Abundance and stability of complexes containing inactive G protein‐coupled receptors and G proteins

Abstract

G protein-coupled receptors interact directly with heterotrimeric G proteins to transduce physiological signals. Early studies of this interaction concluded that GPCRs (R) and G proteins (G) collide with each other randomly after receptor activation, and that R-G complexes are transient. More recent studies have suggested that inactive R and G are preassembled (precoupled) as stable R-G complexes. Here we examine the stability of complexes formed between CFP-labeled α2A adrenoreceptors (C-α2ARs) and G proteins in cells using fluorescence recovery after photobleaching (FRAP). Labeled G proteins diffused in the plasma membrane with equal mobility in the absence and presence of immobile C-α2ARs. Immobile C-α2ARs activated labeled G proteins, demonstrating functional coupling without stable physical association. In contrast, a stable R-G interaction was detected when G proteins were deprived of nucleotides and C-α2ARs were active, as predicted by the ternary complex model. Overexpression of RGS4 accelerated the onset of effector activation but did not detectably alter the interaction between C-α2ARs and G proteins. We conclude that at most a small fraction of C-α2ARs and G proteins exist as R-G complexes at any moment.

WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13

“…In contrast to transmembrane receptors, intracellular proteins, such as the heterotrimeric G proteins, are not directly affected by surface CL in this assay. The mobile fraction of intracellular proteins is reduced only upon interaction with CL-immobilized surface proteins (Qin et al, 2008(Qin et al, , 2011Kilander et al, 2014b). In cells transfected with fluorescently tagged FZD 4 , Ga, and untagged bg, we found that the mobile fraction of FZD 4 was remarkably reduced by surface cross-linking (Fig.…”

“…Based on our data using a live-cell imaging dcFRAP approach, which has been successfully used to study GPCR-G protein interactions in the past (Qin et al, 2008(Qin et al, , 2011Kilander et al, 2014b), we show that FZD 4 interacts with Ga 12/13 but not representatives of other G protein subfamilies. The observed selectivity of FZD 4 for one but not all groups of heterotrimeric G proteins underlines the specificity of the dcFRAP assay and supports the use of overexpressed proteins.…”

“…The procedure was essentially as described in Kilander et al (2014b), Qin et al (2011), andQin et al (2008). Cells were grown on 35-mm extracellular matrix; coated (1:300; Sigma-Aldrich) glass-bottom dishes and assessed using a Zeiss 710 laser-scanning microscope (Zeiss, Jena, Germany).…”

“…1). To assess FZD 4 -G protein interaction, we took advantage of a dual-color FRAP (dcFRAP) protocol that enables simultaneous assessment of lateral mobility of two fluorescently tagged proteins (Phair et al, 2004;Qin et al, 2008Qin et al, , 2011Dorsch et al, 2009;Kilander et al, 2014b) (Fig. 1).…”

Desensitization of Functionalµ-Opioid Receptors Increases Agonist Off-Rate

“…Receptors preassociated with G proteins (precoupled receptors) are thought to signal more rapidly than receptors that are dependent on collision coupling (Nobles et al, 2005;Riven et al, 2006;Philip et al, 2007; reviewed in Lohse et al, 2008;Vilardaga 2010). The stability (and thus the relative number) of the precoupled receptors is reportedly very low for some receptors (a 2 -adrenoceptor; Qin et al, 2008) and substantially greater for others (muscarininc M 3 receptor; Qin et al, 2011). Where functional studies have been done using the activation of GIRK-dependent currents, precoupling has been suggested (Zhang et al, 2002;Lober et al, 2006;Zhou et al, 2012).…”