TROUT RED-BLOOD-CELL ARRESTIN (TRCARR), A NOVEL MEMBER OF THE ARRESTIN FAMILY - CLONING, IMMUNOPRECIPITATION AND EXPRESSION OF RECOMBINANT TRCARR

Arrestins are cytosolic proteins involved in the desensitization of G- protein-coupled receptors. We report the cloning of trout red blood ce ll arrestin which shows 76, 82 and 52% identity with bovine beta-arres tin1, beta-arrestin2 and retinal arrestin respectively. Antibodies wer e generated against the C-terminus of trout red blood cell arrestin. T hese antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by beta-adrenergic stimulation and is then de sensitized whereas the transmembrane signalling pathway is not. To inv estigate the subcellular distribution of arrestin on beta-adrenergic a ctivation and desensitization of the antiporter, precipitation experim ents were carried out on trout erythrocytes. A desensitization-depende nt shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the poss ibility that a small number of cytosolic arrestins might be involved i n the regulation of membrane proteins in trout erythrocyte. Recombinan t trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigat ing the target for arrestin in trout red blood cell, which still remai ns to be identified.