G protein-coupled receptors have been proposed to exist in signalosomes subject to agonistdriven shifts in the assembly-disassembly equilibrium, affected by stabilizing membranelipids and/or cortical actin restricting mobility. We investigated the highly homologous corticotrophin-releasing-factor receptors, CRFR1&2, which differ within their hydrophobic core. Agonist stimulation of CRFR1 and CRFR2 gave rise to similar to concentrationresponse curves for cAMP accumulation, but CRFR2 underwent restricted collision coupling. Both, CRFR1 and CRFR2, formed constitutive oligomers at the cell surface and recruited â- arrestin upon agonist activation (as assessed by fluorescence resonance energy transfer - FRET - microscopy in living cells). However, CRFR2 - but not CRFR1 - failed to undergo agonist-induced internalization. Similarly agonist binding accelerated the diffusion rate of CRFR2 only (detected by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy - FCS) but reduced the mobile fraction, which is indicative of local confinement. Fluorescence intensity distribution analysis (FIDA) demonstrated that the size of CRFR-complexes was not changed. Disruption of the actin cytoskeleton abolished the agonist-dependent increase in CRFR2 mobility, shifted the agonist concentration curve for CRFR2 to the left and promoted agonist-induced internalization of CRFR2. Our observations are incompatible with an agonist-induced change in monomeroligomer equilibrium, but they suggest an agonist-induced redistribution of CRFR2 into a membrane microdomain that affords rapid diffusion but restricted mobility and that is stabilized by the actin cytoskeleton. Our data show that membrane anisotropy can determine the shape and duration of receptor-generated signals in a subtype-specific manner.