Cloning and functional characterization of salamander rod and cone arrestins

PURPOSE. To clone, localize, and determine functional binding characteristi cs of rod and cone arrestins from the retina of the tiger salamander (Ambys toma tigrinum). METHODS. Two arrestins from salamander retina were cloned on the basis of t heir homology to known arrestins from other species. The expression pattern of these arrestins (SalArr1 and SalArr2) in the retina was determined by i mmunocytochemistry and in situ hybridization. Salhrr1 and SalArr2 were expr essed and functionally characterized. RESULTS. Both immunocytochemistry and in situ hybridization show that SalAr r1 and SalArr2 localized specifically to rod and cone photoreceptors, respe ctively. SalArr1 demonstrated a characteristic high selectivity for light-a ctivated phosphorylated rhodopsin (P-Rh*) and significant species selectivi ty, binding preferentially to amphibian rhodopsin over bovine rhodopsin. Mu tant constitutively active forms of SalArr1 demonstrated a 2- to 4-fold inc rease in P-Rh* binding (compared with wild-type protein) and an even more d ramatic (up to 25-fold) increase in binding to unphosphorylated Rh* and dar k P-Rh. Constitutively active SalArr1 mutants also showed a reduced specifi city for amphibian rhodopsin. The ability of Escherichia coli-expressed Sal Arr1, 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* wa s compared. SalArr1 and its mutant form bound to amphibian P-Rh* with high affinity (K-i = 179 and 74 nM, respectively), whereas the affinity of SalAr r2 for P-Rh* was substantially lower (K-i = 9.1 mu M) CONCLUSIONS. SalArr1 and SalArr2 are salamander rod and cone arrestins, res pectively. Crucial regulatory elements in SalArr1 are conserved and play fu nctional roles similar to those of their counterparts in bovine rod arresti n. Rod and cone arrestins are relatively specific for their respective rece ptors.