Rozakis-Adcock M, Kelly P A
Laboratory of Molecular Endocrinology, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 1992 Apr 15;267(11):7428-33.
The prolactin (PRL) receptor, a lactogen- and primate somatogen-binding protein, is a member of an expanding superfamily (cytokine/growth hormone (GH)/PRL) of single membrane-spanning receptors. Two features commonly shared among this group of proteins are the presence of two pairs of cysteines, generally found in the N-terminal region of the extracellular domain, and a WSxWS (WS) motif, frequently located proximal to the transmembrane domain. We have recently shown the 4 cysteines to be critical to the maintenance of the structural and functional integrity of the PRL-receptor. In the present study, we prepared a set of eight chimeric rat PRL/human GH receptors and several alanine mutants, to assess the importance of the Cys-rich domain (residues 12-68) in confering specificity to PRL binding. The role of the WS motif in high affinity binding was also investigated. Binding of 125I-labeled ovine PRL or human GH to membrane preparations from COS-7 cells transiently expressing the mutant receptors have defined a region within the first disulfide loop (residues Arg13, Asp16, Glu18) and the set of lactogen-specific sequences between the two pairs of cysteines as key determinants of PRL-binding specificity, which converge to form a patch on a two-dimensional model of the PRL receptor. We also demonstrate that, although PRL- and GH-specific determinants overlap in certain areas, they are not identical. Finally, substitution of the WS motif with alanine residues precludes high affinity binding to ovine PRL and human GH and suggests that this structural element may provide a target site for the interaction of an accessory protein necessary for the formation of a high-affinity receptor complex.
催乳素（PRL）受体是一种结合催乳素和灵长类生长激素的蛋白质，属于不断扩大的单跨膜受体超家族（细胞因子/生长激素（GH）/PRL）的成员。这类蛋白质通常共有的两个特征是存在两对半胱氨酸，一般位于细胞外结构域的N端区域，以及一个WSxWS（WS）基序，常位于跨膜结构域附近。我们最近发现这4个半胱氨酸对于维持PRL受体的结构和功能完整性至关重要。在本研究中，我们制备了一组8个嵌合大鼠PRL/人GH受体和几个丙氨酸突变体，以评估富含半胱氨酸结构域（第12 - 68位氨基酸残基）在赋予PRL结合特异性方面的重要性。还研究了WS基序在高亲和力结合中的作用。用125I标记的绵羊PRL或人GH与瞬时表达突变受体的COS - 7细胞膜制剂结合，确定了第一个二硫键环内的一个区域（第13位精氨酸、第16位天冬氨酸、第18位谷氨酸）以及两对半胱氨酸之间的一组催乳素特异性序列是PRL结合特异性的关键决定因素，它们在PRL受体的二维模型上汇聚形成一个区域。我们还证明，虽然PRL和GH特异性决定因素在某些区域重叠，但并不相同。最后，用丙氨酸残基取代WS基序会排除与绵羊PRL和人GH的高亲和力结合，这表明该结构元件可能为形成高亲和力受体复合物所需的辅助蛋白相互作用提供一个靶点。
