Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding.
Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding.
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降钙素受体样受体 N 端结构域在配体结合中的作用。
DOI：
10.1021/bi049153f
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Yallampalli,Chandra
中科院分区：
文献类型：
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作者：
Chauhan,Madhu;Rajarathnam,Krishna;Yallampalli,Chandra
降钙素受体样受体 (CRLR) 是一种七跨膜 (7-TM) 结构域 B 类 G 蛋白偶联受体 (GPCR)，需要不同受体活性修饰蛋白 (RAMP) 共表达才能成为功能性降钙素基因相关肽 (CGRP) 受体或肾上腺髓质素 (AM) 受体。据报道，B 类 GPCR 的 N 末端 (Nt) 胞外区域与胰高血糖素和甲状旁腺激素等受体的配体结合。我们假设 CRLR 的 Nt 结构域 (Nt-CRLR) 是一个自主折叠单元，具有明确的结构，并参与配体结合和特异性。为了获得 Nt-CRLR 的结构和功能信息，我们在大肠杆菌中克隆并表达了 Nt-CRLR 作为融合蛋白。过表达的蛋白质形成包涵体，将其重新折叠并纯化，产生可溶性单体蛋白质。 Nt-CRLR 的远紫外 CD 和荧光光谱显示了折叠蛋白的特征。通过研究在Nt-CRLR蛋白存在下125 I-CGRP和125 I-AM与妊娠大鼠子宫膜结合的抑制来确定Nt-CRLR独立于RAMP结合CGRP和AM的能力。我们观察到 Nt-CRLR 以剂量依赖性方式抑制 125I-CGRP 和 125I-AM 与大鼠子宫的结合（IC50 分别为 0.25 和 0.29 μM）。总而言之，我们的数据提供了 Nt-CRLR 是结构化的证据，并且进一步证明结合亲和力的重要部分来自与 Nt 结构域的结合。
Calcitonin receptor-like receptor (CRLR) is a seven-transmembrane (7-TM) domain class B G protein-coupled receptor (GPCR) which requires coexpression of different receptor activity modifying proteins (RAMP) to become a functional calcitonin gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor. The N-terminal (Nt) extracellular region of class B GPCRs in ligand binding has been reported for receptors such as glucagon and parathyroid hormone. We hypothesize that the Nt-domain of CRLR (Nt-CRLR) is an autonomously folded unit possessing a well-defined structure and is involved in ligand binding and specificity. To obtain structural and functional information on the Nt-CRLR, we cloned and expressed the Nt-CRLR as a fusion protein inEscherichia coli. Overexpressed protein formed an inclusion body, which was refolded and purified, resulting in a soluble monomeric protein. Far-UV CD and fluorescence spectra of Nt-CRLR showed characteristics of a folded protein. The ability of Nt-CRLR to bind CGRP and AM independent of RAMPs was determined by studying inhibition of125I-CGRP and125I-AM binding to pregnant rat uterine membrane in the presence of Nt-CRLR protein. We observe that Nt-CRLR inhibits125I-CGRP and125I-AM binding to rat uterus in a dose-dependent fashion (IC50= 0.25 and 0.29 μM, respectively). Taken together, our data provide evidence that Nt-CRLR is structured and further that a significant part of the binding affinity comes from binding to the Nt-domain.