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

Abstract

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.