Cloning and functional characterization of salamander rod and cone arrestins.
OBJECTIVE
To clone, localize, and determine functional binding characteristics of rod and cone arrestins from the retina of the tiger salamander (Ambystoma tigrinum).


METHODS
Two arrestins from salamander retina were cloned on the basis of their homology to known arrestins from other species. The expression pattern of these arrestins (SalArr1 and SalArr2) in the retina was determined by immunocytochemistry and in situ hybridization. SalArr1 and SalArr2 were expressed and functionally characterized.


RESULTS
Both immunocytochemistry and in situ hybridization show that SalArr1 and SalArr2 localized specifically to rod and cone photoreceptors, respectively. SalArr1 demonstrated a characteristic high selectivity for light-activated phosphorylated rhodopsin (P-Rh*) and significant species selectivity, binding preferentially to amphibian rhodopsin over bovine rhodopsin. Mutant constitutively active forms of SalArr1 demonstrated a 2- to 4-fold increase in P-Rh* binding (compared with wild-type protein) and an even more dramatic (up to 25-fold) increase in binding to unphosphorylated Rh* and dark P-Rh. Constitutively active SalArr1 mutants also showed a reduced specificity for amphibian rhodopsin. The ability of Escherichia coli-expressed SalArr1, SalArr2, and an SalArr1-3A (L369A,V370A,F371A) mutant to bind to frog Rh* and P-Rh* and to compete with tritiated SalArr1 for amphibian P-Rh* was compared. SalArr1 and its mutant form bound to amphibian P-Rh* with high affinity (Ki = 179 and 74 nM, respectively), whereas the affinity of SalArr2 for P-Rh* was substantially lower (Ki = 9.1 microM).


CONCLUSIONS
SalArr1 and SalArr2 are salamander rod and cone arrestins, respectively. Crucial regulatory elements in SalArr1 are conserved and play functional roles similar to those of their counterparts in bovine rod arrestin. Rod and cone arrestins are relatively specific for their respective receptors.
Department of Ophthalmology, University of Florida, Gainesville, USA.
W C
WC
Smith
E V
EV
Gurevich
D R
DR
Dugger
S A
SA
Vishnivetskiy
C L
CL
Shelamer
J H
JH
McDowell
V V
VV
Gurevich
D000557:Ambystoma; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D019390:Arrestins; D004247:DNA; D012243:Rhodopsin