ReceptorG Protein Interaction Studied by RET

FRET- and BRET-based assays have been developed to study interactions between G proteins and receptors. To do so, receptors were labeled at the

C-terminus with either CFP or YFP variants (for FRET studies) or luciferase (for BRET studies), and one of the three G protein subunits (a, p, y) was labeled with the corresponding RET acceptor or donor. The detection of a specific RET signal in the absence of receptor stimulation would argue for the existence of precoupling between receptor and G protein.

Several RET-based studies reported that precoupling between certain GPCRs and heterotrimeric G proteins exists in the absence of ligand-induced receptor activation. The most rigorously tested receptor in this respect is the a2A-adrenergic receptor which couples to pertussis toxin-sensitive G proteins and was the object of three independent RET-based studies [30, 63, 64]. Unfortunately, the conclusions drawn from these studies are somewhat contradictory: the formation of complexes between receptors and G proteins in the absence of agonists was proposed from two independent studies [65, 66] , whereas a third study found support for interaction only in the presence of agonist [30] . These contradictory results could only partially be explained by the hypothesis that receptor-G protein precoupling might be receptor specific. In these studies, the receptor G protein precoupling is reduced to the question of whether or not a specific RET signal between labeled receptors and G protein subunits can be detected. All three studies detected RET between receptors and G proteins in the absence of agonists; however, the question of whether this RET signal is specific was answered differently. This case can be valued as an example of the weakness of RET approaches to study steady-state interactions. The choice of control proteins to distinguish between bystander RET and specific FRET is often very difficult, and all three studies used quite different control proteins, none of which qualified for the term "appropriate" as discussed above. A recent study used mobility measurements by means of FRAP [65] in order to study receptor-G protein interaction. This study found no restriction in mobility of fluorescent Gi proteins was found upon experimental immobilization of a2A-adrenergic receptors in living cells. The result supports the results and conclusions of Hein et al. [30] that these receptors interact only in the presence of agonist with G proteins, and that this interaction is short-l ived relative to the lifetime of an active G protein, suggesting the occurrence of catalytic collision coupling. The controversy over precoupling of GPCRs and G proteins is far from being resolved. Although there is solid evidence indicating an interaction between GPCRs and G proteins prior to activation for a number of receptors suggesting that precoupling exists for some GPCRs, more studies are needed to clarify this issue, including those that utilize fluorescence-based approaches. Such assays will help address the important functional consequences of precoupling between inactive receptors and G proteins.

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