Abstract
Arrestin plays a critical role in quenching phototransduction via its ability to specifically interact with the phosphorylated light-activated form of the visual receptor rhodopsin. In an effort to identify the residues involved in interaction with the phosphorylated C terminus of rhodopsin, we introduced point mutations into a basic region in visual arrestin previously implicated in phosphorylation-recognition (residues 163-189). A total of nine point mutations were made, each substituting a neutral hydrophilic residue for a positively charged Lys, Arg, or His. The functional consequences of these mutations were then analyzed by comparing the binding of full-length and truncated wild-type and mutant arrestin to various functional forms of rhodopsin. These studies demonstrate that Arg-171, Arg-175, and Lys-176 in bovine arrestin play a primary role in phosphate interaction, while Lys-166 and Lys-167 likely play a minor role in phosphate binding. In contrast, Lys-163 and His-179 appear to play a regulatory role, while Arg-182 and Arg-189 are not directly involved in arrestin binding to rhodopsin. Arg-175 also appears to function as a phosphorylation-sensitive trigger since charge neutralization by mutagenesis enables arrestin-R175N to bind to light-activated rhodopsin as well as wild-type arrestin binds to phosphorylated light-activated rhodopsin. The implications of these findings for the sequential multisite binding of arrestin to rhodopsin are discussed.
Highlights
In an effort to identify the residues involved in interaction with the phosphorylated C terminus of rhodopsin, we introduced point mutations into a basic region in visual arrestin previously implicated in phosphorylation-recognition
The functional consequences of these mutations were analyzed by comparing the binding of full-length and truncated wild-type and mutant arrestin to various functional forms of rhodopsin. These studies demonstrate that Arg-171, Arg-175, and Lys-176 in bovine arrestin play a primary role in phosphate interaction, while Lys-166 and Lys-167 likely play a minor role in phosphate binding
Arg-175 appears to function as a phosphorylation-sensitive trigger since charge neutralization by mutagenesis enables arrestin-R175N to bind to lightactivated rhodopsin as well as wild-type arrestin binds to phosphorylated light-activated rhodopsin
Summary
DIVERSE FUNCTIONAL ROLES OF POSITIVELY CHARGED RESIDUES WITHIN THE PHOSPHORYLATION-RECOGNITION REGION OF ARRESTIN*. A total of nine point mutations were made, each substituting a neutral hydrophilic residue for a positively charged Lys, Arg, or His. The functional consequences of these mutations were analyzed by comparing the binding of full-length and truncated wild-type and mutant arrestin to various functional forms of rhodopsin. Arg-175 appears to function as a phosphorylation-sensitive trigger since charge neutralization by mutagenesis enables arrestin-R175N to bind to lightactivated rhodopsin as well as wild-type arrestin binds to phosphorylated light-activated rhodopsin. Quenching of the visual transduction cascade involves a rapid activation-dependent phosphorylation of rhodopsin by the enzyme rhodopsin kinase (2) This is followed by the highly selective binding of arrestin to activated phosphorylated rhodopsin, a process that appears to attenuate the activation of transducin (3, 4). The moderate size of this domain as well as the presence of a number of potentially important basic residues makes this region a suitable target for systematic site-directed mutagenesis
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