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J Ahn, S Bungert, LL Molday, RS Molday; Analysis of the Nucleotide Binding Domains of ABCR, the Protein Defective in Stargardt Macular Dystrophy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1400.
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Purpose: ABCR (ABCA4), a member of the ATP binding cassette transporter family, is believed to transport retinal, retinal derivatives or phospholipids across the outer segment disk membrane of rod and cone photoreceptors. The transported substrate has not yet been identified, but all-trans retinal and several other compounds stimulate the ATPase activity of purified and reconstituted ABCR. Our goal is to determine whether the two nucleotide binding domains, NBD1 and NBD2, are equivalent and to identify the structural domains important for the observed retinal-stimulated ATPase activity. Methods: To separate NBD1 and NBD2, ABCR was cleaved in half by mild trypsin digestion of native ABCR from bovine rod outer segments (ROS) or by expression of the two halves as separate polypeptides in transfected COS-1 or HEK 293 cells. ATP binding was measured using the photoactivatable ATP analogue 8-azido-ATP. Another member of the ABCA subfamily, ABCA1, shares 50% amino acid sequence identity and a similar putative membrane topology with ABCR but is involved in apolipoprotein AI-mediated lipid efflux at the plasma membrane. Chimeric proteins composed of ABCR and ABCA1 will help us to identify the domains responsible for retinal binding and ATPase activation. Results: When membranes from ROS or transfected cells were labeled with 8-azido-ATP, the C-terminal half (NBD2) was labeled. Both halves had to be present for ATP binding to NBD2 since no labeling was observed when the C-terminal half was expressed alone. However, the N-terminal half (NBD1) was able to bind ATP only when expressed alone. Mutation of the conserved Walker A lysine in the NBDs had little effect on ATP binding but abolished retinal-stimulated ATPase activity. Conclusion: NBD2 contains the high affinity ATP binding site and the two NBDs are not functionally equivalent. Characterizing the different domains of ABCR is essential for determining the mechanism of ATP-coupled transport in the photoreceptor disk membrane and how mutations in the ABCR gene result in macular degeneration and impaired vision.
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