Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, G␣␤␥ heterotrimers physically dissociate after activation, liberating free G␣ subunits and G␤␥ dimers. This model is supported by evidence obtained in vitro with purified proteins, but its relevance in vivo has been questioned. Here, we show that at least some heterotrimeric G protein isoforms physically dissociate after activation in living cells. G␣ subunits extended with a transmembrane (TM) domain and cyan fluorescent protein (CFP) were immobilized in the plasma membrane by biotinylation and cross-linking with avidin. Immobile CFP-TM-G␣ greatly decreased the lateral mobility of intracellular G␤ 1␥2-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-G␣ oA lost the ability to restrict G␤1␥2-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-G␣ i3 and CFP-TM-G␣ s retained this ability. Activation of cognate G proteincoupled receptors partially relieved the constraint on G␤ 1␥2-YFP mobility induced by immobile CFP-TM-G␣ oA and CFP-TM-G␣i3 but had no effect on the constraint induced by CFP-TM-G␣ s. These results demonstrate the physical dissociation of heterotrimers containing G␣ oA and G␣i3 subunits in living cells, supporting the subunit dissociation model of G protein signaling for these subunits. However, these results are also consistent with the suggestion that G protein heterotrimers (e.g., G␣ s) may signal without physically dissociating.cross-linking ͉ fluorescence recovery after photobleaching ͉ G protein-coupled receptors H eterotrimeric G proteins are known to exist in their inactive state as stable complexes of G␣ subunits and G␤␥ dimers. G␣ subunits cycle between inactive (GDP-bound) and active (GTP-bound) states, and the lifetime of the active state is limited by GTP hydrolysis. Biochemical studies have shown that active G protein heterotrimers dissociate into G␣-GTP and G␤␥ subunits in vitro (1). However, it has been argued that G protein subunits may not dissociate under more physiological conditions (2-5), and recent resonance energy transfer (RET) studies have suggested that G protein activation in cells involves subunit rearrangement rather than dissociation (4, 5). Physical dissociation of G protein heterotrimers has not been shown to occur in living cells. To address this question we developed a method to detect protein association and dissociation (Fig. 1A). In this method, one protein of an interacting pair is immobilized in the plasma membrane by an extracellular cross-linking agent. A decrease in the lateral mobility of a second protein [measured by using fluorescence recovery after photobleaching (FRAP)] indicates a binding interaction between the two. The mobility of this second protein is restored only if the partners dissociate. Here, we use this method to show that some G protein subunits physically dissociate in living cells, whereas other heterotrimers appear t...

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.

Signalling by heterotrimeric G proteins is often isoform-specific, meaning certain effectors are regulated exclusively by one family of heterotrimers. For example, in excitable cells inwardly rectifying potassium (GIRK) channels are activated by Gβγ dimers derived specifically from G i/o heterotrimers. Since all active heterotrimers are thought to dissociate and release free Gβγ dimers, it is unclear why these channels respond primarily to dimers released by G i/o heterotrimers. We reconstituted GIRK channel activation in cells where we could quantify heterotrimer expression at the plasma membrane, GIRK channel activation, and heterotrimer dissociation. We find that G oA heterotrimers are more effective activators of GIRK channels than G s heterotrimers when comparable amounts of each are available. We also find that active G oA heterotrimers dissociate more readily than active G s heterotrimers. Differential dissociation may thus provide a simple explanation for Gα-specific activation of GIRK channels and other Gβγ-sensitive effectors.

RGS proteins accelerate the GTPase activity of heterotrimeric G proteins at the plasma membrane. Association of RGS proteins with the plasma membrane can be mediated by interactions with other membrane proteins and by direct interactions with the lipid bilayer. Here we use fluorescence recovery after photobleaching (FRAP) to characterize interactions between RGS2 and M3 acetylcholine receptors (M3Rs), Ga subunits and the lipid bilayer. Active Ga q and M3Rs both recruited RGS2-EGFP to the plasma membrane. RGS2-EGFP remained bound to the plasma membrane between interactions with active Ga q , but rapidly exchanged between membrane-associated and cytosolic pools when recruited by M3Rs.