Calcitonin and Amylin Receptor Peptide Interaction Mechanisms INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS
Calcitonin and Amylin Receptor Peptide Interaction Mechanisms INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS
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DOI：
10.1074/jbc.m115.713628
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发表时间：
2016-04-15
影响因子：
4.8
通讯作者：
Pioszak, Augen A.
中科院分区：
文献类型：
--
作者：
Lee, Sang-Min;Hay, Debbie L.;Pioszak, Augen A.
受体活性修饰蛋白（RAMP 1 -3）决定了B G类蛋白偶联的降钙素受体（CTR）和CTR样受体（CLR）对降钙素（CT）、淀粉样蛋白（Amy）、降钙素基因相关肽（CGRP）的选择性和肾上腺髓质素（AM）肽。RAMP 1/2部分通过RAMP 1 Trp-84或RAMP 2 Glu-101接触不同的CGRP/AM C-末端残基来改变CGRP/AM的选择性。目前还不清楚RAMP是否使用类似的机制来调节CT和Amy的CTR亲和力，其类似物分别是骨疾病和糖尿病的治疗剂。在此，我们使用纯化的CTR胞外结构域（ECD）和栓系的RAMP 1-和RAMP 2-CTR ECD融合蛋白和拮抗剂肽再现了完整CTR、AMY（1）（CTRRAMP 1）和AMY（2）（CTRRAMP 2）受体的肽选择性。所有三种蛋白质结合鲑鱼降钙素（sCT）。将RAMPs拴系到CTR增强了rAmy、CGRP和AMY拮抗剂AC 413的结合。肽丙氨酸扫描诱变和受体结合的sCT和AC 413的建模支持其在C末端之前的TN（T/V）G基序的共享非螺旋CGRP样构象。在这个基序之后，肽分叉; sCT C-末端Pro对于受体结合至关重要，而AC 413/rAmy C-末端Tyr对结合几乎没有影响。因此，突变体RAMP 1 W84 A-和RAMP 2 E101 A-CTR ECD保留AC 413/rAmy结合。用具有C-末端残基交换的拮抗剂sCT/AC 413/rAmy变体进行的ECD结合和基于细胞的信号传导测定表明，C-末端sCT/rAmy残基身份对亲和力的影响大于选择性。rAmy（8-37）Y37 P对AMY（1）的拮抗作用增强，但仍保持选择性。这些结果揭示了RAMP如何决定CTR和CTR肽选择性的意想不到的差异，并支持RAMP变构调节CTR肽亲和力的假设。
Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY(1) (CTRRAMP1), and AMY(2) (CTRRAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY(1) while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.