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JH McDowell, WC Smith, A Arendt, RL Miller, A Dinculescu, PA Hargrave; The Synthetic Phosphorylated Carboxyl Terminal Region of Rhodopsin Can Be Crosslinked to Arrestin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1392.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To crosslink the synthetic, fully phosphorylated region of bovine rhodopsin comprising residues 330-348 to arrestin in order to determine part of its binding site. Methods: Two analogs of the fully phosphorylated synthetic peptide comprising residues 330-348 of bovine rhodopsin were synthesized: the unacetylated peptide (7P-peptide) and peptide with the amino terminus acetylated (Ac7P-peptide). These analogs were modified at their free amino groups with the bifunctional reagent N-(a-maleimidoacetoxy) succinimide ester (AMAS) that places a sulfhydryl reactive group on the free amino group. The modified peptide was then combined with arrestin and various cysteine mutants of arrestin. SDS-PAGE was performed to determine if the peptide crosslinked to arrestin. Crosslinking was detected by an increase in molecular weight as well as by immunoblots using MAb A11-82P that recognizes phosphorylated rhodopsin. Results: 7P-peptide was crosslinked to arrestin, but only to arrestin mutants that contained the native Cys143. The absence of the other two native cysteine residues had little effect on the crosslinking. In preliminary experiments, mutants with a cysteine residue in an exposed loop of arrestin (i.e. S156C /C143A, S199C/C143A, S156C/S199C/C143A, and S178C/C143A) but lacking Cys143 showed some evidence of crosslinking, but were not as effective as native arrestin or a V177C mutant that contained Cys143. Ac7P-peptide crosslinked to native arrestin but less effectively than 7P-peptide. A four-fold molar excess of the modified 7P-peptide to arrestin, based on the absorbance of the N-(a-maleimido) group, yielded 50% modification of arrestin after a 2 hour incubation while a similar concentration of modified Ac7P-peptide yielded slightly less than 10% modification. Conclusion: Rhodopsin's 7P-peptide can be crosslinked to native arrestin at Cys143. Crosslinking is more effective through the amino terminus of 7P-peptide than through the Lys339 side chain. The data suggest that the cross-linking is somewhat biospecific since crosslinking to the native arrestin was more effective than to mutants lacking Cys143 but containing a cysteine residue in an exposed loop. This suggests that the area near Cys143 comprises part of the binding site for the phosphorylated region of rhodopsin.
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