γ-Aminobutyric acid type A (GABAA) receptors are the most abundant inhibitory neurotransmitter receptors in the mammahan central nervous system. They are ligand- gated chloride ion channels. Each receptor is composed from 5 of the 16 known subunits αl-6, βl-3, δ, γ1-3,δ,π, &thetas; and s. Each subunit type confers different properties to the fully assembled receptor, leading to a diverse range of possible receptor subtypes. However, very few of the theoretically possible subtypes are actually observed in vivo. Here, a series of studies has been undertaken, using the yeast two-hybrid system, to investigate various aspects of GABAA receptor assembly and trafficking, to understand the molecular basis of the observed receptor diversity. Firstly, since GABAA receptor N-terminal domains have been implicated in subunit associations, α1 and β2 subunit N- termini were studied to identify assembly motifs, using 3 different yeast two-hybrid systems, the GAL4, modified LexA and CytoTrap® systems. Secondly, trafficking of receptors and the development and stabilisation of GABAergic synapses were investigated by screening a rat brain cDNA library using the GABAA receptor β3 subunit intracellular loop (β3-IL) and β2 N-terminal domain, respectively, to identify novel interacting proteins. The p2 N-terminus was found to interact with a DnaJ- domain-containing sequence, TIDIL. Thirdly, receptor trafficking was investigated by further characterisation of the previously identified novel protein, GABAA receptor interacting factor (GRIF-1). The binding specificity of GRIF-1 with the GABAA receptor β2-IL was analysed. Structural and functional similarities between GRIF-1 and other members of the novel coiled-coil domain-containing gene family of proteins were investigated. GRIF-1 and a human homologue, KIAA1042, were compared for interactions with the GABAA receptor β2-IL and with kinesin heavy chain (KHC), KIF5C, as a KHC has been shown to associate with Milton, the Drosophila melanogaster orthologue of GRIF-1. Despite the high degree of amino acid homology between GRIF-1 and KIAA1042, only GRIF-1 was found to bind to the GABAA receptorβ2-IL and to KIF5C, suggesting that the subtle differences between GRIF-1 and KIAA1042 lead to differences in functional specificity
