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An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images
Published on: August 31, 2012
Subtype-specific differences in corticotropin-releasing factor receptor complexes detected by fluorescence
Laura Milan-Lobo1, Ingrid Gsandtner, Erwin Gaubitzer
1Medical University of Vienna, Center for Biomolecular Medicine and Pharmacology, Institute of Pharmacology, Waehringerstrasse 13a, A-1090 Vienna, Austria.
Molecular Pharmacology
|September 17, 2009
Summary
Agonist stimulation causes corticotropin-releasing factor receptor 2 (CRFR2) to redistribute into actin-stabilized membrane domains, altering signaling duration. CRFR1 does not exhibit this subtype-specific behavior.
Area of Science:
- Cellular Biology
- Biochemistry
- Pharmacology
Background:
- G protein-coupled receptors (GPCRs) form dynamic signalosomes influenced by membrane lipids and actin cytoskeleton.
- Corticotropin-releasing factor receptors (CRFRs), specifically CRFR1 and CRFR2, are homologous but possess distinct hydrophobic cores.
Purpose of the Study:
- To investigate the agonist-induced dynamic behavior and signaling of CRFR1 and CRFR2.
- To determine the role of the actin cytoskeleton in CRFR trafficking and signaling.
Main Methods:
- Fluorescence resonance energy transfer (FRET) microscopy for assessing receptor oligomerization and beta-arrestin recruitment.
- Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) for measuring receptor diffusion and mobility.
- Actin cytoskeleton disruption using pharmacological agents.
Main Results:
- Both CRFR1 and CRFR2 form constitutive oligomers and recruit beta-arrestin upon agonist stimulation.
- CRFR2, unlike CRFR1, exhibits restricted collision coupling, fails to internalize, and shows agonist-accelerated diffusion with reduced mobile fraction.
- Disruption of the actin cytoskeleton reversed agonist-induced CRFR2 mobility changes and promoted internalization, shifting concentration-response curves.
Conclusions:
- Agonist stimulation induces CRFR2 redistribution into actin-stabilized membrane microdomains, affecting signal duration in a subtype-specific manner.
- Membrane microdomain organization and actin cytoskeleton dynamics play critical roles in modulating GPCR signaling.
- Receptor subtype differences in membrane interactions dictate the shape and duration of cellular responses.
