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Q. Liu, A. Saveliev, J. E. Westfall, R. J. Ferland, E. A. Pierce; A Joubert Syndrome Protein Abelson Helper Integration Site 1 (ahi1) Interacts With the Rp1 Protein in Photoreceptor Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4076.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the RP1 gene cause both dominant and recessive RP. Our previous studieshave shown that RP1 is a photoreceptor microtubule-associated protein that is part of the photoreceptor axoneme, and is required for organization of outer segment structure. In order to help define the mechanism by which RP1 helps control outer segment organization, we used yeast-two hybrid analyses to screen for proteins that interact with RP1.
We constructed 4 over-lapping portions of the Rp1 proteins as baits, and screened a mouse retinal cDNA library using a GAL4-based two-hybrid system. Positive colonies were isolated and verified by specificity testing. The potential interaction between Rp1 and candidate proteins was further evaluated by co-expression and co-immunoprecipitation assays using cultured cells and retinal extracts. Co-localization of candidate interacting proteins with Rp1 in wild type and mutant animals was examined by immunostaining.
A total of 56 specific clones representing 11 candidate interacting proteins were identified. Of these, 2 clones encode the Abelson helper integration site 1 (Ahi1) protein. Immunoprecipitation showed that Ahi1 can co-precipitate Rp1 from retinal extracts and immunostaining studies showed that Ahi1 was located in the transition zone of photoreceptor cells, adjacent to the Rp1 signal in the axoneme. Of particular interest, we found that Rp1 was mislocalized into the synaptic region of photoreceptor cells in Ahi1 knockout mice. Similarly, the Ahi1 protein was partially mislocalized in Rp1-Q662X knockin mice, with the protein detected throughout inner segments as well as in the transition zone.
We have identified Ahi1 as an interacting protein of Rp1 by yeast two-hybrid analyses. Mutations in the AHI1 gene cause the ciliopathy, Joubert syndrome. AHI1 was previously reported to interact with nephrocystin-1 (NPHP1), which is located in the transition zone of photoreceptors and other cilia, and interact with other Joubert syndrome proteins (NPHP2, NPHP3, and NPHP4). These findings highlight the role of RP1 and the cytoskeleton of photoreceptor sensory cilia (PSC) as critical organizers of outer segment structure. They also demonstrate that in order to understand the function of RP1 and the pathogenesis of RP1 disease, we need to consider RP1 in the context of the many other proteins in PSC. We will continue to evaluate the function of the RP1 protein and the significance of its interaction with AHI1 and other cilia proteins.
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