Molecular analysis of human beta-arrestin-1: cloning, tissue distribution, and regulation of expression. Identification of two isoforms generated by alternative splicing.
The cDNA for human beta-arrestin-1 was cloned by polymerase chain reaction (PCR) and identified based on its remarkably high amino acid identity (98.6%) with the bovine sequence. Two alternatively spliced isoforms of human beta-arrestin-1, differing only in the presence or absence of 24 base pairs/8 amino acids within the sequence, were identified and called beta-arrestin-1A and beta-arrestin-1B, respectively. Both isoforms were found in all tissues tested. Southern blot analysis revealed the existence of a single gene for beta-arrestin-1, suggesting that the two isoforms are generated by alternative mRNA splicing. The possible presence of similar isoforms was investigated for the other members of the arrestin/beta-arrestin gene family by PCR. Two isoforms of arrestin were revealed in bovine peripheral blood leukocytes. The expression of beta-arrestin-1 was studied in several human tissues and cell types. High levels of beta-arrestin-1 mRNA and immunoreactivity were found in peripheral blood leukocytes. The possible regulation of the expression of beta-arrestin-1 was also investigated. Our work documents for the first time that the expression of beta-arrestin-1 is modulated by intracellular cAMP. Using two cell types, human endothelial cells and smooth muscle cells, we found that 6-8-h treatments with the cAMP-inducing agents cholera toxin, forskolin, iloprost, and isoproterenol raised beta-arrestin-1 mRNA by 2-4-fold. Forskolin preferentially increased beta-arrestin-1A expression in smooth muscle cells, as assessed by PCR. beta-Arrestin-1 immunoreactivity was 2-3-fold higher in smooth muscle cells exposed to forskolin for 8 h, compared with untreated controls. We conclude that (i) the finding of alternatively spliced isoforms of beta-arrestin-1 and arrestin documents a novel mechanism to generate diversity within the arrestin/beta-arrestin gene family; (ii) the abundant expression of beta-arrestin-1 in peripheral blood leukocytes further supports our previous suggestion of a major role for the beta ARK/beta-arrestin system in regulating receptor-mediated immune functions; (iii) the increased expression of beta-arrestin-1 by cAMP suggests a new mechanism for the regulation of receptor-mediated responses.
Parruti
G
G
Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche Mario Negri, Santa Maria Imbaro, Italy.
Peracchia
F
F
Sallese
M
M
Ambrosini
G
G
Masini
M
M
Rotilio
D
D
De Blasi
A
A
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D019390:Arrestins; D001483:Base Sequence; D015139:Blotting, Southern; D002417:Cattle; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D005136:Eye Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014471:Umbilical Veins; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; D000941:Antigens; D019390:Arrestins; D005136:Eye Proteins; D012333:RNA, Messenger; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D005576:Colforsin; D004247:DNA; D002772:Cholera Toxin