Dynamics of G alpha(q)-protein-p63RhoGEF interaction and its regulation by RGS2

作者：Bodmann Eva Lisa; Rinne Andreas; Brandt Dominique; Lutz Susanne; Wieland Thomas; Grosse Robert; Buenemann Moritz

来源：Biochemical Journal, 2014, 458(1): 131-140.

DOI：10.1042/BJ20130782

• Forster resonance energy transfer (FRET)
• G alpha(q)
• G-protein-coupled receptor (GPCR)
• histaminergic receptor
• p63RhoGEF
• regulator of G-protein signalling 2 (RGS2)

Some G-protein-coupled receptors regulate biological processes via G alpha(12/13)- or G alpha(q/11)-mediated stimulation of RhoGEFs (guanine-nucleotide-exchange factors). p63RhoGEF is known to be specifically activated by G alpha(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 G alpha(q)-CFP and Venus-p63RhoGEF in single living cells. Upon activation of histaminergic H-1 or muscarinic M-3 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 G alpha(q)-p63RhoGEF interactions closely resembled the kinetics of G alpha(q) activity. Analysis of the effect of RGS2 (regulator of G-protein signalling 2) on the dynamics of G alpha(q) activity and their interaction with p63RhoGEF showed that RGS2 is able to accelerate both deactivation of G alpha(q) proteins and dissociation of G alpha(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-G alpha(q-)RGS2 complex.

• 出版日期2014-2-15