Protein-protein interaction and not glycosylation determines the binding selectivity of heterodimers between the calcitonin receptor-like receptor and the receptor activity-modifying proteins
Protein-protein interaction and not glycosylation determines the binding selectivity of heterodimers between the calcitonin receptor-like receptor and the receptor activity-modifying proteins
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DOI：
10.1074/jbc.m102722200
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发表时间：
2001-08-03
影响因子：
4.8
通讯作者：
Bouvier, M
中科院分区：
文献类型：
--
作者：
Hilairet, S;Foord, SM;Bouvier, M
受体活性修饰蛋白（RAMP）和降钙素受体样受体（CRLR）都是产生肾上腺髓质素（AM）和降钙素基因相关肽（CGRP）受体所必需的。在RAMP 1和CRLR共表达时，CRLR的成熟、完全糖基化形式与I-125-CGRP结合相关。相反，RAMP 2和-3促进了较小的核心糖基化CRLR形式的表达，这与AM受体药理学有关。由于核心糖基化是不成熟蛋白质的典型标志，我们测试了核心糖基化CRLR形成AM受体的假设。尽管在与RAMP 2或-3共表达时产生了显著量的核心糖基化CRLR，但交联实验显示I-125-AM仅与完全糖基化形式结合。类似地，I-125-CGRP在与RAMP 1共表达时选择性地识别成熟CRLR种类，表明糖基化并不决定配体结合选择性。我们的研究结果还表明，三个RAMP位于接近的肽结合口袋内的CRLR-RAMP异二聚体，因为I-125-AM和I-125-CGRP被纳入RAMP 2，-3，和-1，分别。交联也稳定了肽-CRLR-RAMP三元复合物，具有预期的配体选择性，表明完全加工的异二聚体代表功能性受体。总体而言，数据表明，直接的蛋白质-蛋白质相互作用决定了CRLR-RAMP复合物的药理学性质。
The receptor activity-modifying proteins (RAMPs) and the calcitonin receptor-like receptor (CRLR) are both required to generate adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors. A mature, fully glycosylated, form of CRLR was associated with I-125-CGRP binding, upon co-expression of RAMP1 and CRLR. In contrast, RAMP2 and -3 promoted the expression of smaller, core-glycosylated, CRLR forms, which were linked to AM receptor pharmacology. Since core glycosylation is classically a trademark of immature proteins, we tested the hypothesis that the core-glycosylated CRLR forms the AM receptor. Although significant amounts of core-glycosylated CRLR were produced upon co-expression with RAMP2 or -3, crosslinking experiments revealed that I-125-AM only bound to the fully glycosylated forms. Similarly, I-125-CGRP selectively recognized the mature CRLR species upon co-expression with RAMP1, indicating that the glycosylation does not determine ligand-binding selectivity. Our results also show that the three RAMPs lie close to the peptide binding pocket within the CRLR-RAMP heterodimers, since I-125-AM and I-125-CGRP were incorporated in RAMP2, -3, and -1, respectively. Cross-linking also stabilized the peptide-CRLR-RAMP ternary complexes, with the expected ligand selectivity, indicating that the fully processed heterodimers represent the functional receptors. Overall, the data indicate that direct protein-protein interactions dictate the pharmacological properties of the CRLR-RAMP complexes.