TRANSCRIPTIONAL REGULATION OF THE HUMAN ALPHA(1A)-ADRENERGIC RECEPTORGENE - CHARACTERIZATION OF THE 5'-REGULATORY AND PROMOTER REGION

We recently cloned cDNAs encoding three subtypes of human alpha(1)-adr energic 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., Parr y-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., Berk owitz, D., and Schwinn, D. A. (1994) Mol. Pharmacol. 45, 171-175). Sev eral lines of evidence indicate that alpha(1a)ARs are important in cli nical diseases such as myocardial hypertrophy and benign prostatic hyp erplasia. Therefore, we initiated studies to understand mechanisms und erlying regulation of alpha(1a)AR gene transcription, A genomic clone containing 6.2 kb of 5'-untranslated region of the human alpha(1a)AR g ene was recently isolated. Ribonuclease protection and primer extensio n assays indicate that alpha(1a)AR gene transcription occurs at multip le initiation sites with the major site located 696 base pairs upstrea m of the ATG, where a classic initiator sequence is located. Transfect ion of luciferase reporter constructs containing varying amounts of 5' -untranslated region into human SK-N-MC neuroblastoma cells indicate t hat a region extending 125 base pairs upstream from the main transcrip tion initiation site contains full alpha(1a)AR promoter activity. Furt hermore, distinct activator and suppressor elements lie 2-3 and 3-5 ki lobase pairs upstream, respectively. Although the alpha(1a)AR promoter contains neither TATA or CAAT elements, gel shift mobility assays tar geting three GC boxes immediately upstream of the main transcription i nitiation site confirm binding of Spl. Activity of the alpha(1a)AR pro moter is cell-specific, demonstrating highest activity in cells endoge nously expressing alpha(1a)ARs. The human alpha(1a)AR gene also contai ns 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 co nclusion, these data represent the first characterization of the alpha (1a)AR gene; our findings should facilitate further studies designed t o understand mechanisms regulating alpha(1)AR subtype-specific express ion in healthy and diseased human tissue.