Bailey S C, Feldman L, Romanowski R R, Davis K L, Sytkowski A J
New England Deaconess Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02215.
Exp Hematol. 1993 Nov;21(12):1535-43.
The binding of erythropoietin (Epo) to its plasma membrane receptor activates signal pathways that result in erythroid cell proliferation and differentiation. To elucidate the structural features of the receptor that are important for hormone binding and signaling, we have developed a series of site-specific antibody probes. These antibodies were raised against synthetic peptides homologous to six exoplasmic domains and one cytoplasmic domain of the murine receptor and were affinity-purified by binding to their respective peptide antigen, immobilized on agarose. Western blot analyses demonstrated that the recombinant receptor expressed transiently in COS-7 cells is synthesized as three protein species of 62, 64, and 66 kd, consistent with previous observations. Importantly, probing the endogenous receptor in both virally transformed erythroleukemia cells and normal erythroid cells demonstrated similar 62- to 66-kd receptor species. The affinity-purified antibodies also recognized several antigenically related proteins. An examination of the capacity of the antireceptor antibodies to block receptor activation by Epo revealed that antibodies to five of the six exoplasmic domains blocked the receptor. This was reversed with excess Epo. Inhibition of receptor activation by antibody probes to five discrete hydrophilic domains suggests that receptor function may be critically dependent on the structural integrity (conformation) of the entire exoplasmic portion.
促红细胞生成素（Epo）与其质膜受体的结合激活信号通路，导致红系细胞增殖和分化。为了阐明受体中对激素结合和信号传导重要的结构特征，我们开发了一系列位点特异性抗体探针。这些抗体是针对与小鼠受体的六个胞外结构域和一个胞质结构域同源的合成肽产生的，并通过与固定在琼脂糖上的各自肽抗原结合进行亲和纯化。蛋白质印迹分析表明，在COS - 7细胞中瞬时表达的重组受体以62、64和66 kd的三种蛋白质形式合成，这与之前的观察结果一致。重要的是，对病毒转化的红白血病细胞和正常红系细胞中的内源性受体进行检测，发现了类似的62至66 kd的受体种类。亲和纯化的抗体还识别了几种抗原相关蛋白。对抗受体抗体阻断Epo激活受体能力的研究表明，六个胞外结构域中有五个结构域的抗体能阻断受体。加入过量的Epo可逆转这种情况。针对五个离散亲水结构域的抗体探针抑制受体激活表明，受体功能可能严重依赖于整个胞外部分的结构完整性（构象）。
