Signaling efficiency of Gαq through its effectors p63RhoGEF and GEFT depends on their subcellular location

Abstract

The p63RhoGEF and GEFT proteins are encoded by the same gene and both members of the Dbl family of guanine nucleotide exchange factors. These proteins can be activated by the heterotrimeric G-protein subunit Gαq. We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined to the cytoplasm. Live-cell imaging studies yielded quantitative information on diffusion coefficients, association rates and encounter times of GEFT and p63RhoGEF. Calcium signaling was examined as a measure of the signal transmission, revealing more efficient signaling through the membrane-associated p63RhoGEF. A rapamycin dependent recruitment system was used to dynamically alter the subcellular location and concentration of GEFT, showing efficient signaling through GEFT only upon membrane recruitment. Together, our results show efficient signal transmission through membrane located effectors, and highlight a role for increased concentration rather than increased encounter times due to membrane localization in the Gαq mediated pathways to p63RhoGEF and PLCβ.

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

“…Confocal images of cells transfected with p63RhoGEF 580 -YFP show that p63RhoGEF 580 is localized to the plasma membrane, as has been described before2223. In contrast, cells transfected with either p63RhoGEF 619 -YFP or YFP-GEFT show only a cytosolic localization of these isoforms (Fig.…”

“…Previous studies have shown that p63RhoGEF 580 is localized to the plasma membrane because of the palmitoylation of one or more cysteines at its the N-terminus2223. In depth sequence analysis of the first 30 amino acids shows that the N-terminus, including cysteines, of p63RhoGEF 580 is well conserved throughout the whole subphylum vertebrata (Fig.…”

“…It has been well established that p63RhoGEF 580 can be activated through G-protein coupled receptors (GPCRs), via direct interaction with the heterotrimeric G-protein subunit Gαq12142231. Activated p63RhoGEF 580 catalyzes the exchange of GDP for GTP on Rho GTPases of the RhoA subfamily12.…”

“…Five putative palmitoylation sites at the N-terminus of p63RhoGEF 580 target this isoform to the plasma membrane2223, while p63RhoGEF 619 resides in the cytoplasm. Interestingly, despite this difference in localization, we observed no difference in basal GEF activity towards RhoA.…”

“…GEFT resides in the cytoplasm, it does not contain the first 106 a.a. of p63RhoGEF 580 , and thus lacks the N-terminal palmitoylation sites. The plasma membrane localization of both these p63RhoGEF isoforms have been shown to be important for their interaction with Gαq2223.…”