Sharma, Seema; (2002) Molecular characterisation of C3, a novel protein putatively associated with GABAA receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Neurotransmitter receptors are thought to be cytoskeletally anchored, clustered and regulated by specifically adapted receptor-associated proteins. Although many such proteins have been characterised for excitatory synapses forming the post-synaptic density, the precise protein architecture of inhibitory synapses remains to be delineated. The GABAA receptors are major mediators of inhibitory neurotransmission in the central nervous system. Previously, the yeast two-hybrid system was used to screen for GABAA receptor-associated proteins and a novel protein, C3, encoded by a partial 1.8 kb cDNA, was found to interact with the GABAA receptor β2 subunit intracellular loop (IL). Further molecular characterisation of C3 is described in this thesis. Using the yeast two-hybrid system with the application of increased selection criteria, the specificity of interaction between GABAA receptor β2 subunits and C3 was corroborated and extended. The full length C3 cDNA sequence was determined by a combination of hybridisation screening of a rat brain cDNA library in conjunction with rapid amplification of cDNA ends (RACE). The full length C3 clone revealed a 2745 bp open reading frame (ORF) encoding a 913 amino acid (aa) protein termed C3 full length (C3 FL). A partial human orthologue for the C3FL, KIAA0549, was identified from database searches. Northern blotting with a [³²P] KIAA0549 probe revealed hybridisation to major and minor mRNA transcripts with the same sizes found for [³²P] C3. There was, however, a qualitatively different tissue distribution between the two probes. The amino acid sequence of the full length KIAA0549 clone, and the KIAA0549 and C3 FL genomic structure were deduced using the human genomic clone, RP11 575 C6. The GABAA receptor β2-IL binding domain was located between C3 FL aa 8-503, as determined by deletion analysis in combination with the yeast two-hybrid assay. C3 FL aa 8-634 was expressed in Escherichia coli as a poly-histidine-tagged fusion protein and immobilised to Ni²⁺ Sepharose. Detergent extracts of rat brain were subjected to C3 FL 8-634-Ni²⁺Sepharose affinity chromatography. An in vitro interaction between C3 FL aa 8-634 and GABAA receptor β2 subunits was demonstrated, confirming that in a different experimental paradigm the two proteins can associate. This, combined with evidence from homologous proteins, suggest that C3FL may function as a GABAA receptor β2 subunit trafficking factor.

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