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Molecular Pharmacology

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Research ArticleArticle

Subtype-Specific Differences in Corticotropin-Releasing Factor Receptor Complexes Detected by Fluorescence Spectroscopy

Laura Milan-Lobo, Ingrid Gsandtner, Erwin Gaubitzer, Dominik Rünzler, Florian Buchmayer, Gottfried Köhler, Antonello Bonci, Michael Freissmuth and Harald H. Sitte
Molecular Pharmacology December 2009, 76 (6) 1196-1210; DOI: https://doi.org/10.1124/mol.109.059139
Laura Milan-Lobo
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Ingrid Gsandtner
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Erwin Gaubitzer
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Dominik Rünzler
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Florian Buchmayer
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Gottfried Köhler
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Antonello Bonci
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Michael Freissmuth
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Harald H. Sitte
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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 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.

Footnotes

  • ↵1 Current affiliation: The Ernest Gallo Clinic and Research Center, Emeryville, California.

  • This work was supported by the Austrian Science Fund/FWF [Grants P17076, P18706, N209]; the Wiener-, Wissenschafts-, Forschungs- und Technologiefonds (MA 05); and the Gallo Research Institute and Clinics.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.109.059139

  • ABBREVIATIONS:

    GPCR
    G protein-coupled receptor
    CFP
    cyan fluorescent protein
    YFP
    yellow fluorescent protein
    CRF
    corticotropin-releasing factor
    GFP
    green fluorescent protein
    CRFR
    corticotropin-releasing factor receptor
    C-SERT-Y
    cyan fluorescent protein-serotonin transporter-yellow fluorescent protein
    ECFP
    enhanced cyan fluorescent protein
    EYFP
    enhanced yellow fluorescent protein
    Y-DAT
    yellow fluorescent protein-dopamine transporter
    FCS
    fluorescence correlation spectroscopy
    FIDA
    fluorescence intensity distribution analysis
    FRAP
    fluorescence recovery after photobleaching
    TM
    transmembrane
    HEK
    human embryonic kidney
    GTPγS
    guanosine 5′-O-(3-thio)triphosphate
    MβCD
    methyl-β-cyclodextrin.

    • Received July 3, 2009.
    • Accepted September 11, 2009.
  • © 2009 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 76 (6)
Molecular Pharmacology
Vol. 76, Issue 6
1 Dec 2009
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Research ArticleArticle

Subtype-Specific Differences in Corticotropin-Releasing Factor Receptor Complexes Detected by Fluorescence Spectroscopy

Laura Milan-Lobo, Ingrid Gsandtner, Erwin Gaubitzer, Dominik Rünzler, Florian Buchmayer, Gottfried Köhler, Antonello Bonci, Michael Freissmuth and Harald H. Sitte
Molecular Pharmacology December 1, 2009, 76 (6) 1196-1210; DOI: https://doi.org/10.1124/mol.109.059139

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Research ArticleArticle

Subtype-Specific Differences in Corticotropin-Releasing Factor Receptor Complexes Detected by Fluorescence Spectroscopy

Laura Milan-Lobo, Ingrid Gsandtner, Erwin Gaubitzer, Dominik Rünzler, Florian Buchmayer, Gottfried Köhler, Antonello Bonci, Michael Freissmuth and Harald H. Sitte
Molecular Pharmacology December 1, 2009, 76 (6) 1196-1210; DOI: https://doi.org/10.1124/mol.109.059139
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