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Abstract：
The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine, and cancer. To date, the network of proteins interacting with CLR (\"CLR interactome\") in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation utilizing anti-human CLR antibody with label-free quantitative nano LC-MS/MS and quantitative in situ proximity ligation assay. By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ proximity ligation assay). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.
摘要：
G蛋白偶联受体（GPCR）降钙素受体样受体（CLR）介导几种细胞类型的基本功能，并与心血管疾病有关。皮肤病,偏头痛和癌症。迄今为止,原代细胞中与CLR相互作用的蛋白质网络（“CLR相互作用组”），这种GPCR在内源性（生理相关）水平上表达，仍然未知。为了解决这个知识差距，我们建立了一种新的综合方法学工作流程/方法，用于对智人CLR相互作用组进行全面/全蛋白质组分析。我们使用原代人真皮淋巴内皮细胞和联合免疫沉淀（IP）利用抗人CLR抗体与无标记的定量纳米液相色谱-串联质谱（纳米LC-MS/MS）和定量原位邻近连接测定（PLA）。通过使用此工作流程，我们在4,902个检测到的细胞蛋白质组成员中鉴定出37种与内源性表达的CLR相互作用的蛋白质(通过定量纳米LC-MS/MS),并揭示了两种激酶和两种转运蛋白与该GPCR的直接相互作用(通过原位PLA).以前尚未将所有确定的相互作用者报告为CLR相互作用组的成员。我们的方法和发现揭示了内源性表达的CLR相互作用组迄今尚未认识到的组成复杂性，并有助于对这种GPCR生物学的基本理解。总的来说，我们的研究提供了首创的综合方法学方法和数据集,作为宝贵的资源和强大的平台/跳板,可在未来的研究中,在一系列细胞类型和疾病的蛋白质组范围内推进生理相关CLR相互作用组的发现和全面表征.
