Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 403, Issue 5, Pages 671-692Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.08.056
Keywords
interleukin-2; receptor assembly; lipid raft; cytoskeleton; FCS
Categories
Funding
- Ministere de l'Education Nationale et de la Recherche
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While interleukin (IL)-2 clearly initiates the sequential assembly of its soluble receptor fragments (sIL-2R) in vitro (with sIL-2R alpha first, sIL-2R beta second, and s gamma c last), the assembly mechanism of full-length subunits (IL-2R) at the surface of living lymphocytes remains to be elucidated Here we demonstrate by fluorescence cross-correlated spectroscopy that native IL-2R beta and and gamma c assemble spontaneously at the surface of hying human leukemia T cells (Kit-225 cell line) in the absence of IL-2 and with 1 1 stoichiometry The dissociation constant of the membrane-embedded IL-2R beta/gamma c complex is measured in situ Forster fluorescence resonance energy transfer analyzed by confocal microscopy of transfected COS-7 cells between combination pairs of various-length receptor chain constructions, using green fluorescent protein derivatives as cytoplasmic carboxy-terminal extensions, showed that IL-2R beta ECFP and gamma c EYFP bind each other through their extracellular domains, and that IL-2 binding brings their transmembrane domains 30 angstrom closer together These observations demonstrate that IL-2R beta/gamma c heterodimers are preformed and that their cytoplasmic domains, carrying Janus kinase (Jak) 1 and Jak3, are pulled and tethered together on cytokine binding, triggering signaling transduction IL-2 binding stabilizes IL-2/IL-2R complexes in membrane nanodomains that promote Jak1/Jak3 phosphorylation The complexes then interact with the cytoskeleton, which slows receptor diffusion (as measured by fluorescence cross-correlated spectroscopy) and promotes STAT (signal transducer and activator of transcription) 5 phosphorylation Separation of IL-2-activated receptors from Triton-lysed cells m detergent-resistant membrane nanodomains by ultracentrifugation on a sucrose gradient confirmed their presence in lipid rafts The release of the IL-2-activated receptor from cytochalasin-treated cells and the IL-2-induced recruitment of actin and tubulin, analyzed by immunoprecipitation, confirmed that the activated receptor interacts with the cytoskeleton Although IL-2R alpha (the third chain that gives the IL-2R beta/gamma c receptor core its high affinity for IL-2) is highly expressed at the cell surface and mainly clustered m membrane microdomains at the surface of Kit-225 cells, the few free IL-2R alpha present bind last to the IL-2/IL-2R beta/gamma c complex and lock IL-2 to its binding site for prolonged action, promoting signal amplification (C) 2010 Elsevier Ltd All rights reserved
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