ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters, which has been implicated in the clearance of all-trans retinal derivatives from rod and cone photoreceptor cells following photoexcitation as part of the visual cycle. Mutations in ABCA4 are known to cause Stargardt macular degeneration and related disorders, associated with a severe loss in vision. Recently, a solid-phase binding assay has been developed to identify retinoids that likely serve as substrates for this transporter. In this procedure, monoclonal antibodies directed either against an epitope within ABCA4 (Rim 3F4 antibody) or against the 9 amino acid 1D4 epitope tag engineered onto the C-terminus of expressed ABCA4 (Rho 1D4 antibody) are covalently bound to a Sepharose matrix. This immunoaffinity matrix is then used to isolate ABCA4 from photoreceptor outer segments or transfected cells. All-trans retinal is added to immobilized ABCA4 in the presence of a phospholipid mixture containing phosphatidylethanolamine. The bound retinoid is then analyzed directly by spectrophotometry or identified by HPLC and/or mass spectrometry following extraction with organic solvents. Using this procedure, it has been shown that unprotonated N-retinylidene-phosphatidylethanolamine binds with high affinity to ABCA4 and is released by the addition of ATP. These procedures and related radiometric assays using titrated retinal have been used to study the binding of N-retinylidene-PE to wild-type and mutant ABCA4 in the absence and presence of nucleotides for structure-function studies.
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