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Stoichiometric Structure-Function Analysis of the Prolactin Receptor Signaling Domain by Receptor Chimeras

机译：催乳素嵌合体的催乳素受体信号传导域的化学计量结构-功能分析

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The intracellular domain of the prolactin (PRL) receptor (PRLr) is required for PRL-induced signaling and proliferation. To identify and test the functional stoichiometry of those PRLr motifs required for transduction and growth, chimeras consisting of the extracellular domain of either the α or β subunit of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellular domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one α- and one β-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimerization of the PRLr intracellular domain. To that end, the extracellular domains of the α- and β-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed α278, α294, α300, α322, or β322; (ii) PRLr tyrosine replacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms. These chimeras were cotransfected into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses. Data from these studies revealed that heterodimeric complexes of the wild type with C-terminal truncation mutants of the PRLr intracellular domain were incapable of ligand-induced signaling or proliferation. Replacement of any single tyrosine residue (Y309F or Y382F) in the dimerized PRLr complex resulted in a moderate reduction of receptor-associated Jak2 activation and proliferation. In contrast,trans replacement of these residues (i.e., αY309F and βY382F) markedly reduced ligand-driven Jak2 activation and proliferation, while cis replacement of both tyrosine residues in a single intracellular domain (i.e., αY309+382F) produced an inactive signaling complex. Analysis of these GM-CSFr–PRLr complexes revealed equivalent levels of Jak2 in association with the mutant receptor chains, suggesting that the tyrosine residues at 309 and 382 do not contribute to Jak association, but instead to its activation. Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes. These data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr complex, contribute to PRL-driven signaling and proliferation. Furthermore, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.
机译：催乳素（PRL）受体（PRLr）的胞内结构域是PRL诱导的信号传导和增殖所必需的。为了鉴定和测试转导和生长所需的PRLr基序的功能化学计量，嵌合体由人粒细胞-巨噬细胞集落刺激因子（GM-CSF）受体（GM-CSFr）的α或β亚基的胞外域组成合成大鼠PRLr的胞内结构域。由于GM-CSF的高亲和力结合是由一个α-和一个β-GM-CSFr的特定配对产生的，因此GM-CSFr / PRLr嵌合体的使用可实现PRLr细胞内结构域的定向二聚化。为此，将α-和β-GM-CSFr的细胞外结构域缀合至以下突变之一：（i）PRLr C-末端截短，称为α278，α294，α300，α322或β322; （ii）PRLr酪氨酸替代品，称为Y309F，Y382F或Y309 + 382F;或（iii）PRLr野生型短，中或长同工型。将这些嵌合体共转染到细胞因子应答的Ba / F3系中，并通过配体结合以及Northern和Western印迹分析证实了它们的表达。这些研究的数据表明，野生型的异二聚体与PRLr细胞内结构域的C端截短突变体不能配体诱导的信号传导或增殖。二聚化的PRLr复合物中任何单个酪氨酸残基（Y309F或Y382F）的置换导致受体相关的Jak2活化和增殖的适度降低。相反，这些残基的 trans 替换（即αY309F和βY382F）显着降低了配体驱动的Jak2激活和增殖，而单个细胞内的两个酪氨酸残基的 cis 替换域（即，αY309+ 382F）产生了无活性的信号复合物。对这些GM-CSFr-PRLr复合物的分析显示，与突变受体链相关的Jak2水平相同，这表明309和382处的酪氨酸残基对Jak缔合没有贡献，而是对其活化起作用。来自已知PRLr受体同工型（短-中间，短-长和中-长）的细胞内结构域的异二聚体配对也产生无活性的受体复合物。这些数据证明二聚化的PRLr复合物的C末端的309和382处的酪氨酸残基以及其他残基有助于PRL驱动的信号传导和增殖。此外，这些发现表明二聚受体复合物中 trans 中Y309和Y382配对的功能要求。

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