Activation of arrestin: requirement of phosphorylation as the negative charge on residues in synthetic peptides from the carboxyl-terminal region of rhodopsin.
OBJECTIVE
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 phosphorylated residues.
McDowell
J H
JH
Departments of Ophthalmology, University of Florida, Gainesville 32610, USA. jmcdowel@ufl.edu
Robinson
P R
PR
Miller
R L
RL
Brannock
M T
MT
Arendt
A
A
Smith
W C
WC
Hargrave
P A
PA
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D019393:Arrestin / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D003544:Cysteic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014785:Vision, Ocular
D019393:Arrestin; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D001224:Aspartic Acid; D018698:Glutamic Acid; D012243:Rhodopsin; D003544:Cysteic Acid; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP