C-Terminal Modification Is Required for GABARAP-Mediated GABAA Receptor Trafficking

Abstract

We investigated the ubiquitin-like modification of GABA A receptor-associated protein (GABARAP) and its function. A fusion protein of GABARAP with v5 in the N terminus and myc in the C terminus was expressed in rat cultured hippocampal neurons and PC12 cells. Western blotting with antibodies to v5 and myc revealed that the C terminus of GABARAP was cleaved off. Cleavage was blocked by mutating the C-terminal Gly116 to Ala, suggesting that G116 is required for the processing. Unlike ubiquitin, GABARAP was not incorporated covalently into higher-molecular-weight protein complexes. Nor was GABARAP degraded by ubiquitinylation through the proteasome, although GABARAP formed noncovalent dimers. Immunofluorescent confocal microscopy demonstrated that recombinantly expressed GABARAP was diffusely localized in PC12 cells. However, prevention of C-terminal processing in the mutant GABARAP G116A resulted in redistribution to the Golgi. In neurons, punctate cytoplasmic staining of GABARAP was seen in soma and processes, whereas GABARAP G116A was limited to soma. Compared with wild-type GABARAP, the colocalization and interaction of GABARAP G116A with GABA A receptors were significantly reduced, resulting in a reduction in expression of receptors in the plasma membrane. When ␣1␤2␥2S-containing GABA A receptors were expressed in oocytes, the increased surface expression of GABA A receptors, as shown by increased GABA currents and surface-accessible GABA A receptor subunit polypeptides resulting from GABARAP coexpression, was prevented by mutation G116A. In addition, the distribution of NSF (N-ethylmaleimide-sensitive factor) was affected in GABARAP G116A -expressing neurons. These results suggest that glycine 116 is required for C-terminal processing of GABARAP and that processing is essential for the localization of GABARAP and its functions as a trafficking protein.

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

“…Indeed, LC3B can be processed by the 20S core proteasome in a ubiquitinindependent manner in vitro and this degradation is inhibited by its interaction with p62, a mediator of autophagic proteolysis [52]. Concerning GABARAP, Chen et al suggest that GABARAP does not undergo the ubiquitinylation process and is not degraded through the proteasome after lactacystin treatment in rat cultured hippocampal neurons [53].…”

Borna Disease Virus P Protein Affects Neural Transmission through Interactions with Gamma-Aminobutyric Acid Receptor-Associated Protein

“…It was reported that GABARAP contains two domains, a larger C-terminal domain (residues 27 to 117) that mediates GABA A -R binding (4,35) and a smaller N-terminal one (residues 1 to 26) that contains a novel tubulin-binding motif (7,32,35). Glycine 116 is required for the C-terminal processing of GABARAP, the localization of the protein, and its functions as a trafficking protein (5).…”

“…(A) In the absence of BDV P protein, GABA A -R is the principal GABA-gated ion channel and plays important roles in neural transmission (17). GABARAP distributes in the cytosol of the cells, interacts with the ␥2 subunit of GABA A -Rs, links GABA A -Rs to microtubules, facilitates the trafficking of GABA A -Rs to the plasma membrane (4,5), and thus enhances GABA-induced currents (10). ER, endoplasmic reticulum.…”

“…GABA A -R-associated protein (GABARAP) originally was identified as a linker between GABA A -Rs and microtubules. Recent study has demonstrated that GABARAP can interact with the ␥2 subunit of GABA A -R and has a functional effect on the regulation of GABA activities by disrupting the trafficking of GABA A -Rs to the cell membranes (4,5).…”

Effects of C-terminal Modifications of GEC1 Protein and γ-Aminobutyric Acid Type A (GABAA) Receptor-associated Protein (GABARAP), Two Microtubule-associated Proteins, on κ Opioid Receptor Expression

“…HA epitope was added 5Ј to the initiation codon and c-Myc epitope immediately 5Ј to the stop codon of the cDNAs in the same vector for expression of HA-tagged or HA-and -Myc-tagged proteins. V5-GABARAP-Myc and V5-GABARAP-AL-Myc were gifts from Dr. Richard W. Olson of UCLA School of Medicine (33). For immunofluorescence microscopy and protein pulldown assays, GEC1 and GABARAP and their mutants were inserted into SalI/ XhoI sites of pCMV-HA vector (Clontech).…”