Activation of arrestin: Requirement of phosphorylation as the negative charge on residues in synthetic peptides from the carboxyl-terminal region of rhodopsin

PURPOSE. TO determine whether substitution of the potential phosphorylation sites of bovine rhodopsin's carboxyl-terminal region with the acidic resid ues aspartic acid, glutamic acid, or cysteic acid promotes the activation o f arrestin. METHODS. Three peptide analogues of the 19-residue carboxyl-terminal region of rhodopsin (330-348) were synthesized: the fully phosphorylated peptide (7P-peptide), the peptide with all potential phosphorylation sites substitu ted with glutamic acid (7E-peptide), and the peptide with the phosphorylati on sites substituted with cysteic acid (7Cya-peptide). The peptides were te sted in assays in which the 7P-peptide had previously been shown to have an effect. Rhodopsin with glutamic acid (Etail) or aspartic acid (Dtail) subs tituted for the phosphorylation sites in rhodopsin were constructed and exp ressed in COS-7 cells and tested in an in vitro assay. RESULTS. Earlier work has demonstrated that the 7P-peptide activates arrest in, showing induction of arrestin binding to light-activated unphosphorylat ed rhodopsin, inhibition of the light-induced phosphodiesterase (PDE) activ ity in rod outer segments (ROS) with excess arrestin, increase in the initi al rapid proteolysis of arrestin by trypsin, and enhanced reactivity of one of arrestin's sulfhydryl groups with inhibition of the reactivity of anoth er. None of these effects was observed in the presence of 7E-peptide or 7Cy a-peptide. The 7Cya-peptide inhibited the PDE activity in ROS, but the same effect was observed both in the presence and the absence of excess arresti n. Because none of the other effects was observed with the 7Cya-peptide, th e authors conclude that the 7Cya-peptide does not activate arrestin, but ac ts, probably nonspecifically, through some other part of the transduction s ystem. Considerable;arrestin-mediated rhodopsin inactivation was observed w ith both the Etail and the Dtail mutant, although these substitutions did n ot yield rhodopsins that were equivalent to phosphorylated rhodopsin. CONCLUSIONS. These results, taken together, suggest that the negative charg e due to phosphates in the carboxyl-terminal region of rhodopsin are requir ed for the full activation of arrestin and that acidic amino acids (carboxy l and sulfonic) do not mimic the negative charge of phosphorylated residues .