Identification of GABA(C) receptor protein homeostasis network components from three tandem mass spectrometry proteomics approaches.
γ-Amino butyric acid type C (GABA(C)) receptors inhibit neuronal firing primarily in retina. Maintenance of GABA(C) receptor protein homeostasis in cells is essential for its function. However, a systematic study of GABA(C) receptor protein homeostasis (proteostasis) network components is absent. Here coimmunoprecipitation of human GABA(C)-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 GABA(C)-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 GABA(C) receptor proteostasis network components is a promising strategy to regulate GABA(C) receptor folding, trafficking, degradation and thus function to ameliorate related retinal diseases.
Center for Proteomics and Bioinformatics and Department of Epidemiology and Biostatistics and ‡Department of Physiology and Biophysics, Case Western Reserve University School of Medicine , 10900 Euclid Avenue, Cleveland, Ohio 44106, United States.
Ya-Juan
YJ
Wang
Dong-Yun
DY
Han
Tracy
T
Tabib
John R
JR
Yates
Ting-Wei
TW
Mu
D001665:Binding Sites; D037281:Calnexin / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D000076249:Heat Shock Transcription Factors; D006706:Homeostasis / Q000235:genetics*; D006801:Humans; D037241:Mannose-Binding Lectins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D018079:Receptors, GABA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D053719:Tandem Mass Spectrometry / Q000379:methods; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D056811:Unfolded Protein Response / Q000235:genetics; D000071717:X-Box Binding Protein 1
D004268:DNA-Binding Proteins; D005136:Eye Proteins; C087200:GABA-C receptor; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; C084004:LMAN1 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D021122:Protein Subunits; D018079:Receptors, GABA; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; D037281:Calnexin