Transcriptional regulation of the human alpha(1a)-adrenergic receptor gene - Characterization of the 5'-regulatory and promoter region

We recently cloned cDNAs encoding three subtypes of human alpha(1)-adrenergic receptors (alpha(1)ARs), alpha(1a), alpha(1b), and alpha(1d) (Schwinn, D. A., Johnston, G. L., Page, S. O., Mosley, M. J., Wilson, K. H., Worman, N. P., Campbell, S., Fidock, M. D., Furness, L. M., Parry-Smith, D. J., Peter, B., and Bailey, D. S. (1995) J. Pharmacol. Exp. Ther. 272, 134-142) and demonstrated predominance of alpha(1a)ARs in many human tissues (Price, D. T., Lefkowitz, R. J., Caron, M. G., Berkowitz, D., and Schwinn, D. A. (1994) Mol. Pharmacol. 45, 171-175). Several lines of evidence indicate that alpha(1a)ARs are important in clinical diseases such as myocardial hypertrophy and benign prostatic hyperplasia. Therefore, we initiated studies to understand mechanisms underlying regulation of alpha(1a)AR gene transcription, A genomic clone containing 6.2 kb of 5'-untranslated region of the human alpha(1a)AR gene was recently isolated. Ribonuclease protection and primer extension assays indicate that alpha(1a)AR gene transcription occurs at multiple initiation sites with the major site located 696 base pairs upstream of the ATG, where a classic initiator sequence is located. Transfection of luciferase reporter constructs containing varying amounts of 5'-untranslated region into human SK-N-MC neuroblastoma cells indicate that a region extending 125 base pairs upstream from the main transcription initiation site contains full alpha(1a)AR promoter activity. Furthermore, distinct activator and suppressor elements lie 2-3 and 3-5 kilobase pairs upstream, respectively. Although the alpha(1a)AR promoter contains neither TATA or CAAT elements, gel shift mobility assays targeting three GC boxes immediately upstream of the main transcription initiation site confirm binding of Spl. Activity of the alpha(1a)AR promoter is cell-specific, demonstrating highest activity in cells endogenously expressing alpha(1a)ARs. The human alpha(1a)AR gene also contains several cis regulatory elements, including several insulin and cAMP response elements. Consistent with these observations, we provide the first evidence that treatment of SK-N-MC cells with insulin and cAMP elevating agents leads to an increase in alpha(1a)AR expression. In conclusion, these data represent the first characterization of the alpha(1a)AR gene; our findings should facilitate further studies designed to understand mechanisms regulating alpha(1)AR subtype-specific expression in healthy and diseased human tissue.