MECHANISM OF PHOSPHORYLATION-RECOGNITION BY VISUAL ARRESTIN AND THE TRANSITION OF ARRESTIN INTO A HIGH-AFFINITY BINDING STATE

Arrestin plays an important role in quenching phototransduction via it s ability to interact specifically with the phosphorylated light-activ ated form of the visual receptor rhodopsin (P-Rh). Previous studies h ave demonstrated that Arg175 in bovine arrestin is directly involved i n the phosphorylation-dependent binding of arrestin to rhodopsin and s eems to function as a phosphorylation-sensitive trigger. In this study , we further probed the molecular mechanism of phosphorylation recogni tion by substituting 19 different amino acids for Arg175. We also asse ssed the effects of mutagenesis of several other highly conserved resi dues within the phosphorylation-recognition region (Val170, Leu172, Le u173, IIe174, Val177, and Gln178). The binding of all of these mutants to P-Rh, light-activated rhodopsin, and truncated rhodopsin, which l acks the carboxyl-terminal phosphorylation sites, was then characteriz ed. Overall, our results suggest that arrestin interaction with the ph osphorylated carboxyl-terminal domain of rhodopsin activates two relat ively independent changes in arrestin: (a) mobilization of additional binding sites and (b) increased affinity of the phosphorylation-recogn ition region for the rhodopsin carboxyl-terminal domain. Together, the se two mechanisms ensure the exquisite selectivity of arrestin toward P-Rh. Mutagenesis of residues that play a major role in binding site mobilization and phosphorylation-recognition enabled us to create ''co nstitutively active'' (phosphorylation-independent) arrestin mutants t hat have high affinity for both P-Rh and light-activated rhodopsin. T he introduction of a negative charge in position 175 was particularly effective in this respect. A detailed molecular model of phosphorylati on-recognition is proposed.