G protein-coupled receptors have been proposed to exist in large signalosomes subject to agonist-driven shifts in the assembly-disassembly equilibrium. Signalosome assembly is also thought to be stabilized by the self-organizing properties of membrane lipids and/or by cortical actin and to result in restricted mobility of receptors. Here I investigated the receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2; these are highly homologous and couple to the same G proteins but differ within their hydrophobic core.
Fluorescence resonance energy transfer (FRET) microscopy showed that CRFRs existed in oligomeric form on the surface of living cells. CRF enhanced FRET in CRFR2 - but not in CRFR1 - suggesting either a conformational change in or an agonist-induced dissociation of CRFR2 oligomers. Agonist binding accelerated the diffusion of CRFR2 - but not of CRFR1 - in both, HEK293 cells and hippocampal neurons detected by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). However,fluorescence intensity distribution analysis (FIDA) demonstrated that agonist binding did not change the size of CRFR-complexes. Disruption of the actin cytoskeleton or extraction of cholesterol abolished the agonist-dependent increase in CRFR2 mobility. These observations are incompatible with an agonist-induced change in monomer-oligomer equilibrium. However, they are consistent with a model in which agonist occupancy facilitates accommodation of cholesterol by the CRFR2 - but not by CRFR1; this conformational change and/or redistribution of CRF2 into lipid microdomains affords rapid diffusion. The difference between the two receptors is likely to arise from their distinct hydrophobic cores.