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Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the α2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits two, β1, and γ2 and a number of GPCRs including M4, α2A, the adenosine Al receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Gα proteins shows specificity: there is interaction between the α2A receptor and Go, but little interaction between the α2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex. [ABSTRACT FROM AUTHOR]

G protein-gated inwardly rectifying K[sup +] (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G proteincoupled receptors (GPCRs) via the inhibitory family of G protein, G[sub i/o], in a membrane-delimited fashion by the direct binding of Gβγ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K[sup +] channel, Kir3.1 + 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein α subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein α subunit. With some combinations of agonist/GPCR, the rate of agonist unbinding is slow and rate-limiting, and deactivation kinetics are not modulated by regulators of G protein-signaling proteins. In another group, channel deactivation is generally faster and limited by the hydrolysis rate of the G protein α subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs. [ABSTRACT FROM AUTHOR]

Examines the role of isoforms of pertussis toxin-sensitive G protein alpha subunits in coupling receptors to the G protein-gated inwardly rectifying potassium channel. Characterization of pertussis toxin-insensitive mutants; Delineating different patterns of receptor stimulation of channel activation.