MOLECULAR ANALYSIS OF HUMAN BETA-ARRESTIN-1 - CLONING, TISSUE DISTRIBUTION, AND REGULATION OF EXPRESSION - IDENTIFICATION OF 2 ISOFORMS GENERATED BY ALTERNATIVE SPLICING

The cDNA for human beta-arrestin-1 was cloned by polymerase chain reac tion (PCR) and identified based on its remarkably high amino acid iden tity (98.6%) with the bovine sequence. Two alternatively spliced isofo rms of human beta-arrestin-1, differing only in the presence or absenc e of 24 base pairs/8 amino acids within the sequence, were identified and called beta-arrestin-1A and beta-arrestin-1B, respectively. Both i soforms were found in all tissues tested. Southern blot analysis revea led the existence of a single gene for beta-arrestin-1, suggesting tha t the two isoforms are generated by alternative mRNA splicing. The pos sible presence of similar isoforms was investigated for the other memb ers of the arrestin/beta-arrestin gene family by PCR. Two isoforms of arrestin were revealed in bovine peripheral blood leukocytes. The expr ession of beta-arrestin-1 was studied in several human tissues and cel l 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 document s for the first time that the expression of beta-arrestin-1 is modulat ed by intracellular cAMP. Using two cell types, human endothelial cell s and smooth muscle cells, we found that 6-8-h treatments with the cAM P-inducing agents cholera toxin, forskolin, iloprost, and isoprotereno l raised beta-arrestin-1 mRNA by 2-4-fold. Forskolin preferentially in creased beta-arrestin-1A expression in smooth muscle cells, as assesse d by PCR. Beta-arrestin-1 immunoreactivity was 2-3-fold higher in smoo th muscle cells exposed to forskolin for 8 h, compared with untreated controls. We conclude that (i) the finding of alternatively spliced is oforms 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 leukoc ytes further supports our previous suggestion of a major role for the betaARK/beta-arrestin system in regulating receptor-mediated immune fu nctions; (iii) the increased expression of beta-arrestin-1 by cAMP sug gests a new mechanism for the regulation of receptor-mediated response s.