The Role of Lateral Diffusion in G-Protein-Coupled Receptor Signaling

Abstract

In the standard model of G-protein-coupled receptor (GPCR) signaling, receptors and G-proteins are free to diffuse laterally within the plane of the plasma membrane, and these molecules encounter each other by random collision. It is possible that formation of the receptor-G-protein (R-G) complex precedes receptor activation, although the dynamics of this process have been challenging to observe in live cells. We have approached this problem by measuring the membrane diffusion and binding reactions of receptors and Gproteins. We examined the functional consequences of immobilizing receptors, G-proteins, and inwardly rectifying potassium (GIRK) channels at the cell surface by biotinylation and avidin crosslinking, and monitored intermolecular binding events reflected by receptor-imposed constraints on G-protein diffusion, as measured by fluorescence recovery after photobleaching. In whole-cell voltage-clamp recordings, we found that both mobile and immobile heterologous p-opioid receptors (MOR) activated endogenous GIRK channels in cerebellar granule neurons with kinetics and agonist sensitivity resembling native synaptic responses. In HEK 293 cells, immobile GIRK channels were activated by multiple populations of immobilized receptors and Gproteins. Immobilization of MOR constrained the apparent mobility of freely diffusing fluorescent Gaj/o-family proteins, indicating measurable binding reactions between the two protein types, but had no effect on the diffusion of unrelated membrane proteins or Gaq subunits. Transient binding reactions were highly specific, as determined by competition with unlabeled binding partners. RG binding was disrupted by receptor agonist or GTPase-deficient G-protein mutants. Neither receptor antagonists nor pertussis toxin blocked basal R-G binding. Furthermore, the Ga subunit amino terminus (amino acids 1-31) was sufficient for mediating R-G binding. Our results provide evidence for the free diffusion of receptors and Gproteins, as well as a pre-signaling binding-dissociation equilibrium between them that is altered upon activation. The frequency of collisions between receptors and G-proteins does not limit the rate of signaling in neurons, but by diffusion receptors can “swap” G-proteins that are not stably associated with GIRK channels. A three-stage sequential fit model of R-G coupling is suggested.