Kinetics of recruitment and allosteric activation of ARHGEF25 isoforms by the heterotrimeric G-protein Gαq

Rho GTPases are master regulators of the eukaryotic cytoskeleton. The activation of Rho GTPases is governed by Rho guanine nucleotide exchange factors (GEFs). Three RhoGEF isoforms are produced by the gene ARHGEF25; p63RhoGEF(580), GEFT and a recently discovered longer isoform of 619 amino acids (p63RhoGEF(619)). The subcellular distribution of p63RhoGEF(580) and p63RhoGEF(619) is strikingly different in unstimulated cells, p63RhoGEF(580) is located at the plasma membrane and p63RhoGEF(619) is confined to the cytoplasm. Interestingly, we find that both P63RhoGEF(580) and p63RhoGEF(619) activate RhoGTPases to a similar extent after stimulation of Gαq coupled GPCRs. Furthermore, we show that p63RhoGEF(619) relocates to the plasma membrane upon activation of Gαq coupled GPCRs, resembling the well-known activation mechanism of RhoGEFs activated by Gα(12/13). Synthetic recruitment of p63RhoGEF(619) to the plasma membrane increases RhoGEF activity towards RhoA, but full activation requires allosteric activation via Gαq. Together, these findings reveal a dual role for Gαq in RhoGEF activation, as it both recruits and allosterically activates cytosolic ARHGEF25 isoforms.