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

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posted on 06.12.2013, 00:00 by Ya-Juan Wang, Dong-Yun Han, Tracy Tabib, John R. Yates, Ting-Wei Mu
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.