Wilkinson, Helen Louise; (2000) The identification of a novel protein interacting with GABAA receptors using the yeast two-hybrid system. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

γ-Aminobutyric acidA (GABAA) receptors are members of the ligand-gated ion channel superfamily. They are hetero-oligomeric proteins which are composed of varying combinations of subunits to form functional receptors. The fifteen known GABAA receptor genes code for multiple subunit classes which are categorised by their respective amino acid sequence homologies i.e. α1-α6, β1-β3, γ1-γ3, δ, ε and π. The anchoring and clustering of neurotransmitter receptors at positions apposed to the site of neurotransmitter release is important for the efficient functioning of chemical synapses. The interaction of specific proteins with neurotransmitter receptors has been demonstrated to be necessary for this anchoring and clustering process. We therefore used the yeast two-hybrid system to identify potential GABAA receptor anchoring proteins. The DNA encoding the GABAA receptor β2 intracellular loop was used as the bait to screen a rat brain cDNA library of 1.8×10^6 recombinants. Three clones encoding protein sequences which interact with the intracellular loop of the GABAA receptor β2 subunit have been identified. One of these contained an insert of 1.9 kb which database searches have shown to be a novel sequence. The searches did however show a 84% DNA identity to the human expressed sequence tag (EST) zm75b03.rl. Northern blot analysis showed that the clone hybridised with three transcript sizes of 6.2 kb, 4.2 kb and 2.9 kb. The larger transcript was expressed in heart, brain, spleen, lung, liver, skeletal muscle and kidney but not in testis. The smallest transcript was expressed primarily in the testis and to a much lesser extent in the heart, brain, spleen, liver, lung, skeletal muscle and kidney. The 1.9 kb clone was radiolabelled and used as a probe to identify other hybridising clones enabling the determination of further nucleotide sequence. The creation of a GST fusion protein of the GABAA receptor β2 intracellular loop and a polyHisCS fusion protein allowed antibodies to C3 to be produced and 'pull-down' assays to be performed to further characterise the interaction between C3 and the GABAA receptor β2 intracellular loop.

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