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W. Smith, M. Ascano, S. Gregurick, P. Robinson; New Elements Responsible for Regulating the Selectivity of Arrestin for Phosphorylated Photoactivated Rhodopsin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1175.
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Purpose: A recent computational model of arrestin/rhodopsin binding demonstrates an interaction of the C–terminal peptide of rhodopsin with Lys–15, Arg–29, Lys–300, and His–301 of arrestin . Of these, only Lys–15 has been previously implicated . In this study, we performed site–directed mutagenesis of these four residues to empirically assess their role in the interaction of arrestin with rhodopsin. Methods: Native bovine visual arrestin and arrestin mutants (K15E, R29E, K300A, K300E, H301A, H301E, and K300A/H301A) were expressed in yeast with an N–terminal His–tag. Purified proteins were evaluated in three ways: a) binding to rhodopsin in a centrifugation assay, b) inhibition of transducin activation in a GTPγS binding assay, and c) susceptibility to proteolysis in the presence of a peptide that mimics the fully phosphorylated C–terminus of rhodopsin (Rho–7PP). Results: Charge reversal of Arg–29 (R29E) produced an arrestin that binds equally well to photoactivated rhodopsin that is not phosphorylated (R*). Trypsinolysis of R29E showed rapid cleavage of the carboxy–terminus, even in the absence of Rho–7PP. Binding of arrestin to light–activated phosphorhodopsin (R*P) was significantly reduced in two mutants–– K15E (50% of native) and K300E (80% of native). These same two mutations, along with R29E and K300A, also impaired arrestin’s reduction of transducin activation. The C–terminus of H301A and H301E mutants was more sensitive to trypsinolysis in the presence of Rho–7PP. Conclusions: Our findings suggest that the studied residues play different roles in the interaction of arrestin with rhodopsin. Arg–29 appears to have a role in maintaining selectivity of arrestin for phosphorylated rhodopsin since R29E binds photoactivated rhodopsin regardless of its phosphorylation state. Because Arg–29 does not project towards the polar core of arrestin, we hypothesize that the Arg–29 mutation affects the positioning of arrestin’s regulatory C–terminus, which is in keeping with its susceptibility to trypsinolysis. His–301 appears to also have a role in maintaining the selectivity for R*P, although not to the same extent as Arg–29. Lys–300 apparently influences the activation of arrestin since K300A and K300E can bind R*P, but are unable to adopt a conformation that allows quenching of transducin activation. 1. Ling Y, Ascano M, Robinson P, Gregurick SK (2004) Biophys. J. 86: 2445–2454. 2. Vishnivetskiy SA, Schubert C, Climaco GC, Gurevich YV, Velez MG, Gurevich VV (2000) J. Biol. Chem. 275: 41049–41057.
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