CDB15:0000904 IL2 — IL2RB

ABSTRACT: The structural basis of the high affinity interleukin-2 receptor which was previously reconstituted in a cultured murine T cell line, EL4 by expressing either wild-type Tac antigen complementary DNA (cDNA) or a chimeric cDNA was characterized. The chimeric cDNA encodes a membrane portion whose extracellular portion consists of that of Tac antigen whereas transmembrane and cytoplasmic portions consists of those the human insulin beta chain. The Tac antigen/anti-Tac antibody complex was treated by chemical crosslinking reagents, purified by goat anti-mouse immunoglobulin (Ig), and was analysed by SDS-PAGE. We here demonstrated the presence in mouse EL4 transfectants of a novel membrane protein which is closely associated with the products of transfected cDNAs in the absence of interleukin-2. The protein is 75 kDa in size and is detected in cells which express high affinity interleukin-2 receptor but not in cells which only express low affinity interleukin-2 receptor. The transmembrane region and the cytoplasmic region of Tac antigen is not necessary for the formation of the complex consisting of Tac antigen and 75 kDa molecule, indicating that a murine 75 kDa molecule associates with Tac antigen extra-cellularly.

ABSTRACT: Although activated human T and B lymphocytes express both high-affinity and low-affinity membrane receptors for interleukin-2 (IL-2), the structural features that distinguish these receptors have remained unresolved. The high-affinity receptors appear to mediate IL-2 induced T cell growth and internalization of IL-2, whereas no function has yet been ascribed to the low-affinity receptors. The Tac antigen is an IL-2 binding protein of relative molecular mass 55,000 (Mr 55K) that participates in the formation of both high- and low-affinity receptors. But Tac complementary DNA transfection and membrane fusion studies have suggested that additional T-cell components are required to produce high-affinity IL-2 receptors. In this study, we report the identification of a second human IL-2 binding protein that (1) has an Mr of approximately 70K, (2) lacks reactivity with the anti-Tac antibody, (3) binds IL-2 with intermediate affinity and (4) is present on the surface of resting T cells, large granular lymphocytes (natural killer cells), and certain T and B cell lines in the absence of the Tac antigen. Chemical crosslinking of 125I-labelled IL-2 bound to high-affinity IL-2 receptors produces labelling of both the p70 protein and the Tac antigen and the anti-Tac antibody blocks the crosslink detection of both of these proteins. Expression of Tac cDNA in a T cell line expressing the p70 protein, but lacking both Tac and high-affinity receptors, results in the reconstitution of high-affinity IL-2 receptors in these cells. Together, these findings suggest that the high-affinity human IL-2 receptor may be a membrane complex composed of at least the p70 protein and Tac antigen.

ABSTRACT: Biologically relevant interleukin-2 receptors (IL-2Rs) are present in two affinity states on responsive cells. High affinity receptors (HAR) apparently exist as heterotrimers (alpha, beta and gamma) while the other functional complex, the intermediate affinity receptor (IAR), is comprised of beta and gamma chains. The mechanisms by which the beta and gamma subunits contribute the formation of HAR and IAR are still unclear. Soluble forms of the beta and gamma chains were cloned, epitope-tagged, expressed in insect cells and purified. IL-2 binding and neutralization of IL-2 bioactivity by beta and gamma extracellular domains (ectodomains) was analyzed by several biochemical and biological approaches. The results indicate that beta clearly binds IL-2 with low affinity (KI-KD = 3 microM) whereas gamma binding is detectable, but of very low affinity (apparent KI > 15 microM) in the absence of beta. Interestingly, combinations of beta and gamma ectodomains interact to bind IL-2 with higher affinity and greater stability than either chain alone. An apparent stable binding complex is formed when beta, gamma, and IL-2 are combined. Ligand binding by the beta and gamma chains in solution is specific for IL-2 and is of sufficient affinity and stability to effectively neutralize IL-2 in biological assays (binding IC50 = biological IC50). Direct analyses of binding kinetics by surface plasmon resonance reveals that the increased affinity and biological neutralizing ability of beta gamma, as compared to beta, is due to a very slow dissociation rate contributed by the gamma ectodomain. While IL-2R beta and gamma cytoplasmic and transmembrane domains are not essential for interactive binding of IL-2, they may contribute to IL2 binding affinity. The recognition and association of IL-2 by the beta gamma IAR appears to be contributed primarily by the beta chain while the stability and dissociation is likely dominated by the gamma chain. It is anticipated that the gamma subunit functions in a similar manner when participating in high affinity IL-4 and IL-7 binding.