Dynamics of receptor/G protein coupling in living cells

Abstract

The interaction of activated G protein‐coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)‐based assay, we measure directly and in living cells the interaction of YFP‐labeled α2A‐adrenergic receptors with CFP‐labeled G proteins. Upon agonist stimulation, a small, concentration‐dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Gα. Simultaneously recorded G protein‐regulated inwardly rectifying K+ channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Gα mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild‐type G proteins interacts with the activated receptor at any time. Our data suggest that α2A‐adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…GTPγS induced separation of the receptor and G protein as did GDP (at half the rate), making it unlikely that RG separation is sufficient for activation. These results are compatible with a cell activation model regulated by the G protein conformational changes rather than disassembly of the Gαβγ heterotrimer [7,9,19].…”

“…The data also strongly suggest that the G protein heterotrimer appears to remain intact within the time frame of signal initiation (Table 1). Interestingly our kinetics for the dissociation of FPR-GFP or wild type FPR from Gα i in the detergent solubilized system are much faster than was reported in cells from experiments involving FRET between β 2 AR and Gα s [19]. We do not know whether this reflects differences between the intact cells and detergent, the difference between specific receptors and G proteins, the possibility that their measurement has contributions from a higher order topographical redistribution, or the possibility that ligand dissociation and rebinding [44][45][46] is rate-limiting in their measurement of RG dissociation.…”

“…Furthermore, other experimental evidence has led to questions about the absolute necessity of including subunit dissociation as an element of activation [5][6][7][8][9]19].…”

“…The locally high concentrations of signaling components would permit subsecond kinetics observed with GPCR signaling [19,49]. One can envision a case where a ligand activated receptor can hop from one G protein heterotrimer to the next, thereby activating several G proteins during the lifetime of a bound ligand.…”

“…The reason why we did not observe subunit dissociation could be due to the absence of effectors that could potentially be responsible for turning ON a switch mechanism that may cause the rapid dissociation of the α subunit. As a case in point, under basal conditions, GDP, which has a reported affinity of 10 −11 M [28] for the α subunit, is readily ejected in a millisecond time frame [19] from the binding pocket upon a transient contact between the ligand activated receptor during the GDP/ GTP nucleotide exchange step of GPCR activation. This point is made in recognition of the incomplete nature of our model system.…”