Ramsden, SFL; (2012) The role of gephyrin, collybistin and novel GEFs in the synaptic clustering of inhibitory receptors. Doctoral thesis (PhD), UCL (University College London).

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Abstract

GABAA and glycine receptors are clustered at inhibitory synapses via interactions with the scaffolding protein gephyrin. In turn, gephyrin is translocated to synapses by collybistin, a guanine nucleotide exchange factor (GEF) for Cdc42. Mutations in the human collybistin gene (ARHGEF9) give rise to a range of symptoms including anxiety, epilepsy and intellectual disability. However collybistin knockout mice revealed a selected loss of clustering of distinct GABAA receptor subtypes, whilst glycine receptors clustering remained intact. This suggests that other GEFs might also contribute to gephyrin and inhibitory receptor clustering. This thesis describes the identification and charactertisation of two novel GEFs that were indentified in a yeast two-hybrid screen using a gephyrin bait: IQSEC2 and IQSEC3. Full-length IQSEC2 did not interact with gephyrin in vitro, and is located at excitatory synapses in vivo, so is unlikely to have a role in GABAAR and GlyR clustering. However, four missense mutations in IQSEC2 were found in families with X-linked intellectual disability (XLID) that impair either calmodulin binding to the IQ-like domain (R359C) or ArfGEF activity (R758Q, Q801P and R863W). By contrast, full-length IQSEC3 did interact with gephyrin in vitro and co-localised with gephyrin and inhibitory receptors in vivo, making this ArfGEF a plausible clustering factor. I also show that a gene fusion affecting IQSEC3 may also result in autosomal intellectual disability associated with behavioural defects. Lastly, I examined the interactions between inhibitory GABAAR and GlyR subunits with gephyrin, mapping binding sites for gephyrin on the GABAAR α3 subunit. My analysis revealed that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3-M4 domain and the motif SMDKAFITVL at the N-terminus of the gephyrin E domain. Site-directed mutagenesis of the gephyrin E-domain revealed that GABAAR α3 and GlyR β subunits bind to an overlapping site on the gephyrin E-domain.

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