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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor
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Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

机译：神经激肽1受体N联糖基化位点改变的功能后果

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摘要

The neurokinin 1 receptor (NK1R), a G protein-coupled receptor involved in diverse functions including pain and inflammation, has two putative N-linked glycosylation sites, Asn-14 and Asn-18. We studied the role of N-linked glycosyiation in the functioning of the NK1R by constructing three receptor mutants: two single mutants (Asn → Gln-14 and Asn → Gln-18) and a double mutant, lacking both glycosylation sites. Using a lentiviral transfection system, the mutants were stably transfected into NCM 460 cells, a nontrans-formed human colonic epithelial cell line. We observed that the magnitude of glycosylation as estimated by changes in gel migration depends on the number of glycosylation sites available, with the wild-type receptor containing the greatest amount of glycosylation. All mutant receptors were able to bind to substance P and neurokinin A ligand with similar affinities; however, the double mutant, nonglycosylated NK1R showed only half the B_(max) of the wild-type NK1R. In terms of receptor function, the ablation of both N-linked glycosylation sites did not have a profound effect on the receptors' abilities to activate the MAP kinase families (p42/p44, JNK, and p38), but did affect SP-induced IL-8 secretion. All mutants were able to internalize, but the kinetics of internalization of the double mutant receptor was more rapid, when compared with wild-type NK1R. Therefore, glycosylation of NK1R may stabilize the receptor in the plasma membrane. These results contribute to the ongoing elucidation of the role of glycosylation in G protein-coupled receptors and the study of the neurokinin receptors in particular.
机译：神经激肽1受体（NK1R）是一种G蛋白偶联受体，具有多种功能，包括疼痛和炎症，具有两个假定的N联糖基化位点Asn-14和Asn-18。我们通过构建三个受体突变体：两个单个突变体（Asn→Gln-14和Asn→Gln-18）和一个双突变体，缺少两个糖基化位点，研究了N-联糖基化在NK1R功能中的作用。使用慢病毒转染系统，将突变体稳定转染到NCM 460细胞（一种未转化的人结肠上皮细胞系）中。我们观察到，通过凝胶迁移的变化估计的糖基化程度取决于可用糖基化位点的数量，其中野生型受体含有最大量的糖基化。所有的突变受体都能够以相似的亲和力结合P物质和神经激肽A配体。但是，双突变体非糖基化的NK1R仅显示野生型NK1R的B_（max）的一半。就受体功能而言，两个N联糖基化位点的消融对受体激活MAP激酶家族（p42 / p44，JNK和p38）的能力没有重大影响，但确实影响了SP诱导的IL -8分泌物。所有突变体都能够内化，但是与野生型NK1R相比，双重突变体受体的内化动力学更快。因此，NK1R的糖基化可以稳定质膜中的受体。这些结果有助于继续阐明糖基化在G蛋白偶联受体中的作用，尤其是对神经激肽受体的研究。

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