Identification of Clathrin Heavy Chain as a Direct Interaction Partner for the γ-Aminobutyric Acid Type A Receptor Associated Protein

Abstract

Gamma-aminobutyric acid type A receptors (GABAA receptors) are the major sites of GABA-mediated fast synaptic inhibition in the central nervous system. Variation of the cell surface receptor count is postulated to be of importance in modulating inhibitory synaptic transmission. The GABAA receptor associated protein (GABARAP) is a ubiquitin-like modifier, implicated in GABAA receptor clustering, trafficking, and turnover. GABARAP pull-down experiments with brain lysate identified clathrin heavy chain to be GABARAP-associated. Phage display screening of a randomized peptide library for GABARAP ligands yielded a sequence motif which characterizes the peptide binding specificity of GABARAP. Sequence database searches with this motif revealed clathrin heavy chain as a protein containing the identified sequence motif within its residues 510-522, supporting the result of the pull-down experiments. Calreticulin, which was identified recently as a GABARAP ligand, contains a very similar sequence motif. We demonstrate that calreticulin indeed competes with clathrin heavy chain for GABARAP binding. Finally, employing nuclear magnetic resonance spectroscopy, we mapped the GABARAP residues responsible for binding to clathrin. The hereby mapped GABARAP regions overlap very well with the homologue residues in yeast Atg8 that were recently shown to be important for autophagy. Together with the knowledge that GABARAP and clathrin are known to be involved in GABAA receptor trafficking within the cell, this strongly suggests a clear physiological relevance of the direct interaction of GABARAP with clathrin heavy chain.

Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABA A receptors

“…In addition to GABAergic synapses, somatodendritic membranes also harbor ion channels involved in regulating excitability (37). Moreover, GABARAP has multiple partners and thus likely has functions in addition to mediating GABA A receptor localization (27,38). Therefore, giant ankyrin-G and its GABARAP partner likely modulate cell surface behavior of yet to be discovered somatodendritic membrane proteins such as potassium and calcium channels.…”

Nix directly binds to GABARAP: A possible crosstalk between apoptosis and autophagy

“…16 Besides the GABA A receptor, GABARAP binds to a broad range of proteins, which are all associated with vesicle transport and fusion events, receptor transport processes, and also with autophagy and apoptosis. 12,[17][18][19][20][21][22][23][24][25][26] Indeed, among other functions, GABARAP has been demonstrated to be involved in autophagic processes. Thus, the protein level of the membrane-bound form of GABARAP (GABARAP-II) is upregulated during starvation and it was shown to colocalize with LC3-II, which is commonly used as a marker protein for autophagosomal membranes.…”

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein

“…We also confirmed previously published GABARAPL1 and/or GABARAP protein-interactions. For example, we found the clathrin heavy chain protein, a GABARAP cellular partner that is essential in the formation of coated vesicles [25]. In the brain, neurons regulate the number of postsynaptic receptors, such as GABA A receptors, by internalization in clathrincoated vesicles.…”

“…Of the various potential GABARAPL1 protein partners identified, eleven were confirmed by both MS and MS/MS analysis (Table 1). Among all identified proteins, we recovered tubulin, a protein already known to interact in vitro with GABARAPL1 [8] and clathrin heavy chain, a protein described to associate with GABARAP [25]. The HSP90b protein corresponding to the 84 kDa heat shock protein (Q1PSW2) identified in band n 4 (Fig.…”