Dynamics of G{alpha}q-protein-p63RhoGEF interaction and its regulation by RGS2

Some G‑protein‑coupled receptors regulate biological processes via Gα12/13‑ or Gαq/11‑mediated stimulation of Rho guanine nucleotide exchange factors (RhoGEFs). p63RhoGEF is known to be specifically activated by Gαq/11 and mediates a major part of the acute response of vascular smooth muscle cells to Angiotensin II treatment. In order to gain information about the dynamics of receptor-mediated activation of p63RhoGEF we developed a FRET-based assay to study interactions between Gαq-CFP and Venus-p63RhoGEF in single living cells. Upon activation of histaminergic H1 or muscarinic M3 receptors a robust FRET signal occurred that allowed for the first time to analyze the kinetics of this interaction in detail. On- and offset kinetics of Gαq-p63RhoGEF interactions closely resembled the kinetics of Gαq‑activity. Analysis of the effect of RGS2 on the dynamics of Gαq-activity and their interaction with p63RhoGEF showed that RGS2 is able to accelerate both deactivation of Gαq proteins as well as dissociation of Gαq and p63RhoGEF to a similar extent. Furthermore we were able to detect activation-dependent FRET between RGS2 and p63RhoGEF and observed a reduced p63RhoGEF mediated downstream signaling in the presence of RGS2. In summary, these observations support the concept of a functional, activation-dependent p63RhoGEF-Gαq-RGS2 complex.

Source: BJ Cell - December 4, 2013 Category: Biochemistry Authors: E Bodmann, A Rinne, D Brandt, S Lutz, T Wieland, R Grosse, M Bünemann Tags: BJ Signal Source Type: research