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首页> 外文期刊>Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research >Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor Type 1 membrane translocation and function.
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Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor Type 1 membrane translocation and function.

机译：细胞内环1中第217位的半胱氨酸在人PTH受体1型膜的转运和功能中起着关键作用。

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PTHR1 mutants lacking endogenous cysteines in transmembrane and intracellular domains were generated. Mutant receptors were tested for their biological activities and mRNA and cell surface expression levels. C217 in intracellular loop 1 was determined to play a critical role in cell surface translocation and function of the receptor. INTRODUCTION: Elucidating the role of different domains of PTH receptor 1 (PTHR1) is essential for understanding the mechanism of ligand-receptor interactions. Here we present a study directed at determining the importance of cysteine residues present in the intracellular and transmembrane (TM) domains of the receptor. MATERIALS AND METHODS: Mutant receptors were generated by site-directed mutagenesis. Biological activities were characterized by adenylyl cyclase and competition binding assays. RT-PCR, ELISA, and immunofluorescence microscopy were carried out to determine receptor mRNA and protein expression levels. RESULTS: Mutations C460L and C462L in TM7, C568L in the C-terminal intracellular domain of the receptor, and removal of C397 in intracellular loop (ICL)3 by insertion of cleavage sites for Factor Xa did not affect binding affinity of PTH or agonist-induced adenylyl cyclase activity, although maximal responses (IC(max) and EC(max)) were decreased. However, mutations C217L in ICL1 or both C217L and C568L simultaneously resulted in a decrease in binding and loss of adenylyl cyclase activity. RT-PCR results showed that the observed changes in binding and activity were not caused by changes in mRNA expression. Next, we determined cell surface and total expression of the wildtype and mutant receptors by ELISA. We found that mutations of C460/C462 to L moderately decreased transfer of receptors to the cell surface. However, mutation of C217 to L in the ICL1 drastically reduced cell surface expression. Immunofluorescence and confocal microscopy studies confirmed reduced cell surface expression of receptors containing the C217L mutation. Similar results were obtained when replacing C217 and C460/C462 of the receptor with A instead of L. CONCLUSIONS: Our studies indicate that the cysteine at position 217 in ICL1 plays a critical role in translocation to the cell surface and biological function of PTHR1.
机译：PTHR1突变体缺乏跨膜和细胞内域内源性半胱氨酸。测试突变受体的生物学活性以及mRNA和细胞表面表达水平。已确定细胞内环1中的C217在细胞表面易位和受体功能中起关键作用。简介：阐明PTH受体1（PTHR1）不同结构域的作用对于理解配体-受体相互作用的机制至关重要。在这里，我们提出了一项研究，旨在确定受体的细胞内和跨膜（TM）域中存在的半胱氨酸残基的重要性。材料与方法：突变受体是通过定点诱变产生的。通过腺苷酸环化酶和竞争结合测定来表征生物活性。进行RT-PCR，ELISA和免疫荧光显微镜检查以确定受体mRNA和蛋白质表达水平。结果：TM7的C460L和C462L突变，受体的C末端细胞内结构域中的C568L突变，以及通过插入Xa因子切割位点去除细胞内环（ICL）3中的C397均不影响PTH或激动剂-诱导腺苷酸环化酶活性，尽管最大响应（IC（最大）和EC（最大））降低。但是，ICL1中的C217L突变或C217L和C568L都同时导致结合减少和腺苷酸环化酶活性降低。 RT-PCR结果表明，观察到的结合和活性变化不是由mRNA表达的变化引起的。接下来，我们通过ELISA确定了细胞表面以及野生型和突变型受体的总表达。我们发现，C460 / C462突变为L会适度降低受体向细胞表面的转移。但是，ICL1中C217突变为L大大降低了细胞表面表达。免疫荧光和共聚焦显微镜研究证实了含有C217L突变的受体的细胞表面表达降低。当用A代替L代替受体的C217和C460 / C462时，获得了相似的结果。结论：我们的研究表明ICL1中217位的半胱氨酸在PTHR1向细胞表面的转运和生物学功能中起关键作用。

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