Identification of GABA_C Receptor Protein Homeostasis Network Components from Three Tandem Mass Spectrometry Proteomics Approaches

γ-Amino butyric acid type C (GABAC) receptors inhibit neuronal firing primarily in retina. Maintenance of GABAC receptor protein homeostasis in cells is essential for its function. However, a systematic study of GABAC receptor protein homeostasis (proteostasis) network components is absent. Here coimmunoprecipitation of human GABAC-ρ1-receptor complexes was performed in HEK293 cells overexpressing ρ1 receptors. To enhance the coverage and reliability of identified proteins, immunoisolated ρ1-receptor complexes were subjected to three tandem mass spectrometry (MS)-based proteomic analyses, namely, gel-based tandem MS (GeLC–MS/MS), solution-based tandem MS (SoLC–MS/MS), and multidimensional protein identification technology (MudPIT). From the 107 identified proteins, we assembled GABAC-ρ1-receptor proteostasis network components, including proteins with protein folding, degradation, and trafficking functions. We studied representative individual ρ1-receptor-interacting proteins, including calnexin, a lectin chaperone that facilitates glycoprotein folding, and LMAN1, a glycoprotein trafficking receptor, and global effectors that regulate protein folding in cells based on bioinformatics analysis, including HSF1, a master regulator of the heat shock response, and XBP1, a key transcription factor of the unfolded protein response. Manipulating selected GABAC receptor proteostasis network components is a promising strategy to regulate GABAC receptor folding, trafficking, degradation and thus function to ameliorate related retinal diseases.C型γ-氨基丁酸（γ-Aminobutyric acid type C, GABAC）受体主要通过抑制视网膜神经元的放电活动发挥生理调控作用。维持细胞内GABAC受体的蛋白质稳态，是其正常行使功能的必要前提。然而，目前针对GABAC受体蛋白质稳态（proteostasis）网络组分的系统性研究仍付阙如。本研究在过表达ρ1受体的HEK293细胞中，对人源GABAC-ρ1受体复合物开展了免疫共沉淀（coimmunoprecipitation）实验。为提升鉴定蛋白的覆盖度与可靠性，我们对免疫分离得到的ρ1受体复合物进行了三类基于串联质谱（tandem mass spectrometry, MS）的蛋白质组学分析，分别为凝胶基串联质谱（GeLC–MS/MS）、液相串联质谱（SoLC–MS/MS）以及多维蛋白质鉴定技术（MudPIT）。从鉴定得到的107种蛋白质中，我们构建了GABAC-ρ1受体蛋白质稳态网络组分，涵盖具备蛋白质折叠、降解及转运功能的各类蛋白。我们针对典型的ρ1受体互作蛋白展开了验证分析，包括参与糖蛋白折叠的凝集素分子伴侣钙连蛋白（calnexin），以及糖蛋白转运受体LMAN1；同时通过生物信息学分析，筛选出调控细胞内蛋白质折叠的全局效应因子，包括热休克反应的核心调控因子热休克因子1（HSF1），以及未折叠蛋白反应（unfolded protein response）的关键转录因子X盒结合蛋白1（XBP1）。靶向调控筛选得到的GABAC受体蛋白质稳态网络组分，有望通过调节GABAC受体的折叠、转运与降解过程，进而改善其相关视网膜疾病的临床症状。