TY - JOUR T1 - Subtype-Specific Differences in Corticotropin-Releasing Factor Receptor Complexes Detected by Fluorescence Spectroscopy JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1196 LP - 1210 DO - 10.1124/mol.109.059139 VL - 76 IS - 6 AU - Laura Milan-Lobo AU - Ingrid Gsandtner AU - Erwin Gaubitzer AU - Dominik Rünzler AU - Florian Buchmayer AU - Gottfried Köhler AU - Antonello Bonci AU - Michael Freissmuth AU - Harald H. Sitte Y1 - 2009/12/01 UR - http://molpharm.aspetjournals.org/content/76/6/1196.abstract N2 - G protein-coupled receptors have been proposed to exist in signalosomes subject to agonist-driven shifts in the assembly disassembly equilibrium, affected by stabilizing membrane lipids and/or cortical actin restricting mobility. We investigated the highly homologous corticotropin-releasing factor receptors (CRFRs), CRFR1 and -2, which are different within their hydrophobic core. Agonist stimulation of CRFR1 and CRFR2 gave rise to similar concentration-response curves for cAMP accumulation, but CRFR2 underwent restricted collision coupling. Both CRFR1 and CRFR2 formed constitutive oligomers at the cell surface and recruited β-arrestin upon agonist activation (as assessed by fluorescence resonance energy transfer microscopy in living cells). However, CRFR2, but not CRFR1, failed to undergo agonist-induced internalization. Likewise, agonist binding accelerated the diffusion rate of CRFR2 only (detected by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) but reduced the mobile fraction, which is indicative of local confinement. Fluorescence intensity distribution analysis 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 monomer-oligomer 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.© 2009 The American Society for Pharmacology and Experimental Therapeutics ER -