Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors
2022; Elsevier BV; Volume: 298; Issue: 10 Linguagem: Inglês
10.1016/j.jbc.2022.102423
ISSN1083-351X
AutoresYajuan Wang, Xiao-Jing Di, Ting‐Wei Mu,
Tópico(s)Autophagy in Disease and Therapy
ResumoGamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry analysis utilizing stable isotope labeling by amino acids in cell culture. Furthermore, we performed comparative proteomics by using both WT α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for WT α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. In addition, we verified endogenous interactions between α1 subunits and selected interactors by using coimmunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21 (tripartite motif containing-21), an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy. Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry analysis utilizing stable isotope labeling by amino acids in cell culture. Furthermore, we performed comparative proteomics by using both WT α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for WT α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. In addition, we verified endogenous interactions between α1 subunits and selected interactors by using coimmunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21 (tripartite motif containing-21), an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy. Normal organismal physiology depends on the maintenance of protein homeostasis (proteostasis) in each cellular compartment (1Hartl F.U. Bracher A. Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis.Nature. 2011; 475: 324-332Crossref PubMed Scopus (2322) Google Scholar, 2Balch W.E. Morimoto R.I. Dillin A. Kelly J.W. Adapting proteostasis for disease intervention.Science. 2008; 319: 916-919Crossref PubMed Scopus (1836) Google Scholar, 3Hetz C. Adapting the proteostasis capacity to sustain brain healthspan.Cell. 2021; 184: 1545-1560Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 4Powers E.T. Gierasch L.M. The proteome folding problem and cellular proteostasis.J. Mol. Biol. 2021; 433167197Crossref PubMed Scopus (15) Google Scholar), which dictates a delicate balance between protein synthesis, folding, assembly, trafficking, and degradation while minimizing misfolding and aggregation (5Bukau B. Weissman J. Horwich A. Molecular chaperones and protein quality control.Cell. 2006; 125: 443-451Abstract Full Text Full Text PDF PubMed Scopus (1227) Google Scholar, 6Hetz C. Zhang K. Kaufman R.J. Mechanisms, regulation and functions of the unfolded protein response.Nat. Rev. Mol. Cell Biol. 2020; 21: 421-438Crossref PubMed Scopus (770) Google Scholar, 7Needham P.G. Guerriero C.J. Brodsky J.L. Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases.Cold Spring Harbor Perspect. Biol. 2019; 11: a033928Crossref PubMed Scopus (72) Google Scholar). For one specific client protein, its interaction with a network of proteins, especially its proteostasis network components, in the crowded cellular environment is critical to maintain its proteostasis. However, how the proteostasis network orchestrates the biogenesis of multisubunit multispan ion channel proteins is poorly understood. The current limited knowledge about such protein quality control (QC) machinery is gained from the study of various classes of membrane proteins, including cystic fibrosis transmembrane (TM) conductance regulator (8Peters K.W. Okiyoneda T. Balch W.E. Braakman I. Brodsky J.L. Guggino W.B. et al.CFTR folding consortium: methods available for studies of CFTR folding and correction.Methods Mol. Biol. 2011; 742: 335-353Crossref PubMed Scopus (27) Google Scholar), T-cell receptors (9Feige M.J. Behnke J. Mittag T. Hendershot L.M. Dimerization-dependent folding underlies assembly control of the clonotypic αβT cell receptor chains.J. Biol. Chem. 2015; 290: 26821-26831Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), sodium channels (10Buck T.M. Jordahl A.S. Yates M.E. Preston G.M. Cook E. Kleyman T.R. et al.Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein.Biochem. J. 2017; 474: 357-376Crossref PubMed Scopus (17) Google Scholar), potassium channels (11Delaney E. Khanna P. Tu L. Robinson J.M. Deutsch C. Determinants of pore folding in potassium channel biogenesis.