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; p63RhoGEF580, GEFT and a recently discovered longer isoform of 619 amino acids (p63RhoGEF619). The subcellular distribution of p63RhoGEF580 and p63RhoGEF619 is strikingly different in unstimulated cells, p63RhoGEF580 is located at the plasma membrane and p63RhoGEF619 is confined to the cytoplasm. Interestingly, we find that both P63RhoGEF580 and p63RhoGEF619 activate RhoGTPases to a similar extent after stimulation of Gαq coupled GPCRs. Furthermore, we show that p63RhoGEF619 relocates to the plasma membrane upon activation of Gαq coupled GPCRs, resembling the well-known activation mechanism of RhoGEFs activated by Gα12/13. Synthetic recruitment of p63RhoGEF619 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.