Quantitative interactome proteomics identifies a proteostasis network for GABA(A) receptors

Gamma-aminobutyric acid type A (GABA(A)) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABA(A) receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABA(A) receptors. However, how the proteostasis network orchestrates GABA(A) receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABA(A) 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 GABA(A) 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 GABA(A) 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 GABA(A) receptor proteostasis to ameliorate related neurological diseases such as epilepsy.