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Investigations of GABAA receptor phosphorylation and receptor-protein interactions.

McAinsh, K.; (2006) Investigations of GABAA receptor phosphorylation and receptor-protein interactions. Doctoral thesis , UCL (University College London). Green open access

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Abstract

GABAA receptors mediate the majority of fast synaptic inhibition in the adult mammalian brain, and play a critical role in controlling neuronal excitability during development. The functional properties and stability of cell-surface GABAA receptors are key determinants of the efficacy of GABAergic neurotransmission. I therefore employed a broad spectrum of biochemical, and cell and molecular biological techniques to identify molecular interactions that may be involved in regulating the activity and number of GABAA receptors at the neuronal surface. In this thesis, I identified a novel interaction between the GABAA receptor and the calcium/calmodulin-dependent protein kinase II (CaMKII). Affinity-purification assays revealed that CaMKII forms a native complex with the GABAA receptor in brain, and that the kinase binds non-selectively to the major intracellular domain (ICD) of various GABAA receptor subunits. The interaction between CaMKII and the receptor (3 subunits was found to be dependent upon phosphorylation of the kinase at T286, but appeared to be independent of subunit phosphorylation. Furthermore, a CaMKII binding site was identified in the ICD of the receptor (3 subunits, between residues 304 and 323. Further work demonstrated that CaMKII selectively phosphorylates the ICDs of the receptor 3 and y subunits, and identified protein phosphatases that dephosphorylate the receptor 33 subunit at the CaMKII sites. Depolarisation of cultured immature cortical neurons was found to increase the level of enzymatically-active CaMKII that associates with the ICD of the receptor 33 subunit, and to trigger CaMKII-dependent phosphorylation of the receptor 33 subunit at both the S408 and S409 residues. Work presented in this thesis also identified multivalent interactions between GABAA receptor y subunits and the AP2 adaptor complex, a major component of the endocytic machinery. The ICD of each receptor y subunit was found to interact with the native AP2 complex from brain, and to bind selectively and directly to the u2- adaptin of AP2. Two distinct binding sites for jx2-adaptin were identified in the ICD of the y2S subunit. The N-terminal half of the ICD was found to interact with sub- domain B of u2-adaptin via a putative basic-rich motif. In contrast, the C-terminal half of the ICD was found to bind sub-domain A of u2-adaptin, likely via a tyrosine- based motif. Both u2-adaptin binding motifs were found to be common to all the y subunit isoforms.

Type: Thesis (Doctoral)
Title: Investigations of GABAA receptor phosphorylation and receptor-protein interactions.
Identifier: PQ ETD:593014
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by Proquest.
URI: https://discovery.ucl.ac.uk/id/eprint/1445690
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