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The Journal of Neuroscience, June 20, 2007, 27(25):6655-6663; doi:10.1523/JNEUROSCI.0919-07.2007

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Cellular/Molecular
C-Terminal Modification Is Required for GABARAP-Mediated GABA_A Receptor Trafficking

Zi-Wei Chen, Chang-Sheng S. Chang, Tarek A. Leil, and Richard W. Olsen

Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1735

Correspondence should be addressed to Dr. Richard W. Olsen, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California, Los Angeles, CHS 23-338, 650 Charles Young Drive South, Los Angeles, CA 90095-1735. Email: rolsen{at}mednet.ucla.edu

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 1ß22S-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.

Key words: GABA_A receptors; GABARAP; trafficking; ubiquitinylation-like modification; NSF; inhibitory neurotransmission

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Received March 1, 2007; revised May 8, 2007; accepted May 9, 2007.

Correspondence should be addressed to Dr. Richard W. Olsen, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California, Los Angeles, CHS 23-338, 650 Charles Young Drive South, Los Angeles, CA 90095-1735. Email: rolsen{at}mednet.ucla.edu

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