Hanley, Jonathan Gordon; (1998) Identification of GABAC receptor-interacting proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Ionotropic GABA receptors are the major sites of fast synaptic inhibition in the brain and retina, and can be divided into two subtypes based on physiology, pharmacology and subunit composition; GABAA and GABAC receptors. The clustering of neurotransmitter receptors at synaptic sites is critical for efficient neurotransmission; glycine receptors are clustered in synapses by gephyrin which anchors the receptor to microtubules; nicotinic acetylcholine receptors are clustered at the neuromuscular junction by rapsyn which is thought to link the receptors to the actin cytoskeleton. No such mechanism has been identified for a GABA receptor. In this study, the yeast two-hybrid system was used to isolate molecules which might play a role in clustering ionotropic GABA receptors at synaptic sites. GABAC receptors were studied because of their simpler subunit composition, comprising ρ(1-3) subunits, in contrast to the more complex structure of GABAA receptors composed of α(1-6), β(1-3), γ(1-3), δ, ϵ and π. The large intracellular loop between transmembrane regions three and four was considered to be the most likely part of the molecule to interact with a cytoskeletal anchoring protein, and would possibly also yield other interesting binding partners. Therefore, a bait comprising the large intracellular domain of ρ1 was constructed and used to screen a retinal yeast two-hybrid cDNA library. This yielded three positive interacting clones; a C-terminal variant of the glycine transporter GLYT-1, a region of microtubule-associated protein MAP1B, and a novel protein. The majority of the work in this thesis concentrates on the ρ1-MAP1B interaction. Polyclonal antibodies were raised against ρ1 intracellular loop and against the GLYT-1 C-terminal variant as part of this study. The interaction with MAP1B was confirmed by an in vitro overlay assay, affinity- purification ("pull-down") assays and immunoprecipitation from retina. Immunofluorescence microscopy on retinal sections and dissociated bipolar cells demonstrates colocalisation of ρ1-containing GABA receptors and MAP1B at synapses in the inner plexiform layer. Furthermore, MAP1B influences the subcellular distribution of ρ1 subunit in transiently transfected COS cells. The binding site for ρ1 on MAP1B was mapped to a region just N-terminal to the proposed microtubule-binding domain, and the reciprocal site on ρ1 was found to be a region close to transmembrane domain 4. This study demonstrates that MAP1B is a strong candidate for a GABAC receptor synaptic anchoring protein, and also that a glycine transporter and a novel, uncharacterised protein also interact with these receptors.

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