Dynamics of Gα_q-protein–p63RhoGEF interaction and its regulation by RGS2

Some G-protein-coupled receptors regulate biological processes via Gα_12/13- or Gα_q/11-mediated stimulation of RhoGEFs (guanine-nucleotide-exchange factors). p63RhoGEF is known to be specifically activated by Gα_q/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 Gα_q–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 the analysis of the kinetics of this interaction in detail. On- and off-set kinetics of Gα_q–p63RhoGEF interactions closely resembled the kinetics of Gα_q activity. Analysis of the effect of RGS2 (regulator of G-protein signalling 2) on the dynamics of Gα_q activity and their interaction with p63RhoGEF showed that RGS2 is able to accelerate both deactivation of Gα_q proteins and dissociation of Gα_q 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 signalling in the presence of RGS2. In summary, these observations support the concept of a functional activation-dependent p63RhoGEF–Gα_q–RGS2 complex.