RESUMEN
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.
Asunto(s)
Interleucina-2/metabolismo , Conformación Proteica , Receptores de Interleucina-2/metabolismo , Unión Competitiva , ADN Complementario/genética , Humanos , Cinética , Ligandos , Fragmentos de Péptidos/metabolismo , Unión Proteica , Receptores de Interleucina-2/química , Receptores de Interleucina-2/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales CultivadasRESUMEN
To determine how adequately patients who participate as subjects for clinical investigation understand their commitment, 50 patients admitted to the Clinical Research Center of the University of Washington were interviewed. Their responses were compared with the detailed statements in the consent forms for the 23 specific protocols under which they were being studied. In spite of careful controls in accord with guidelines of a review committee, only half the patients were able to describe accurately their expectations of the studies to which they had given signed consent.