Galpha q allosterically activates and relieves autoinhibition of p63RhoGEF

Galpha(q) directly activates p63RhoGEF and closely related catalytic domains found in Trio and Kalirin, thereby linking G(q)-coupled receptors to the activation of RhoA. Although the crystal structure of G alpha(q) in complex with the catalytic domains of p63RhoGEF is available, the molecular mechanism of activation has not yet been defined. In this study, we show that membrane translocation does not appear to play a role in G alpha(q)-mediated activation of p63RhoGEF, as it does in some other RhoGEFs. G alpha(q) instead must act allosterically. We next identify specific structural elements in the PH domain that inhibit basal nucleotide exchange activity, and provide evidence that G alpha(q) overcomes this inhibition by altering the conformation of the alpha 6-alpha N linker that joins the DH and PH domains, a region that forms direct contacts with RhoA. We also identify residues in G alpha(q) that are important for the activation of p63RhoGEF and that contribute to G alpha subfamily selectivity, including a critical residue in the G alpha(q) C-terminal helix, and demonstrate the importance of these residues for RhoA activation in living cells.