Modulation of growth factor receptor function by isoform heterodimerization
Modulation of growth factor receptor function by isoform heterodimerization
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DOI：
10.1073/pnas.93.12.5947
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发表时间：
1996-06-11
影响因子：
11.1
通讯作者：
Clevenger, CV
中科院分区：
文献类型：
--
作者：
Chang, WP;Clevenger, CV
催乳素（PRL）依赖性信号的激活是配体诱导的受体二聚化（PRLr）的结果。虽然三种PRLr异构体（短、中、长）已经被表征，并且在prl应答组织中有不同的共表达，但配体诱导的PRLr异构体异二聚化的功能影响尚未被研究。合成了人粒细胞-巨噬细胞集落刺激因子受体(GM-CSFr) α亚基或β亚基胞外结构域和大鼠PRLr中间或短亚型（PRLr- 1或PRLr- s）胞内结构域的嵌合体。由于GM-CSF的高亲和力结合是由一个α和β GM-CSFr对的细胞外结构域介导的，因此使用GM-CSFr/PRLr嵌合体特异性地指导了配体-受体复合物中PRLr细胞内结构域的二聚化。Northern blot和免疫沉淀分析表明，这些构建体稳定转染到Ba/F3细胞系中，流式细胞术显示，植红蛋白结合的人GM-CSF与转染物特异性结合，证实了嵌合受体在细胞表面的表达。当测试它们对GM-CSF的增殖能力时，只有表达GM-CSFr/ prlr - 1同源二聚体的嵌合转染显示出显著的[H-3]胸腺嘧啶掺入。表达GM-CSFr/PRLr-S同型二聚体或GM-CSFr/PRLr-S+I异源二聚体的转染物刺激GM-CSF均不能诱导增殖。与这些数据一致，gm - csf诱导的两种磷酸酪氨酸激酶Jak2和Fyn的激活仅在同源二聚体GM-CSFr/ prlr - 1中观察到。这些结果表明，PRLr-S作为显性阴性亚型发挥作用，下调由受体复合物介导的信号传导和增殖。因此，在PRLr- 1的羧基端激活Jak2和Fyn所必需的结构基序，在PRLr- s中不存在，在二聚体PRLr复合物的每个成员中都是必需的。
Activation of prolactin (PRL)-dependent signaling occurs as the result of ligand-induced dimerization of receptor (PRLr). Although three PRLr isoforms (short, intermediate, and long) have been characterized and are variably coexpressed,in PRL-responsive tissues, the functional effects of ligand-induced PRLr isoform heterodimerization have not been examined, To determine whether heterodimeric PRLr complexes were capable of ligand-induced signaling and cellular proliferation, chimeras consisting of the extracellular domain of either the alpha or beta subunit of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFr) and the intracellular domain of the rat intermediate or short PRLr isoforms (PRLr-I or PRLr-S) were synthesized. Because high affinity binding of GM-CSF is mediated by the extracellular domain of one alpha and beta GM-CSFr pair, use of GM-CSFr/PRLr chimera specifically directed the dimerization of the PRLr intracellular domains within ligand-receptor complexes. Stable transfection of these constructs into the Ba/F3 line was demonstrated by Northern blot and immunoprecipitation analyses, Flow cytometry revealed specific binding of a phycoerythrin-conjugated human GM-CSF to the transfectants, confirming cell surface expression of the chimeric receptors. When tested for their ability to proliferate in response to GM-CSF, only chimeric transfectants expressing GM-CSFr/PRLr-I homodimers demonstrated significant [H-3]thymidine incorporation. GM-CSF stimulation of transfectants expressing either GM-CSFr/PRLr-S homodimers or GM-CSFr/PRLr-S+I heterodimers failed to induce proliferation. Consistent with these data, the GM-CSF-induced activation of two phosphotyrosine kinases, Jak2 and Fyn, was observed only in homodimeric GM-CSFr/PRLr-I transfectants. These results show that the PRLr-S functions as a dominant negative isoform, down-regulating both signaling and proliferation mediated by the receptor complex. Thus, structural motifs necessary for Jak2 and Fyn activation within the carboxy terminus of the PRLr-I, absent in the PRLr-S, are required in each member of the dimeric PRLr complex.