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.

Introduction

Interleukin (IL)-2 is a major cytokine that possesses inflammatory and immune properties.¹ It exerts its effects on many cell types, including T and B lymphocytes, monocytes, and natural killer cells. Its role in lymphocyte proliferation, activation-induced cell death, and homeostasis regulation has been extensively studied.²

The effects of IL-2 on its target cells are mediated through cytoplasmic signaling pathways such as the Janus kinase (Jak)/STAT (signal transducer and activator of transcription), PI3K/Akt, and MAPK pathways involving specific lymphocyte surface receptors made up of two chains (IL-2Rβ/γc) or three chains (IL-2Rα, IL-2Rβ, and γc).² IL-2Rβ (CD122; 67–75 kDa) is shared by both IL-2 receptors and IL-15 receptors; it has a large intracytoplasmic domain (286 residues) and plays a critical role in signal transduction.³ The last component γc (CD132; 58–64 kDa) has a rather short cytoplasmic domain (89 residues); this chain is shared by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors.2, 4 Recent evidence indicates that it is also implicated in the growth hormone receptor.⁵ When IL-2Rβ and γc associate, they form an intermediate-affinity receptor common to IL-2 and IL-15, with K_d values of about 1 and 0.25 nM, respectively.⁶ Expression of the third chain proprietary for IL-2 or IL-15, namely IL-2Rα (CD25; 55 kDa) or IL-15Rα, leads to the formation of a three-component high-affinity receptor from the IL-2Rβ/γc core that is specific for IL-2 or IL-15 in the range 10–15 pM.⁶ As single chains, IL-2Rα and IL-15Rα bind their cognate cytokine with dissociation constants of about 19 and 35 pM, respectively, and the contribution of their short cytoplasmic domains (13 and 39 residues, respectively) to cytokine-induced signal transduction is still under debate.7, 8 The cytoplasmic domains of IL-2Rβ and γc constitutively bind Jak1 and Jak3, respectively,⁹ which phosphorylate each other on cytokine binding then phosphorylate IL-2Rβ (Tyr536), providing a single binding site mainly for either STAT5a or STAT5b, but also for STAT1 or STAT3.¹⁰ STATs are then phosphorylated by Jak pairs, dissociate from IL-2R, dimerize,¹¹ bind importins, and translocate through pores in the nucleus, where they regulate cytokine-dependent gene transcription.

The structure of the IL-2/IL-2R complex was solved from the soluble extracellular fragments IL-2Rα, IL-2Rβ, and γc.¹² Its architecture and organization are similar to those of the human growth hormone and erythropoietin receptor complexes,¹³ with addition of the IL-2Rα chain to the dimer core, capping IL-2 and making it out of reach of the IL-2Rβ and γc chains. No structural data are yet available for IL-2Rβ and γc cytoplasmic domains or for any homologous protein domain.

The assembly pathway of the three solubilized extracellular domains sIL-2Rα, sIL-2Rβ, and sγc has been studied in vitro by isothermal calorimetry¹⁴ and surface plasmon resonance¹⁵ in the presence and in the absence of IL-2. sIL-2Rβ/sγc association is not detectable in vitro when the protein partner concentration is in the submicromolar range and when the soluble receptor assembly starts from the IL-2/sIL-2Rα nucleus. However, we observed in previous studies that IL-2Rβ/γc coimmunoprecipitated in the absence of IL-2 from cell lysates, and that the cytokine stabilizes preassociated hetererodimers.¹⁶ Human leukemia CD4 T-cell lines (Kit-225) express all three receptor subunits IL-2Rα, IL-2Rβ, and γc. The assembly of IL-2Rβ and γc has also been detected by Förster fluorescence resonance energy transfer (FRET) in the absence of IL-2 between labeled monoclonal antibody (mAb) pairs at the surface of fixed Kit-225 cells analyzed by flow cytometry.17, 18 As the affinity of IL-2 for IL-2Rβ/γc is 20-fold its affinity for IL-2Rα expressed at the cell surface, any extrapolation of the receptor assembly pathway observed in solution to membrane-embedded chains is questionable even in Kit-225 cell lines where IL-2Rα has 10-fold the abundance of IL-2Rβ and γc. Indeed, most IL-2Rα are observed packed in membrane microdomains (diameter, 200–1000 nm) at the Kit-225 cell surface,¹⁹ and fewer chains remain available for formation of the ternary complex receptor outside these domains.

In this work, we aimed to describe the IL-2R assembly mechanism at the surface of living cells and the IL-2-induced conformational change that it undergoes. We took full advantage of recent developments in time-resolved fluorescence microimaging to analyze the IL-2R chain assembly by FRET²⁰ and fluorescence autocorrelated spectroscopy (FCS)/fluorescence cross-correlated spectroscopy (FCCS).²¹ The degree of freedom of receptor chains at the plasma membrane was investigated by FCS: lipid-dependent compartmentalization and cytoskeleton-dependent confinement were extrapolated from their lateral diffusion rate offset before and after IL-2 binding.22, 23, 24 Experiments on transiently transfected COS-7 cells expressing tagged proteins as controls were initiated and then repeated on lymphocytes using labeled antibodies to detect receptor chains. Our work here on human leukemia CD4 T-cell lines demonstrates that: (1) the IL-2Rβ/γc heterodimer is formed prior to IL-2 binding; (2) IL-2 changes the conformation of IL-2Rβ/γc; and (3) IL-2 binding slows the receptor diffusion rate and increases its confinement time through formation of the signaling complex on the cytoplasmic domains and interaction with the cytoskeleton meshwork. We show here that there is a functional link between receptor compartmentalization in lipid rafts and Jak1/3 phosphorylation, and between receptor confinement by the cytoskeleton and STAT5 phosphorylation. These experimental results are discussed in the light of molecular models.