Brandon, Nicholas John; (1999) Identification of interactions between signalling molecules and GABAA receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

GABAA receptors are the main sites of inhibitory neurotransmission in the brain. They are also the sites of action of a number of clinically important drugs such as the benzodiazepines and barbiturates. Phosphorylation has been identified as an important mechanism by which neurons control the function of these receptors. GABAA receptors are hetero-pentamers which are assembled from the subunit classes α, β, γ, σ and ϵ. Subunits have a distinctive topology which includes 4 transmembrane domains, extracellular amino-, and carboxy termini, and a large intracellular domain between transmembrane regions 3 and 4. In vivo most benzodiazepine sensitive receptors are believed to be composed of α, β, and γ subunits. Intracellular domains of receptor subunits contain consensus phosphorylation sites for a range of protein kinases, including the cAMP-dependent kinase (PKA), the Ca2+-phospholipid dependent kinase (PKC) and the non-receptor tyrosine kinase src. Studies with subunit cDNAs, transiently expressed in heterologous cells, has identified principally the β and γ2 subunits as substrates in vivo for these kinases. To date there have been few studies on the phosphorylation of the native receptor. Using polyclonal antibodies, which were raised for this study, the tyrosine phosphorylation of the γ2 subunit has been able to be studied for the first time in neurones. Using an antibody which recognises the first 29 amino-terminal amino-acids of the γ2 subunit, this protein was shown to be tyrosine phosphorylated in adult rat brain lysate. It has been previously shown that the tyrosine kinase src phosphorylates the residues Y365 and Y367 of the γ2 subunit in HEK293 cells, causing upregulation of receptor function. The second antibody successfully raised in this study recognises the γ2 subunit when it is specifically phosphorylated on these two tyrosine residues. This allowed a more direct analysis of γ2 subunit tyrosine phosphorylation in adult rat brain lysate, and cultured cortical neurons. Results indicated that in cortical neurons phosphorylation of the γ2 subunit, on Y365 and Y367, is under very tight regulation. At present, there is little known as to how kinases or any other signalling molecules interact with GABAA receptors in neurones. Previous studies have identified serine and tyrosine kinase activities co-purifying with the receptor from brain preparations. To identify how protein kinases and related signalling molecules are targetted to the GABAA receptor, the intracellular domains of receptor subunits were used as baits to probe rat brain extracts. This approach has identified a very intimate association between PKC, the 'Receptor for Activated C-kinase', RACK-1, and the GABAA receptor β1 and β3 subunits. These interactions were initially identified by analysis of proteins bound to receptor fusion proteins in in vitro 'pull-down' assays. Both molecules were identified as binding directly and independently to the receptor by 'gel-overlay' assays. Confirmation of the in vivo existence of this complex was performed by co-precipitation of RACK-1 and PKC with the GABAA receptor from transfected HEK293 cells and from cultured cortical neurons. The functional significance of the RACK-1 interaction was investigated electrophysiologically in HEK293 cells, by disrupting the binding of RACK-1 with a polypeptide corresponding to its binding site on the GABAA β1 subunit. Finally, in cortical neurons, the β3 subunit was shown to be highly basally phosphorylated, due to the activity of closely associated PKC.

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