Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

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 quantita-tive immunoprecipitation-tandem mass spectrometry analysis utilizing stable isotope labeling by amino acids in cell culture. Furthermore, we performed comparative proteomics by using both WT ??1 subunit and a misfolding-prone ??1 subunit car-rying the A322D variant as the bait proteins. We identified 125 interactors for WT ??1-containing receptors, 105 proteins for ??1(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, traf-ficking 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 ??1 subunits and selected interactors by using coimmunoprecipi-tation in mouse brain homogenates. Moreover, we showed that TRIM21 (tripartite motif containing-21), an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone ??1(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.

Automated summary

This study identified the interactomes of GABAA receptors and revealed potential components and pathways involved in the biogenesis and degradation of GABAA receptors. TRIM21 was found to positively regulate the degradation of misfolding-prone GABAA receptor subunits. This research provides insights into the molecular mechanisms of GABAA receptor proteostasis and offers possibilities for the treatment of neurological diseases.