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UBC Theses and Dissertations

Identification and characterization of a novel retinal protein, ANKRD33, and its interacting partner HPCAL-1 Rostamirad, Shabnam

Abstract

The outer segment is a specialized region of rod and cone photoreceptor cells located in vertebrate retina. It features stacks of membranous discs containing visual pigment molecules, and the membranous structures undergo continuous renewal process. In order to better understand the cellular mechanisms in the outer segment, Kwok et al. (2008) used tandem mass spectrometry on bovine rod outer segment preparations, and identified many proteins of unknown function, one of which was ankyrin repeat domain 33 (ankrd33). Ankrd33 belongs to the ankyrin repeat protein class, which has been described to be involved in a variety of functions, such as cell-cell signalling and cytoskeleton structure. However, the function and localization of Ankrd33 have not been previously investigated. In this project, ankrd33 was cloned from bovine retinal cDNA and RT-PCR experiments showed a retina specific gene expression of this protein. In addition, monoclonal antibodies were raised against N and C-termini of ankrd33. These antibodies were used to localize the protein in retina. In addition, they were used to identify the interacting partners of ankrd33 in photoreceptors. Ankrd33 was found to be exclusively expressed in the outer segments of photoreceptor cells and co-immunoprecipitation studies identified hippocalcin like 1 protein (HPCAL-1) as one of the interacting partners. HPCAL-1 belongs to the family of proteins called neuronal calcium sensors (NCS) which are EF-hand containing Ca2+-binding proteins and expressed in different neuronal cells. These proteins are involved in calcium modulation of numerous cellular activities based on the cell type and their interacting partners. As a first step in identifying the possible cellular pathways that ankrd33 and HPCAL-1 might be involved in, monoclonal antibodies were produced against the full length of HPCAL-1 and were used for immunofluorescence studies and in vitro confirmation of the interaction between the two proteins. Immunofluorescence studies showed labelling of ankr33 and HPCAL-1 in rod and cone outer segment with cone outer segment having a stronger signal. These results showed that ankrd33 and HPCAL-1 are both highly expressed in the cone outer segment and these proteins may be involved in calcium dependent-cone specific pathways.

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