Abstract
Visual arrestin plays an important role in regulating light responsiveness via its ability to specifically bind to the phosphorylated and light-activated form of rhodopsin. Previously, we utilized an in vitro translation system to express and characterize the full-length (404 amino acids) and two truncated forms of visual arrestin. Here we have extended these studies to include a total of 33 different truncation and deletion mutants of arrestin, ranging from 69 to 391 amino acids in length. Mutants were produced by cutting within the open reading frame of the bovine arrestin cDNA with selective restriction enzymes followed by in vitro translation of the transcribed truncated mRNAs. Mutant arrestin binding to dark, light-activated, dark phosphorylated, and light-activated phosphorylated rhodopsin as well as to opsin, phosphoopsin, and truncated rhodopsin was then extensively characterized. In addition, the sensitivity of arrestin/rhodopsin interactions to conditions of increasing ionic strength was measured. These studies suggest the localization of multiple functional domains within the arrestin molecule that include: 1) a "phosphorylation recognition" domain, which interacts with the phosphorylated carboxyl terminus of rhodopsin, was localized predominately between residues 158-185; 2) an "activation recognition" domain, which interacts with those portions of the rhodopsin molecule that change conformation upon light activation, was found to consist of at least three regions within the first 191 residues of the arrestin molecule; 3) a hydrophobic interaction domain, localized between residues 191 and 365, appears to be mobilized upon binding of arrestin to activated phosphorylated rhodopsin; 4) a regulatory domain, localized in the COOH-terminal region of arrestin (residues 365-391), was found to play a role in controlling the conformational change in arrestin necessary for mobilization of the hydrophobic interaction domain; and 5) The NH2 terminus of arrestin (residues 2-16) was found to be important for interacting with the regulatory COOH-terminal region as well as maintaining the conformation of the NH2-terminal half of arrestin. A mechanism which ensures strict arrestin binding selectivity toward phosphorylated light-activated rhodopsin is proposed.
Highlights
Visual arrestin plays an importantrole in regulating The ability of an organism to regulate the intensity of a light responsiveness via its ability to bind response in the presence of a continuous stimulus plays an to thephosphorylated and light-activated formof rho- important role in cell function
Following light actiboxyl terminus of rhodopsin, was localized predomi- vation, rhodopsin kinase phosphorylates metarhodopsin I1 at nately between residues 158-185; 2a)n ”activation multiple sites on its carboxyl-terminal domain [3, 4]. This recognition” domain, which interacts with those por- phosphorylation reduces the ability of rhodopsin to tions of the rhodopsin molecule that change confor- interact with transducin and promotes the association of mation upon light activation, was found to consist of at least three regions within the first 191 residues of the arrestin molecule; 3) a hydrophobic interaction domain, localized between residues 191 and 365,appears tobe mobilized uponbinding of arrestin toactivated phosphorylated rhodopsin; 4) a regulatory domain, localized in theCOOH-terminal region of arrestin,was found to play a role in arrestin
Anotherstrategy for generating mutant arrestins involved initial linearization of the plasmid with HindIII, followedby a partial digestion with restriction enzymes that have two to four sites within the arrestin ORF (Fig. 1).In vitro transcription of the resulting plasmids generated a mixture of truncatedand full-length mRNAs, the position of truncation being determined by the position of the cutwithin the transcribed DNA strand [38]
Summary
STRICT SELECTIVITY TOWARD LIGHT-ACTIVATED (Receivedfor publication, January 20, 1993). We have utilized in vitro translation immediately dissolved in diethylpyrocarbonate-treated water, and t o express visual arrestin and characterize its binding to reprecipitated by addition of 0.1 volume of 3 M sodium acetate, pH rhodopsin[28] These studiesdemonstrated that in uitro translated arrestin is fully functionalin terms of its ability to bind to the light-activated phosphorylated form of rhodopsin.Twotruncatedarrestinswerealso in uitro. The in vitro translated arrestins (0.2-1 pmol) were incubated in 30 mM potassium HEPES, 2 mM MgC12,150 mM potassium acetate, pH 7.5 (buffer B) with 0.3-0.6 pg (7.5-15 pmol) of the various rhodopsins in a volume of 50 pl for 5 min a t 37 "C either in the dark or with illumination (room light). The samples were kept on ice and at thaeppropriate timewere loaded onto a 2-ml Sepharose 2B column equilibrated with buffer A to separate bound and free arrestins
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