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 4620-4625Crossref PubMed Scopus (10) Google Scholar), and nicotinic acetylcholine receptors (12Kweon H.J. Gu S. Witham E. Dhara M. Yu H. Mandon E.D. et al.NACHO engages N-glycosylation ER chaperone pathways for α7 nicotinic receptor assembly.Cell Rep. 2020; 32108025Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). We have been using gamma-aminobutyric acid type A (GABAA) receptors as an important membrane protein substrate to clarify its biogenesis pathway (13Fu Y.L. Wang Y.J. Mu T.W. Proteostasis maintenance of cys-loop receptors.Adv. Protein Chem. Struct. Biol. 2016; 103: 1-23Crossref PubMed Scopus (11) Google Scholar), which is currently understudied. GABAA receptors are the primary inhibitory neurotransmitter-gated ion channels in mammalian central nervous systems (14Macdonald R.L. Olsen R.W. GABA(A) receptor channels.Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1802) Google Scholar) and provide most of the inhibitory tone to balance the tendency of excitatory neural circuits to induce hyperexcitability, thus maintaining the excitatory–inhibitory balance (15Akerman C.J. Cline H.T. Refining the roles of GABAergic signaling during neural circuit formation.Trends Neurosci. 2007; 30: 382-389Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). There are 19 known GABAA receptor subunits in mammals, including α1–6, β1–3, γ1–3, θ, ε, π, δ, and ρ1–3. Loss of function of GABAA receptors is a prominent cause of genetic epilepsies, and recent advances in genetics have identified a growing number of epilepsy-associated variants in over ten genes that encode the subunits of GABAA receptors, including over 150 variants in those encoding major synaptic subunits (α1, β2, and γ2 subunits) (Fig. 1A, top right figure) (16Fu X. Wang Y.J. Kang J.Q. Mu T.W. GABAA Receptor Variants in Epilepsy. Exon Publications, Brisbane (AU)2022Crossref Google Scholar, 17Hernandez C.C. Macdonald R.L. A structural look at GABA(A) receptor mutations linked to epilepsy syndromes.Brain Res. 2019; 1714: 234-247Crossref PubMed Scopus (49) Google Scholar, 18Hirose S. Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.Prog. Brain Res. 2014; 213: 55-85Crossref PubMed Scopus (90) Google Scholar, 19Noebels J.L. The biology of epilepsy genes.Annu. Rev. Neurosci. 2003; 26: 599-625Crossref PubMed Scopus (237) Google Scholar, 20Steinlein O.K. ion channel mutations in neuronal diseases: a genetics perspective.Chem. Rev. 2012; 112: 6334-6352Crossref PubMed Scopus (26) Google Scholar). GABAA receptors belong to the Cys-loop superfamily of ligand-gated ion channels, sharing common structural characteristics with other Cys-loop receptor members (21Dougherty D.A. Cys-loop neuroreceptors: structure to the rescue?.Chem. Rev. 2008; 108: 1642-1653Crossref PubMed Scopus (94) Google Scholar). A functional GABAA receptor is composed of five subunits. Each subunit has a large extracellular (or the endoplasmic reticulum [ER] luminal) N terminus, four TM helices (TM1–TM4, with TM2 domain lining the interior of the pore), and a short extracellular (or the ER luminal) C terminus (Fig. 1A) (22Laverty D. Desai R. Uchański T. Masiulis S. Stec W.J. Malinauskas T. et al.Cryo-EM structure of the human α1β3γ2 GABA(A) receptor in a lipid bilayer.Nature. 2019; 565: 516-520Crossref PubMed Scopus (198) Google Scholar, 23Zhu S. Noviello C.M. Teng J. Walsh Jr., R.M. Kim J.J. Hibbs R.E. Structure of a human synaptic GABAA receptor.Nature. 2018; 559: 67-72Crossref PubMed Scopus (314) Google Scholar). GABA binding to GABAA receptors in the extracellular domain induces conformational changes, opens the ion pore to conduct chloride, hyperpolarizes the plasma membrane, and inhibits neuronal firing in mature neurons. To function properly, GABAA receptors need to fold into their native structures and assemble correctly to form a pentamer on the ER membrane and traffic efficiently through the Golgi en route to the plasma membrane (Fig. 1A). Misfolded GABAA receptors are recognized by the cellular protein QC machinery. ER-associated degradation (ERAD) is one major cellular pathway to target misfolded GABAA receptors to the cytosolic proteasome for degradation (7Needham P.G. Guerriero C.J. Brodsky J.L. Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases.Cold Spring Harbor Perspect. Biol. 2019; 11: a033928Crossref PubMed Scopus (72) Google Scholar, 24Olzmann J.A. Kopito R.R. Christianson J.C. The mammalian endoplasmic reticulum-associated degradation system.Cold Spring Harbor Perspect. Biol. 2013; 5: a013185Crossref PubMed Scopus (245) Google Scholar, 25Di X.J. Wang Y.J. Han D.Y. Fu Y.L. Duerfeldt A.S. Blagg B.S. et al.Grp94 protein delivers γ-aminobutyric acid type A (GABAA) receptors to Hrd1 protein-mediated endoplasmic reticulum-associated degradation.J. Biol. Chem. 2016; 291: 9526-9539Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 26Gallagher M.J. Shen W.Z. Song L.Y. Macdonald R.L. Endoplasmic reticulum retention and associated degradation of a GABA(A) receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the alpha 1 subunit.J. Biol. Chem. 2005; 280: 37995-38004Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 27Poliquin S. Kang J.Q. Disruption of the ubiquitin-proteasome system and elevated endoplasmic reticulum stress in epilepsy.Biomedicines. 2022; 10: 647Crossref PubMed Scopus (7) Google Scholar). Another potential degradation pathway is to target the aggregation-prone GABAA receptors to the lysosome through autophagy, ER-phagy, or ER-to-lysosome-associated degradation (28Fregno I. Molinari M. Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways.Crit. Rev. Biochem. Mol. Biol. 2019; 54: 153-163Crossref PubMed Scopus (82) Google Scholar, 29Wilkinson S. ER-Phagy: shaping up and destressing the endoplasmic reticulum.FEBS J. 2019; 286: 2645-2663PubMed Google Scholar, 30Arancibia-Cárcamo I.L. Yuen E.Y. Muir J. Lumb M.J. Michels G. Saliba R.S. et al.Ubiquitin-dependent lysosomal targeting of GABA(A) receptors regulates neuronal inhibition.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 17552-17557Crossref PubMed Scopus (65) Google Scholar, 31Davis K.M. Sturt B.L. Friedmann A.J. Richmond J.E. Bessereau J.L. Grant B.D. et al.Regulated lysosomal trafficking as a mechanism for regulating GABAA receptor abundance at synapses in Caenorhabditis elegans.Mol. Cell. Neurosci. 2010; 44: 307-317Crossref PubMed Scopus (14) Google Scholar). Maintenance of a delicate balance between GABAA receptor folding, trafficking, and degradation utilizing its interacting proteins is critical for its function. However, the GABAA receptor interactome, especially the proteostasis network that orchestrates GABAA receptor biogenesis in the ER, has not been studied systematically in the literature despite recent advances about the trafficking of GABAA receptors beyond the ER (32Ge Y. Kang Y. Cassidy R.M. Moon K.M. Lewis R. Wong R.O.L. et al.Clptm1 limits forward trafficking of GABA(A) receptors to scale inhibitory synaptic strength.Neuron. 2018; 97: 596-610.e8Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 33Han W. Li J. Pelkey K.A. Pandey S. Chen X. Wang Y.X. et al.Shisa7 is a GABA(A) receptor auxiliary subunit controlling benzodiazepine actions.Science. 2019; 366: 246-250Crossref PubMed Scopus (47) Google Scholar, 34Han W. Shepard R.D. Lu W. Regulation of GABA(A)Rs by transmembrane accessory proteins.Trends Neurosciences. 2021; 44: 152-165Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 35Lorenz-Guertin J.M. Jacob T.C. GABA type a receptor trafficking and the architecture of synaptic inhibition.Developmental Neurobiol. 2018; 78: 238-270Crossref PubMed Scopus (38) Google Scholar, 36Nakamura Y. Morrow D.H. Modgil A. Huyghe D. Deeb T.Z. Lumb M.J. et al.Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.J. Biol. Chem. 2016; 291: 12394-12407Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 37Heller E.A. Zhang W. Selimi F. Earnheart J.C. Ślimak M.A. Santos-Torres J. et al.The biochemical anatomy of cortical inhibitory synapses.PLoS One. 2012; 7e39572Crossref Scopus (44) Google Scholar). Here, we used quantitative immunoprecipitation–tandem mass spectrometry (IP–MS/MS) analysis utilizing stable isotope labeling by amino acids in cell culture (SILAC) in human embryonic kidney 293T (HEK293T) cells to identify the interactomes for both WT and a misfolding-prone GABAA receptor. Endogenous interactions between selected interactors and GABAA receptors were verified in mouse brain homogenates. Furthermore, bioinformatics analysis enabled us to assemble a proteostasis network model for the cellular folding, assembly, degradation, and trafficking pathways of GABAA receptors. We employed a proteomics-based approach to identify the interactomes of GABAA receptors in HEK293T cells by carrying out a quantitative IP–MS/MS analysis utilizing SILAC (Fig. 1B) (38Mann M. Functional and quantitative proteomics using SILAC.Nat. Rev. Mol. Cell Biol. 2006; 7: 952-958Crossref PubMed Scopus (775) Google Scholar). Since HEK293T cells do not own endogenous GABAA receptors, precise control of the subtypes and variants of GABAA receptors can be achieved by exogenously expressing their subunits (39Thomas P. Smart T.G. HEK293 cell line: a vehicle for the expression of recombinant proteins.J. Pharmacol. Toxicol. Methods. 