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L. Molday, R.S. Molday; Protein–Protein Interactions Involving ABCA4, the ABC Transporter Linked to Stargardt Macular Degeneration . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1701.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: ABCA4, also known as ABCR, is a member of the superfamily of ABC transporters that has been implicated in the transport of N–retinylidene–phosphatidylethanolamine across the disc membranes. Mutations in the gene encoding ABCA4 have been linked to a number of retinal degenerative diseases including Stargardt macular degeneration, cone–rod dystrophy, inverse retinitis pigmentosa and age–related macular degeneration. To more fully understand the role of ABCA4 in retinoid processing and retinal degenerative diseases, we have initiated a study to identify proteins in rod outer segments (ROS) that interact with ABCA4. Methods: ROS were isolated from bovine retina by sucrose density centrifugation. The ROS were solubilized in CHAPS or Triton X–100 and passed through an immunoaffinity column consisting of the anti–ABCA4 Rim 3F4 monoclonal antibody covalently bound to Sepharose. Proteins that co–purified with ABCA4 on the affinity matrix were analyzed by SDS gel electrophoresis, Western blotting and MALDI–TOF mass spectrometry. Results: Two proteins migrating on SDS gels with apparent molecular masses of 44 kDa and 36 kDa co–purified with ABCA4 on a Rim 3F4 – Sepharose affinity matrix. MALDI–TOF mass spectrometry of tryptic fragments identified the 44 kDa protein as a variant of arrestin. Western blotting further showed that this protein was the spliced form of arrestin known as p44 arrestin. Little full–length arrestin co–purified with ABCA4. Western blots labeled with anti–rhodopsin antibodies identified the 36 kDa protein as rhodopsin. The effect of detergents, light, retinoid and nucleotides on the interaction of ABCA4 with arrestin p44 and rhodopsin is currently under investigation. Conclusions: Our results indicate that in the presence of CHAPS and Triton X–100, rhodopsin and arrestin p44 coprecipitate with ABCA4. These proteins possibly acting as a complex may regulate the ATP–dependent retinoid transport activity of ABCA4.
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