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A Dinculescu, JH McDowell, S Amici, D Dugger, N Richards, PA Hargrave, WC Smith; An Insertional Mutagenesis and Immunochemical Analysis of Visual Arrestin Interaction with Rhodopsin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1391.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Visual arrestin inactivates the phototransduction cascade by specifically binding to light-activated, phosphorylated rhodopsin. Our goal was to further probe the structural determinants of arrestin function by using a combined insertional mutagenesis and immunochemical approach. Methods: Recombinant arrestins containing the ten amino acid c-myc tag (EQKLISEEDL) at different positions were generated in yeast, purified on affinity columns and tested for binding to rhodopsin in centrifugation assays. Those mutants in which the insertion did not abolish their function were used in competition experiments with the anti-myc antibody. Partial tryptic proteolysis was performed on all myc-tagged arrestins to test if the digestion pattern was similar to that of wild-type protein. Results: When the myc tag was placed on the C-terminus after amino acid 399, between amino acids 99-100 or 162-163 in arrestin, binding to rhodopsin was found to be very similar to wild-type. Insertions in the 68-78 loop have resulted in proteins for which binding to the receptor was either markedly decreased or completely abolished. Partial tryptic proteolysis revealed a digestion pattern similar to wild-type protein for all mutants. The binding of light-activated, phosphorylated rhodopsin to the 162myc163 or 72myc73 arrestins in solution was completely inhibited in the presence of less than 2-fold molar excess anti-myc antibody relative to arrestin. In contrast, the antibody did not block the interaction of the 399myc or 99myc100 arrestins with the receptor and was found specifically in the arrestin-rhodopsin pellets for light-reactions. Conclusion: The results indicate that an interactive surface for rhodopsin is located on or near the concave region of the N-domain of arrestin.
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