Evans, B.R.; (2006) Ush1c: expression and interactions in the retina. Doctoral thesis , University of London.

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Abstract

Usher syndrome is an autosomal recessive disease, displaying pathology of the auditory and visual systems. The three clinical phenotypes are linked to eleven different loci, and differ in the severity and age of onset of sensorineural hearing loss, vestibular areflexia and Retinitis Pigmentosa. The expression pattern of Ush1c which encodes a PDZ domain containing protein (Harmonin) has been established in the murine ear. This study examined the localisation of the protein in the murine eye, detecting Ush1c mRNA from E12.5 and localising the protein to the newborn photoreceptors from P2. In the developed retina, harmonin was localised to the photoreceptor outer segments. Further immunoreactivity was noted in unfixed tissue, showing harmonin colocalising with the USH1B and 1D proteins to the photoreceptor inner segments and outer plexiform layer. The USH1B and USH1D proteins are known to interact with and be involved in the correct localisation of harmonin in the ear. Yet in the eye, harmonin localisation in fixed tissue was maintained in the outer segments in the absence of either USH1 protein. To further examine the protein interactions of harmonin, a Yeast Two-Hybrid study was undertaken using a bovine retinal library. Six possible interacting proteins were identified and confirmed by yeast mating. Several of these positive interactors, although interacting with the whole harmonin protein, did not interact with any of its PDZ binding domains. One such protein was phosducin, a small acidic cytosolic protein. Its potential as a binding partner for harmonin in the retina was confirmed by a pull down assay and immunolocalisation studies. Phosducin is known to interact with proteins associated with glutamate receptors at the photoreceptor ribbon synapses. Its interaction with harmonin would suggest a role for harmonin in the outer plexiform layer and more specifically at the ribbon synapses of the retina.

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