Pham Vi, Zhu Yue, Dal Maso Emma, Reynolds Christopher A, Deganutti Giuseppe, Atanasio Silvia, Hick Caroline A, Yang Dehua, Christopoulos Arthur, Hay Debbie L, Furness Sebastian G B, Wang Ming-Wei, Wootten Denise, Sexton Patrick M
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia.
The National Center for Drug Screening and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
ACS Pharmacol Transl Sci. 2019 Mar 18;2(3):183-197. doi: 10.1021/acsptsci.9b00010. eCollection 2019 Jun 14.
Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY receptors (AMYR) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMYR is thus important for peptide drug targeting of this receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by calcitonin (CT) and related peptides (Dal Maso, E., . (2019) The molecular control of calcitonin receptor signaling. , 31-51). That work revealed ligand- and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMYRs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMYR, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the receptor.
胰淀素与胰岛素在胰岛β细胞中共表达，对胃排空和食物摄入有显著影响。据推测，胰淀素对饱腹感的作用涉及降钙素受体（CTR）和受体活性调节蛋白3（RAMP3）的异二聚体——AMY受体（AMYR）。因此，了解通过AMYR进行信号传导的分子控制对于该受体的肽类药物靶向治疗很重要。我们之前使用丙氨酸扫描诱变来研究CTR细胞外表面对降钙素（CT）和相关肽引发的结合和信号传导的贡献（达尔·马索，E.，.（2019年）降钙素受体信号传导的分子控制。，31 - 51）。该研究揭示了突变的配体和途径特异性效应，细胞外环（ECL）2和3在单个肽介导的信号传导的不同传播中尤为重要。在本研究中，我们在与RAMP3共表达以形成AMYRs的背景下，对CTR的ECL2和ECL3进行了等效的丙氨酸扫描，以检查肽在cAMP积累和细胞外信号调节激酶（ERK）磷酸化（pERK）中的功能亲和力和效力。确定了突变对三种主要不同类型的CT肽（鲑鱼CT（sCT）、人CT（hCT）和猪CT（pCT））以及大鼠胰淀素（rAmy）或人α - CGRP（降钙素基因相关肽，hCGRP）的代表性肽的影响，其效力通过RAMP相互作用而增强。我们证明，当与RAMP3复合形成AMYR时，CTR ECL2和ECL3在信号传导传播中的动态性质发生了根本改变，尽管仅预测与ECL2有直接相互作用。此外，这项工作表明这些环在受体信号传导中的作用高度依赖于肽，说明即使肽序列的细微变化也可能改变受体下游的信号输出。
