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J. Hugh McDowell, Phyllis R. Robinson, Ron L. Miller, Michael T. Brannock, Anatol Arendt, W. Clay Smith, Paul A. Hargrave; Activation of Arrestin: Requirement of Phosphorylation as the Negative Charge on Residues in Synthetic Peptides from the Carboxyl-Terminal Region of Rhodopsin. Invest. Ophthalmol. Vis. Sci. 2001;42(7):1439-1443.
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purpose. To determine whether substitution of the potential phosphorylation
sites of bovine rhodopsin’s carboxyl-terminal region with the acidic
residues aspartic acid, glutamic acid, or cysteic acid promotes the
activation of 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
substituted with glutamic acid (7E-peptide), and the peptide with the
phosphorylation sites substituted with cysteic acid (7Cya-peptide). The
peptides were tested in assays in which the 7P-peptide had previously
been shown to have an effect. Rhodopsin with glutamic acid (Etail) or
aspartic acid (Dtail) substituted for the phosphorylation sites in
rhodopsin were constructed and expressed in COS-7 cells and tested in
an in vitro assay.
results. Earlier work has demonstrated that the 7P-peptide activates
arrestin, showing induction of arrestin binding to light-activated
unphosphorylated rhodopsin, inhibition of the light-induced
phosphodiesterase (PDE) activity in rod outer segments (ROS) with
excess arrestin, increase in the initial rapid proteolysis of arrestin
by trypsin, and enhanced reactivity of one of arrestin’s sulfhydryl
groups with inhibition of the reactivity of another. None of
these effects was observed in the presence of 7E-peptide or
7Cya-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 arrestin. Because none of the other effects was observed with
the 7Cya-peptide, the authors conclude that the 7Cya-peptide does not
activate arrestin, but acts, probably nonspecifically, through some
other part of the transduction system. Considerable arrestin-mediated
rhodopsin inactivation was observed with both the Etail and the Dtail
mutant, although these substitutions did not yield rhodopsins that were
equivalent to phosphorylated rhodopsin.
conclusions. These results, taken together, suggest that the negative charge due to
phosphates in the carboxyl-terminal region of rhodopsin are required
for the full activation of arrestin and that acidic amino acids
(carboxyl and sulfonic) do not mimic the negative charge of
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