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Experimental and Computational Studies of the Desensitization Process in the Bovine Rhodopsin-Arrestin Complex

Y. Ling ^*, M. Ascano ^*, P. Robinson ^* and S. K. Gregurick 

^* Department of Biological Sciences, Department of Chemistry and Biochemistry, University of Maryland, Baltimore, Maryland

Correspondence: Address reprint requests to Asst. Prof. Susan K. Gregurick, Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250. Tel.: 410-455-8698; E-mail: greguric{at}umbc.edu.

The deactivation of the bovine G-protein-coupled receptor, rhodopsin, is a two-step process consisting of the phosphorylation of specific serine and threonine residues in the cytoplasmic tail of rhodopsin by rhodopsin kinase. Subsequent binding of the regulatory protein arrestin follows this phosphorylation. Previous results find that at least three phosphorylatable sites on the rhodopsin tail (T³⁴⁰) and at least two of the S³³⁸, S³³⁴, or S³⁴³ sites are needed for complete arrestin-mediated deactivation. Thus, to elucidate the details of the interaction between rhodopsin with arrestin, we have employed both a computational and an in vitro experimental approach. In this work, we first simulated the interaction of the carboxy tail of rhodopsin with arrestin using a Monte Carlo simulated annealing method. Since at this time phosphorylation of specific serines and threonines is not possible in our simulations, we substitute either aspartic or glutamic acid residues for the negatively charged phosphorylated residues required for binding. A total of 17 simulations were performed and analysis of this shows specific charge-charge interactions of the carboxy tail of rhodopsin with arrestin. We then confirmed these computational results with assays of comparable constructed rhodopsin mutations using our in vitro assay. This dual computational/experimental approach indicates that sites S³³⁴, S³³⁸, and T³⁴⁰ in rhodopsin and K¹⁴ and K¹⁵ on arrestin are indeed important in the interaction of rhodopsin with arrestin, with a possible weaker S³⁴³ (rhodopsin)/K¹⁵ (arrestin) interaction.

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