Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

Ya-Juan Wang; Xiao-Jing Di; Ting-Wei Mu

doi:10.1016/j.jbc.2022.102423

Quantitative interactome proteomics identifies a proteostasis network for GABA_A receptors

J Biol Chem. 2022 Oct;298(10):102423. doi: 10.1016/j.jbc.2022.102423. Epub 2022 Aug 27.

Authors

Ya-Juan Wang¹, Xiao-Jing Di², Ting-Wei Mu³

Affiliations

¹ Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA; Center for Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. Electronic address: yajuan.wang@case.edu.
² Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
³ Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. Electronic address: tingwei.mu@case.edu.

Abstract

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.

Keywords: GABA(A) receptor; SILAC; assembly; degradation; epilepsy; folding; interactome; proteostasis; trafficking.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
Endoplasmic Reticulum / genetics
Endoplasmic Reticulum / metabolism
Mice
Proteomics
Proteostasis*
Receptors, GABA-A* / metabolism
gamma-Aminobutyric Acid / metabolism

Substances

gamma-Aminobutyric Acid
Receptors, GABA-A

Grants and funding

R01 NS117176/NS/NINDS NIH HHS/United States