Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay
Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay
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DOI：
10.1073/pnas.93.8.3455
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发表时间：
1996-04-16
影响因子：
11.1
通讯作者：
Dickerson, IM
中科院分区：
文献类型：
--
作者：
Luebke, AE;Dahl, GP;Dickerson, IM
使用表达克隆策略分离编码赋予非洲爪蟾卵母细胞降钙素基因相关肽（CGRP）反应性的蛋白质的cDNA，通过使用基于囊性纤维化跨膜电导调节器（CFTR）的测定来筛选豚鼠Corti器官（哺乳动物听觉器官）cDNA文库。CFTR是磷酸化后被激活的氯离子通道；该通道活性被用作 CGRP 诱导的细胞内激酶激活的传感器，通过使用这种卵母细胞 CFTR 测定筛选了来自豚鼠 Corti 器官的 cDNA 文库，鉴定出包含编码小亲水蛋白的开放阅读框的 cDNA，该亲水蛋白被推测为 CGRP 受体或 CGRP 受体复合物的组成部分。该 CGRP 受体组分蛋白赋予 CFTR 测定 CGRP 特异性激活，因为在应用降钙素、胰淀素、神经肽 Y、血管活性肠肽或 β-内啡肽时未检测到激活。原位杂交证明 CGRP 受体组分蛋白在 Corti 器官的外毛细胞中表达，并与含有 CGRP 的传出神经末梢共定位。
An expression-cloning strategy was used to isolate a cDNA that encodes a protein that confers calcitonin gene-related peptide (CGRP) responsiveness to Xenopus laevis oocytes, A guinea pig organ of Corti (the mammalian hearing organ) cDNA library was screened by using an assay based on the cystic fibrosis transmembrane conductance regulator (CFTR), The CFTR is a chloride channel that is activated upon phosphorylation; this channel activity was used as a sensor for CGRP-induced activation of intracellular kinases, A cDNA library from guinea pig organ of Corti was screened by using this oocyte-CFTR assay, A cDNA was identified that contained an open reading frame coding for a small hydrophilic protein that is presumed to be either a CGRP receptor or a component of a CGRP receptor complex. This CGRP receptor component protein confers CGRP-specific activation to the CFTR assay, as no activation was detected upon application of calcitonin, amylin, neuropeptide Y, vasoactive intestinal peptide, or beta-endorphin, In situ hybridization demonstrated that the CGRP receptor component protein is expressed in outer hair cells of the organ of Corti and is colocalized with CGRP-containing efferent nerve terminals.