids 252-288 in the linker region between the RGS domain and the Dbl homology domain or of the last 150 C-terminal amino acids resulted in non- additive reduction of in vitro exchange activity. In contrast, p115 RhoGEF pieces lacking this extended C terminus were over 5-fold more active than t he full-length exchange factor in vivo. These results suggest that p115 Rho GEF is inhibited in the cellular milieu through modification or interaction of inhibitory factors with its C terminus. Endogenous p115 RhoGEF that was immunoprecipitated from cells stimulated with lysophosphatidic acid or sph ingosine 1-phosphate was more active than when the enzyme was immunoprecipi tated from untreated cells. This indicates an additional and potentially no vel long lived mechanism for regulation of p115 RhoGEF by G protein-coupled receptors.