title: Subtype-specific differences in corticotropin-releasing factor receptor complexes detected by fluorescence spectroscopy.
abstract: 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 beta-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.
authors.affiliation: Medical University of Vienna, Center for Biomolecular Medicine and Pharmacology, Institute of Pharmacology, Waehringerstrasse 13a, A-1090 Vienna, Austria.
authors.forename: Laura
authors.initials: L
authors.lastname: Milan-Lobo
authors.forename: Ingrid
authors.initials: I
authors.lastname: Gsandtner
authors.forename: Erwin
authors.initials: E
authors.lastname: Gaubitzer
authors.forename: Dominik
authors.initials: D
authors.lastname: Rünzler
authors.forename: Florian
authors.initials: F
authors.lastname: Buchmayer
authors.forename: Gottfried
authors.initials: G
authors.lastname: Köhler
authors.forename: Antonello
authors.initials: A
authors.lastname: Bonci
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D029845:Amphibian Proteins / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D005372:Filipin / Q000494:pharmacology; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D051379:Mice; D036361:Peptide Hormones / Q000378:metabolism; D018019:Receptors, Corticotropin-Releasing Hormone / Q000096:biosynthesis / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D053778:Thiazolidines / Q000494:pharmacology; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1
chemical_list: D029845:Amphibian Proteins; D019390:Arrestins; D019086:Bridged Bicyclo Compounds, Heterocyclic; C101531:CRF receptor type 2; D036361:Peptide Hormones; D018019:Receptors, Corticotropin-Releasing Hormone; D053778:Thiazolidines; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1; C025727:sauvagine; D005372:Filipin; D003346:Corticotropin-Releasing Hormone; D002784:Cholesterol; D000242:Cyclic AMP; C037067:latrunculin A