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 its ability to interact specifically with the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Previous studies have demonstrated that Arg175 in bovine arrestin is directly involved in the phosphorylation-dependent binding of arrestin to rhodopsin and seems to function as a phosphorylation-sensitive trigger. In this study, we further probed the molecular mechanism of phosphorylation recognition by substituting 19 different amino acids for Arg175. We also assessed the effects of mutagenesis of several other highly conserved residues within the phosphorylation-recognition region (Val170, Leu172, Leu173, Ile174, Val177, and Gln178). The binding of all of these mutants to P-Rh*, light-activated rhodopsin, and truncated rhodopsin, which lacks the carboxyl-terminal phosphorylation sites, was then characterized. Overall, our results suggest that arrestin interaction with the phosphorylated carboxyl-terminal domain of rhodopsin activates two relatively independent changes in arrestin: (a) mobilization of additional binding sites and (b) increased affinity of the phosphorylation-recognition region for the rhodopsin carboxyl-terminal domain. Together, these 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 "constitutively active" (phosphorylation-independent) arrestin mutants that have high affinity for both P-Rh* and light-activated rhodopsin. The introduction of a negative charge in position 175 was particularly effective in this respect. A detailed molecular model of phosphorylation-recognition is proposed.