Influence of Gαq on the Dynamics of M3-Acetylcholine Receptor–G-Protein–Coupled Receptor Kinase 2 Interaction

Abstract

G-protein-coupled receptor kinase 2 (GRK2) is a serine/ threonine kinase with an important function in the desensitization of G-protein-coupled receptors. Based on its ability to bind G-protein bg subunits as well as activated Ga q subunits, it can be considered as an effector for G-proteins. The recruitment of GRK2 to activated receptors is well known to be mediated by Gbg together with negatively charged membrane phospholipids. In the current study, we address the role of Ga q on the interaction of GRK2 with activated G q -protein-coupled receptors. Therefore, we established new Förster resonance energy transfer (FRET)-based assays to study the interaction of GRK2 with the M 3 -acetylcholine (M 3 -ACh) receptor as well as G q -protein subunits with high spatiotemporal resolution in single living human embryonic kidney 293T cells. M 3 -ACh receptor stimulation with 10 mM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding affinity toward Ga q [GRK2(D110A)] and Gbg [GRK2(R587Q)] were used to determine the specific role of G q -protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Ga q enhances the extent and stability of the GRK2-M 3 -ACh receptor interaction, and that not only Gbg but also Ga q can target GRK2 to the membrane. This reveals an important role of Ga q in efficient recruitment of GRK2 to M 3 -ACh receptors. Furthermore, interactions between Ga q and GRK2 were associated with a prolongation of the interaction between GRK2 and the M 3 -ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Ga q influences signaling and desensitization.

Dynamics of G protein effector interactions and their impact on timing and sensitivity of G protein-mediated signal transduction

“…Interestingly, following receptor-mediated G protein-activation G␤␥ bound first with a delay of less than a second to GRK2, whereas G␣q joined the complex with a delay of about 3-4 s. The dissociation kinetics after agonist withdrawal was comparable to those of G protein reassembly-kinetics. We could not observe any major acceleration of G protein deactivation by GRK2 and therefore conclude that the RGS domain of GRK2 does not serve as a GTPase-activating protein (GAP) for G␣q (Wolters et al, 2015) in intact cells. In accordance with previous studies on the role of G␤␥, we found that the recruitment of GRK2 to the receptor via G␣q was not sufficient to allow for GRK2-mediated arrestin recruitment.…”

“…However, not all RGS domains induce an enhancement of the GTPase activity of G␣ subunits, but rather may serve as a G␣-binding element (Shankaranarayanan et al, 2008). FRET assays used by Wolters et al (2015) offer the possibility to study the kinetics of individual steps of the receptor-mediated G protein cascade in a very similar experimental setting and therefore help to analyze effects of regulatory proteins on G protein dynamics in intact cells. We found that activation of M 3 AChR led to a robust interaction of GRK2 with G␣q, as well as with G␤␥.…”

“…Therefore, the development of a Förster resonance energy transfer-(FRET-) based assays to study G protein activity in intact cells was a major break through Frank et al, 2005;Janetopoulos et al, 2001). It enabled the study of real-time kinetics of G protein activity of various subtypes in intact cells (see also (Bodmann et al, 2014;Hein et al, 2005;Wolters et al, 2015)). These FRET assays have also been employed for real-time monitoring conformational changes underlying GPCR activation (Hoffmann et al, 2012Rochais et al, 2007;Vilardaga et al, 2003) as well as for studying receptor G protein interactions (Hein et al, 2006(Hein et al, , 2005Wolters et al, 2015) in single intact cells.…”

“…It enabled the study of real-time kinetics of G protein activity of various subtypes in intact cells (see also (Bodmann et al, 2014;Hein et al, 2005;Wolters et al, 2015)). These FRET assays have also been employed for real-time monitoring conformational changes underlying GPCR activation (Hoffmann et al, 2012Rochais et al, 2007;Vilardaga et al, 2003) as well as for studying receptor G protein interactions (Hein et al, 2006(Hein et al, , 2005Wolters et al, 2015) in single intact cells. Recently, new insights into the dynamics of G protein-effector interactions and how they affect dynamics and sensitivity of receptor-mediated G protein activity have been gained by developing FRET-based assays to study these interactions in intact cells.…”

Engineered Hyperphosphorylation of the β2-Adrenoceptor Prolongs Arrestin-3 Binding and Induces Arrestin Internalization

“…In experiments in which FRET amplitudes are shown, relative expression levels of fluorescently labeled proteins were determined as described before (Wolters et al, 2015). In brief, both fluorophores were excited individually and background fluorescence was subtracted for each channel.…”