2005; 51: 187-200Crossref PubMed Scopus (483) Google Scholar). Furthermore, we performed comparative proteomics by using both WT α1 subunit and a well-characterized misfolding-prone α1 subunit carrying the A322D variant as the bait proteins to determine the potential difference between them (40Gallagher M.J. Ding L. Maheshwari A. Macdonald R.L. The GABA(A) receptor alpha 1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12999-13004Crossref PubMed Scopus (85) Google Scholar). The A322D variant introduces an extra negative charge in the third TM (TM3) helix of the α1 subunit, causing its substantial misfolding and excessive degradation by ERAD (40Gallagher M.J. Ding L. Maheshwari A. Macdonald R.L. The GABA(A) receptor alpha 1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12999-13004Crossref PubMed Scopus (85) Google Scholar, 41Di X.J. Han D.Y. Wang Y.J. Chance M.R. Mu T.W. SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.Chem. Biol. 2013; 20: 1456-1468Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). HEK293T cells stably expressing either WT α1β2γ2 or α1(A322D)β2γ2 GABAA receptors were labeled with heavy media, whereas HEK293T cells that were transfected with empty vector (EV) plasmids were cultured in normal light media. The same amount of light and heavy cell lysates was mixed. The α1 or α1(A322D) complexes were immunoprecipitated using a monoclonal antibody against the N-terminal region of the α1 subunit before being subjected to SDS-PAGE and in-gel digestion and tandem MS analysis. Coomassie blue–stained gels showed numerous clearly visible bands in samples expressing GABAA receptors (Fig. 1C, lanes 2 and 3), which represented potential interacting proteins for α1 subunit–containing GABAA receptors, indicating efficient coimmunoprecipitations. In addition, Western blot analysis detected α1 subunits effectively at ∼50 kDa postimmunoprecipitation in samples expressing GABAA receptors (Fig. 1D, lanes 2 and 3), whereas no α1 band was observed in the EV control sample (Fig. 1D, lane 1), supporting the efficient isolation of the α1 subunit–containing GABAA receptor complexes. The α1 subunit–containing GABAA receptor interactomes were identified using the SILAC ratio with arbitrary yet strict criteria to remove potential false positives. To be included as an interactor, it must (1) have a SILAC ratio of WT α1/EV or α1(A322D)/EV to be at least 1.30; (2) have a p < 0.05; and (3) have a Benjamini and Hochberg correction (42Benjamini Y. Hochberg Y. Controlling the false discovery rate - a practical and powerful approach to multiple testing.J. R. Stat. Soc. Ser. B-Methodological. 1995; 57: 289-300Google Scholar) of false discovery rate (FDR) of no more than 0.10. The top right green area in Figure 2, A contains high-confidence interactors for GABAA receptors. As a result of the stringent criteria, the WT α1-containing GABAA receptor interactome contains 125 proteins, the α1(A322D)-containing GABAA receptor interactome contains 105 proteins, and 54 proteins overlap within two interactomes (Fig. 2B). These 176 interactors for GABAA receptors are used for the following bioinformatics analysis (see Table S1 for protein list). Since GABAA receptors inhibit neuronal firing in the mammalian central nervous system, we first compared the expression abundance of their interactors between HEK293 cells and the nervous system. ProteomicsDB (https://www.proteomicsdb.org/) provides an MS-based navigation of the human proteome from tissues, cell lines, and body fluids, enabling the comprehensive mapping of the protein abundance of GABAA receptor interactors (43Wilhelm M. Schlegl J. Hahne H. Gholami A.M. Lieberenz M. Savitski M.M. et al.Mass-spectrometry-based draft of the human proteome.Nature. 