Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization.
Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization.
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DOI：
10.1016/j.bpc.2020.106477
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发表时间：
2020-12
影响因子：
3.8
通讯作者：
Pioszak AA
中科院分区：
文献类型：
--
作者：
Lee S;Pioszak AA
肽激素胰淀素受体是降钙素受体（CTR）和称为受体活性修饰蛋白（RAMP）的辅助蛋白的复合物。CTR的可溶性胞外域（ECD）是肽类激素降钙素的重要结合位点。RAMP也具有ECD，并且CTR ECD与RAMP ECD的结合增强肽激素胰淀素的亲和力。然而，RAMP ECD缔合如何增强胰淀素亲和力的机制仍然难以捉摸。在这里，我们报告的证据支持直接的拮抗胰淀素类似物AC 413和RAMP 2 ECD之间的分子相互作用。我们用荧光偏振法（FP）测定了FITC标记的肽对纯化受体ECD的亲和力。我们首先发现，将RAMP 2 ECD添加到麦芽糖结合蛋白（MBP）标记的CTR ECD和工程化的MBP标记的RAMP 2 ECD-CTR ECD融合蛋白（MBP-RAMP 2-CTR ECD融合物）增强了AC 413亲和力。这表明这些重组ECD系统代表功能性胰淀素受体。有趣的是，AC 413 C-末端残基Tyr 25（Y25）至Pro突变消除了其对MBP-RAMP 2-CTR ECD融合体的选择性亲和力，表明AC 413 C-末端残基在胰淀素受体选择性中的关键作用。我们的RAMP 2 ECD：CTR ECD复合物的结构模型预测了AC 413 C-末端残基Y25与RAMP 2 Glu 101（E101）的分子相互作用。我们的FP肽结合测定显示MBP-RAMP 2-CTR ECD融合物的RAMP 2 E101 A突变使AC 413亲和力降低7倍，而具有Y25 P突变的AC 413的亲和力变化最小。一致地，AC 413对无MBP的RAMP 2-CTR ECD融合蛋白的结合亲和力也因RAMP 2 E101 A突变而显著降低。总之，我们的结果支持AC 413 C-末端残基Y25和RAMP 2 E101之间的分子相互作用，扩大了我们对辅助蛋白RAMP 2如何增强肽激素胰淀素对其受体的亲和力的理解。
The peptide hormone amylin receptor is a complex of the calcitonin receptor (CTR) and an accessory protein called receptor activity-modifying proteins (RAMPs). The soluble extracellular domain (ECD) of CTR is an important binding site of peptide hormone calcitonin. RAMPs also have an ECD and the association of CTR ECD with RAMP ECD enhances the affinity of peptide hormone amylin. However, the mechanism of how RAMP ECD association enhances amylin affinity remains elusive. Here, we report evidence supporting direct molecular interaction between an antagonistic amylin analog AC413 and RAMP2 ECD. We measured FITC-labeled peptide affinity for purified receptor ECD using fluorescence polarization (FP). We first found that RAMP2 ECD addition to maltose-binding protein (MBP)-tagged CTR ECD and an engineered MBP-tagged RAMP2 ECD-CTR ECD fusion protein (MBP-RAMP2-CTR ECD fusion) enhanced AC413 affinity. This suggests that these recombinant ECD systems represent functional amylin receptors. Interestingly, AC413 C-terminal residue Tyr25 (Y25) to Pro mutation eliminated its selective affinity for the MBP-RAMP2-CTR ECD fusion suggesting the critical role of the AC413 C-terminal residue in amylin receptor selectivity. Our structural model of the RAMP2 ECD:CTR ECD complex predicted molecular interaction of AC413 C-terminal residue Y25 with RAMP2 Glu101 (E101). Our FP peptide-binding assay showed that the RAMP2 E101A mutation of MBP-RAMP2-CTR ECD fusion decreased AC413 affinity by 7-fold, while the affinity of AC413 with the Y25P mutation was minimally changed. Consistently, AC413 binding affinity for the MBP-free RAMP2-CTR ECD fusion protein was also markedly decreased by the RAMP2 E101A mutation. Together, our results support the molecular interaction between the AC413 C-terminal residue Y25 and RAMP2 E101 expanding our understanding of how the accessory protein RAMP2 enhances affinity of peptide hormone amylin for its receptor.
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影响因子：
2.9
作者：
Roehrl, MHA;Wang, JY;Wagner, G
通讯作者：
Wagner, G
影响因子：
4.8
作者：
Lee, Sang-Min;Hay, Debbie L.;Pioszak, Augen A.
通讯作者：
Pioszak, Augen A.
影响因子：
--
作者：
Bower, Rebekah L.;Yule, Lauren;Hay, Debbie L.
通讯作者：
Hay, Debbie L.
DOI：
10.1016/s1056-8719(00)00074-5
发表时间：
1999-12-01
影响因子：
1.9
作者：
Armour, SL;Foord, S;Chen, WJ
通讯作者：
Chen, WJ
影响因子：
--
作者：
Liang, Yi-Lynn;Belousoff, Matthew J.;Sexton, Patrick M.
通讯作者：
Sexton, Patrick M.