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 cell arrestin which shows 76, 82 and 52% identity with bovine beta-arrestin1, beta-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These 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 desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on beta-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant 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 investigating the target for arrestin in trout red blood cell, which still remains to be identified.
Jahns
R
R
Département de Biologie Cellulaire et Moléculaire du CEA, Villefranche-sur Mer, France.
Borgese
F
F
Lindenthal
S
S
Straub
A
A
Motais
R
R
Fiévet
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019393:Arrestin; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D014337:Trout / Q000097:blood
D000941:Antigens; D019393:Arrestin; D005136:Eye Proteins; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D012243:Rhodopsin; D007545:Isoproterenol