Title

Characterization of novel GABAA receptor-associated proteins

Date of Completion

January 2004

Keywords

Biology, Molecular|Biology, Neuroscience

Degree

Ph.D.

Abstract

The γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the mammalian brain. In order to begin to understand the mechanisms that direct the targeting and localization, clustering, and trafficking of brain GABAARs, I sought to identify cytosolic proteins that interact with the intracellular portion of the GABAAR subunits. Therefore, I used the yeast two-hybrid technique to screen an adult rat brain cDNA library using, as bait, the large intracellular loop (IL), of the GABAAR β3 subunit. I found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2), a protein known to be involved in vesicular and protein trafficking, interacts with the β subunits of the GABAA receptors. The native BIG2 and GABAARs both coprecipitated from detergent extracts with either anti-GABAAR or anti-BIG2 antibodies. Double label immunofluorescence of cultured hippocampal neurons showed that BIG2 concentrates in the trans-Golgi network and is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABAARs. The results are consistent with the hypotheses that the interaction of BIG2 with the GABAAR β subunits plays a role in the exocytosis and trafficking of assembled GABA AR to the cell surface. ^ I also isolated a clone corresponding to a novel splice form of the glutamate receptor interacting protein 1 (GRIP1). This splice form, called GRIP1c 4–7, contains 4 PDZ domains that are identical to PDZ domains 4–7 of GRIP1. GRIP1c 4–7 also contains 35 amino acids at the N-terminus and 12 amino acids at the C-terminus that differ from those of GRIP1a/b. Based on these peptide sequences, I have also isolated additional long and short GRIP1 splice variants called GRIP1d and GRIP1e 4–7. GRIP1c 4–7 interacted with GluR2/3 subunits of the AMPA receptor and with gephyrin but not with the GABA AR α1 subunit in detergent extracts of rat brain membranes. In low-density hippocampal cultures, GRIP1c 4–7 clusters colocalized with components of both GABAergic and glutamatergic synapses. GRIP1c 4–7-specific antibodies recognized a 75 kDa Mr protein that is enriched in a postsynaptic density (PSD) fraction isolated from brain. These results indicate that GRIP1c 4–7 plays functional role(s) in both GABAergic and glutamatergic synapses. ^