Rozakis-Adcock M, Kelly P A
Laboratory of Molecular Endocrinology, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada.
J Biol Chem. 1991 Sep 5;266(25):16472-7.
The recent isolation and sequencing of the rat liver prolactin (PRL) receptor cDNA (clone F3) revealed that the receptor is a small molecular weight protein (nonglycosylated form, Mr 33,000; glycosylated form, Mr 42,000) comprised of 291 amino acids. A second form of the PRL receptor exists (591 amino acids) that contains a much longer cytoplasmic domain. In the present study, site-directed point mutations of the 5 conserved cysteine (Cys) residues and of the three potential N-linked glycosylation sites in the extracellular domain of the rat PRL receptor were constructed to assess their involvement in hormone binding. In addition, a truncation mutant (T delta 237) lacking 55 of 57 intracellular amino acids was constructed to determine the influence of the cytoplasmic domain on ligand-receptor interactions. Binding studies of transiently transfected COS-7 cells demonstrated that serine substitution of the first 4 Cys residues (Cys12, Cys22, Cys51, and Cys62) completely eliminated binding of 125I-ovine PRL and 125I-U5 and -U6, two monoclonal antibodies that bind the receptor molecule outside the PRL-binding domain. RNA blot analysis of the transfected cells showed that both the wild-type and mutant clones had similar levels of expression of receptor mRNA. Immunoblot analysis demonstrated that lack of PRL binding in these mutants was not due to incomplete processing of the protein, since the fully glycosylated Mr 42,000 form of the receptor was seen. Mutation of Cys184 had no effect on affinity or dimerization capacity of the receptor, suggesting the 5th cysteine is not directly involved in the binding domain. Carbohydrate groups of some receptors have been shown to be involved in ligand-receptor interactions as well as intracellular trafficking. This does not appear to be the case for the PRL receptor, since there was no corresponding decrease in affinity for PRL or cell surface receptor expression, following mutation of each of the 3 asparagine residues to aspartate. Interestingly, T delta 237 showed a 4-5-fold increase in affinity for PRL as well as a marked increase in the number of receptor sites. Whole cell binding assays also demonstrated that loss of the cytoplasmic domain lead to inefficient recycling of the receptor. These studies suggest that the first 4 conserved Cys residues are crucial for ligand binding.(ABSTRACT TRUNCATED AT 400 WORDS)
最近对大鼠肝脏催乳素（PRL）受体cDNA（克隆F3）的分离和测序显示，该受体是一种小分子量蛋白质（非糖基化形式，Mr 33,000；糖基化形式，Mr 42,000），由291个氨基酸组成。存在PRL受体的第二种形式（591个氨基酸），其含有长得多的胞质结构域。在本研究中，构建了大鼠PRL受体胞外结构域中5个保守半胱氨酸（Cys）残基和3个潜在N-连接糖基化位点的定点突变，以评估它们在激素结合中的作用。此外，构建了一个缺失57个胞内氨基酸中55个的截短突变体（T delta 237），以确定胞质结构域对配体-受体相互作用的影响。对瞬时转染的COS-7细胞的结合研究表明，前4个Cys残基（Cys12、Cys22、Cys51和Cys62）的丝氨酸替代完全消除了125I-羊催乳素以及125I-U5和-U6（两种结合PRL结合域外受体分子的单克隆抗体）的结合。对转染细胞的RNA印迹分析表明，野生型和突变型克隆的受体mRNA表达水平相似。免疫印迹分析表明，这些突变体中PRL结合的缺乏不是由于蛋白质加工不完全，因为可以看到受体的完全糖基化Mr 42,000形式。Cys184的突变对受体的亲和力或二聚化能力没有影响，表明第5个半胱氨酸不直接参与结合域。一些受体的碳水化合物基团已被证明参与配体-受体相互作用以及细胞内运输。PRL受体似乎并非如此，因为在将3个天冬酰胺残基中的每一个突变为天冬氨酸后，对PRL的亲和力或细胞表面受体表达没有相应降低。有趣的是，T delta 237对PRL的亲和力增加了4-5倍，并且受体位点的数量也显著增加。全细胞结合试验还表明，胞质结构域的缺失导致受体的再循环效率低下。这些研究表明，前4个保守的Cys残基对配体结合至关重要。（摘要截短于400字）
