title: IL-2 induces conformational changes in its preassembled receptor core, which then migrates in lipid raft and binds to the cytoskeleton meshwork.
abstract: While interleukin (IL)-2 clearly initiates the sequential assembly of its soluble receptor fragments (sIL-2R) in vitro (with sIL-2Rα first, sIL-2Rβ second, and sγc 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-2Rβ and γc assemble spontaneously at the surface of living 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-2Rβ/γc complex is measured in situ. Förster 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-2Rβ:ECFP and γc:EYFP bind each other through their extracellular domains, and that IL-2 binding brings their transmembrane domains 30 Å closer together. These observations demonstrate that IL-2Rβ/γc 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 in 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-2Rα (the third chain that gives the IL-2Rβ/γc receptor core its high affinity for IL-2) is highly expressed at the cell surface and mainly clustered in membrane microdomains at the surface of Kit-225 cells, the few free IL-2Rα present bind last to the IL-2/IL-2Rβ/γc complex and lock IL-2 to its binding site for prolonged action, promoting signal amplification.
authors.affiliation: Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, Paris, France.
authors.forename: Anne-Hélène
authors.initials: AH
authors.lastname: Pillet
authors.forename: Vincent
authors.initials: V
authors.lastname: Lavergne
authors.forename: Virginie
authors.initials: V
authors.lastname: Pasquier
authors.forename: Franck
authors.initials: F
authors.lastname: Gesbert
authors.forename: Jacques
authors.initials: J
authors.lastname: Thèze
authors.forename: Thierry
authors.initials: T
authors.lastname: Rose
mesh_terms: D000818:Animals; D001665:Binding Sites; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007376:Interleukin-2 / Q000737:chemistry* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D015375:Receptors, Interleukin-2 / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D007376:Interleukin-2; D008164:Luminescent Proteins; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins