Kuziel W A, Ju G, Grdina T A, Greene W C
Howard Hughes Medical Institute, Department of Medicine, Durham, NC.
J Immunol. 1993 Apr 15;150(8 Pt 1):3357-65.
Binding of IL-2 to the high affinity IL-2R results in the formation of a stable complex consisting of IL-2 and at least three distinct, noncovalently associated receptor subunits: IL-2R alpha (p55), IL-2R beta (p70-75), and IL-2R gamma (p64). Ligand binding also stimulates the rapid endocytosis of this receptor complex. To gain further insight into the function of the various subunits of the multicomponent high affinity IL-2R complex, we have carried out binding, internalization, and proliferation assays using an IL-2 analog designated F42A that contains a single Phe for Ala amino acid substitution at position 42. This mutation markedly reduces the intrinsic affinity of the resultant IL-2 analog for the low affinity IL-2R alpha subunit although having little or no effect on binding to the IL-2R beta (or IL-2R beta/gamma) intermediate affinity receptor. We have confirmed that F42A does not bind to the IL-2R alpha chain when expressed alone on MT-1 cells or in the presence of the large or small excess of IL-2R beta chains present on either YT-1 cells or forskolin-induced YT-1 cells, respectively. However, although F42A does not interact with the large number of low affinity IL-2R alpha chains present on HUT 102B2 cells, this ligand does bind to the small number of IL-2R beta chains present on these cells with at least 10-fold higher than expected affinity. These findings indicate that excess IL-2R alpha chains may exert positive effects on binding perhaps by changing the conformation of IL-2R beta. In F42A-stimulated internalization assays on forskolin-induced YT-1 cells, the IL-2R alpha chain is consistently endocytosed together with the IL-2R beta subunit indicating that IL-2R alpha is stably associated with the F42A-IL-2R beta complex even though the alpha-subunit contributes little if any affinity to the F42A binding reaction. In proliferation assays on mouse IL-3-dependent pro-B BA/F3 cells stably coexpressing transfected human IL-2R alpha and IL-2R beta subunits (but a mouse IL-2R gamma subunit), F42A proved to be a more effective agonist of growth than wild-type IL-2. These results suggest that the interaction between wild-type IL-2 but not F42A and the IL-2R alpha subunit in this mixed species high affinity receptor complex may induce an unfavorable receptor conformation leading to diminished rather than enhanced growth signal transduction.
白细胞介素-2（IL-2）与高亲和力IL-2受体结合，导致形成一种稳定的复合物，该复合物由IL-2和至少三个不同的、非共价结合的受体亚基组成：IL-2Rα（p55）、IL-2Rβ（p70 - 75）和IL-2Rγ（p64）。配体结合还会刺激该受体复合物的快速内吞作用。为了更深入了解多组分高亲和力IL-2R复合物各亚基的功能，我们使用一种名为F42A的IL-2类似物进行了结合、内化和增殖测定，该类似物在第42位氨基酸处含有一个苯丙氨酸被丙氨酸取代的单氨基酸替换。这种突变显著降低了所得IL-2类似物对低亲和力IL-2Rα亚基的内在亲和力，尽管对与IL-2Rβ（或IL-2Rβ/γ）中亲和力受体的结合几乎没有影响。我们已经证实，当单独在MT-1细胞上表达或分别在YT-1细胞或福斯可林诱导的YT-1细胞上存在大量或少量过量的IL-2Rβ链时，F42A不与IL-2Rα链结合。然而，尽管F42A不与HUT 102B2细胞上存在的大量低亲和力IL-2Rα链相互作用，但这种配体确实与这些细胞上存在的少量IL-2Rβ链结合，其亲和力至少比预期高10倍。这些发现表明，过量的IL-2Rα链可能通过改变IL-2Rβ的构象对结合产生积极影响。在福斯可林诱导的YT-1细胞上进行的F42A刺激的内化测定中，IL-2Rα链始终与IL-2Rβ亚基一起被内吞，这表明IL-2Rα与F42A - IL-2Rβ复合物稳定结合，尽管α亚基对F42A结合反应的亲和力贡献很小或没有贡献。在稳定共表达转染的人IL-2Rα和IL-2Rβ亚基（但为小鼠IL-2Rγ亚基）的小鼠IL-3依赖性前B细胞BA/F3细胞上进行的增殖测定中，F42A被证明是比野生型IL-2更有效的生长激动剂。这些结果表明，在这种混合物种高亲和力受体复合物中，野生型IL-2而非F42A与IL-2Rα亚基之间的相互作用可能诱导不利的受体构象，导致生长信号转导减弱而非增强。