2014; 509: 582-587Crossref PubMed Scopus (1377) Google Scholar). Hierarchical clustering analysis showed that these interactors have comparable protein levels between HEK293 cells and human brain tissues (Fig. 2C and Table S2). For example, many molecular chaperones, such as Hsp90s (Hsp90AA1, Hsp90AB1, and Hsp90B1), Hsp70s (HspA5 and HspA8), Hsp40s (DNAJA1, DNAJA2, and DNAJB11), calnexin (CANX), and Hsp47 (SerpinH1), are abundantly expressed in both systems. In addition, tissue-based map of the human proteome based on quantitative transcriptomics (https://www.proteinatlas.org/) enabled us to compare RNA levels of GABAA receptor interactors between HEK293 cells, a human neuronal SH-SY5Y cell line, and human brain tissues (44Sjöstedt E. Zhong W. Fagerberg L. Karlsson M. Mitsios N. Adori C. et al.An atlas of the protein-coding genes in the human, pig, and mouse brain.Science. 2020; 367eaay5947Crossref PubMed Scopus (285) Google Scholar, 45Uhlén M. Fagerberg L. Hallström B.M. Lindskog C. Oksvold P. Mardinoglu A. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 3471260419Crossref PubMed Scopus (8150) Google Scholar). Hierarchical clustering analysis showed that many interactors, including molecular chaperones and ubiquitin-dependent degradation factors, such as UBA1, UBR5, UBE3C, SEL1L, and VCP, have similar RNA expression patterns between these systems (Fig. 2D and Table S2). These results are consistent with the report that most proteins are well conserved among human tissues (45Uhlén M. Fagerberg L. Hallström B.M. Lindskog C. Oksvold P. Mardinoglu A. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 3471260419Crossref PubMed Scopus (8150) Google Scholar). Previously, proteomic analyses were carried out to identify the interacting proteins for GABAA receptor subunits using knockin mice carrying Venus (a GFP variant)-tagged α1 subunit (37Heller E.A. Zhang W. Selimi F. Earnheart J.C. Ślimak M.A. Santos-Torres J. et al.The biochemical anatomy of cortical inhibitory synapses.PLoS One. 2012; 7e39572Crossref Scopus (44) Google Scholar), pHluorin (a pH-sensitive GFP variant)-Myc-tagged α2 subunit (36Nakamura Y. Morrow D.H. Modgil A. Huyghe D. Deeb T.Z. Lumb M.J. et al.Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.J. Biol. Chem. 2016; 291: 12394-12407Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), or His6-FLAG-YFP-tagged γ2 subunit (32Ge Y. Kang Y. Cassidy R.M. Moon K.M. Lewis R. Wong R.O.L. et al.Clptm1 limits forward trafficking of GABA(A) receptors to scale inhibitory synaptic strength.Neuron. 2018; 97: 596-610.e8Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). GFP-affinity purification from the cerebral cortex using Venus-tagged α1 subunits as bait led to the identification of 18 proteins in the inhibitory synaptic complexes, including 11 GABAA receptor subunits, five scaffolding and adhesion proteins (gephrin [GPHN], neuroligin 2 [NLGN2], neuroligin 3 [NLGN3], collybistin [ARHGEF9], and neurexin 1 [NRXN1]), and two proteins with less known functions (neurobeachin [NBEA] and LHFPL4) (37Heller E.A. Zhang W. Selimi F. Earnheart J.C. Ślimak M.A. Santos-Torres J. et al.The biochemical anatomy of cortical inhibitory synapses.PLoS One. 2012; 7e39572Crossref Scopus (44) Google Scholar). However, because of the use of size-exclusion chromatography during the protein complex purification process, proteins that are potentially involved in the cellular folding and trafficking of GABAA receptors were not well identified. GFP-trap purification from hippocampus and cortex using pHluorin-Myc-tagged α2 subunits as bait resulted in the identification of 174 proteins, including 14 GABAA receptor subunits, known GABAA receptor interactors such as gephrin, neuroligins, and collybistin, and 149 novel binding partners (36Nakamura Y. Morrow D.H. Modgil A. Huyghe D. Deeb T.Z. Lumb M.J. et al.Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.J. Biol. Chem. 2016; 291: 12394-12407Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The novel interactors were further categorized into five groups, including G protein–coupled receptors, ion channels, and transporters (26 interactors), factors that regulate protein trafficking, stability, and cytoskeletal anchoring (38 interactors), factors that regulate phosphorylation and GTP exchange (26 interactors), miscellaneous enzymes (27 interactors), and miscellaneous proteins (32 interactors) (36Nakamura Y. Morrow D.H. Modgil A. Huyghe D. Deeb T.Z. Lumb M.J. et al.Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.J. Biol. Chem. 2016; 291: 12394-12407Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Comparison between the α2 subunit–containing GABAA receptor interactome and our α1 subunit–containing GABAA receptor interactome showed overlapping interactors, including factors that regulate protein folding and degradation (DNAJA1, RPN2, SQSTM1, USP9X, and DDB1), transporters (SLC25A3, SLC25A4, and SLC25A5), and miscellaneous proteins (PHB2 and IMMT). Furthermore, tandem affinity purification from whole brain homogenates using His6-FLAG-YFP-tagged γ2 subunits as bait gave rise to the identification of 11 known associated proteins (GABAA receptor subunits, gephrin and neuroligin 2) and 39 novel binding partners (32Ge Y. Kang Y. Cassidy R.M. Moon K.M. Lewis R. Wong R.O.L. et al.Clptm1 limits forward trafficking of GABA(A) receptors to scale inhibitory synaptic strength.Neuron. 2018; 97: 596-610.e8Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The novel interactors were further classified into five groups, including ion channels (two interactors), factors that regulate protein folding and trafficking (six interactors), mitochondria proteins (six interactors), miscellaneous enzymes (seven interactors), and miscellaneous proteins (18 interactors). Comparison between the γ2 subunit–containing GABAA receptor interactome and our α1 subunit–containing GABAA receptor interactome showed overlapping interactors, including a factor that regulate protein degradation (PSMC2), an enzyme (PTPLAD1), and a miscellaneous protein (EMD). Among the three GABAA receptor proteomic analyses using knockin mice, six synaptic proteins were recognized from at least two studies, including gephrin, neuroligin 2, neuroligin 3, collybistin, neurobeachin, and LHFPL4. However, such synaptic proteins were not identified in our α1 subunit–containing GABAA receptor interactome possibly because of their lack of expression in HEK293T cells. The only other overlapping interactor from at least two knockin mice proteomic studies is CACNA1E, the α1E subunit of R-type voltage-dependent calcium channels, which could indicate that different subunits utilize differentiating cellular interaction networks. Moreover, the limited interactome overlapping from these proteomic analyses could arise from using different purification procedures to isolate the GABAA receptor–containing complex, such as using different detergents. Nonetheless, in addition to certain GABAA receptor subunits, our α1 subunit–containing GABAA receptor interactome showed 13 overlapping interactors with one other proteomic study, providing promising candidates for future investigation. To annotate cellular component for GABAA receptor interactors, we used Database for Annotation, Visualization and Integrated Discovery (DAVID) to carry out Gene Ontology (GO) analysis (46Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (26401) Google Scholar, 47Huang da W. Sherman B.T. Lempicki R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.Nucleic Acids Res. 2009; 37: 1-13Crossref PubMed Scopus (10682) Google Scholar). Since many proteins reside in more than one cellular location, we only choose one primary subcellular location for them manually. To aid such an assignment, we also integrate subcellular location information from UniProt Database (https://www.uniprot.org/) and GeneCards: The Human Gene Database (https://www.genecards.org/). GABAA receptor interactors are distributed in various cellular locations, including the nucleus (33 interactors), ER (25 interactors), Golgi (2 interactors), proteasome (4 interactors), lysosome (1 interactor), mitochondria (21 interactors), cytoskeleton (23 interactors), cytosol (59 interactors), plasma membrane (5 interactors), and extracellular space (3 interactors) since biogenesis and function of GABAA receptors require their interactions with a network of proteins throughout the cell (Fig. 2E and Table S3). Because of the essential role of the ER in protein QC, 25 interactors (14 for WT α1-containing GABAA receptor only, three for α1(A322D) variant–containing GABAA receptor only, and eight for both) are located to this organelle. Eight overlapping interactors in the ER include molecular chaperones (CANX, HspA5, DNAJB11, Hsp90B1, and SerpinH1), factors involved in N-linked glycosylation (RPN1 and DPM1), and a Ca2+-bi
Referência(s)