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Mechanisms of transcriptional regulation of the human beta(3)-adrenergic receptor were studied using SK-N-MC cells, a human neuroblastoma cell line that expresses beta(3)- and beta(1)-adrenergic receptors endogenously. Deletions spanning different portions of a 7-kb 5'-flanking region of the human beta(3)-adrenergic receptor gene were linked to a luciferase reporter and transfected in SK-N-MC, CV-1, and HeLa cells. Maximal luciferase activity was observed when a 200-bp region located between -6.5 and -6.3 kb from the translation start site was present. This region functioned only in SK-N-MC cells. Electrophoretic mobility shift assays of nuclear extracts from SK-N-MC, CV-1, and HeLa cells using double stranded oligonucleotides spanning different portions of the 200-bp region as probes and transient transfection studies revealed the existence of three cis-acting regulatory elements: A) -6.468 kb-AGGTGGACT--6.458 kb, B) -6.448 kb-GCCTCTCTGGGGAGCAGCTTCTCC-6.428 kb, and C) -6.405 kb-20 repeats of CCTT-6.385 kb. These elements act together to achieve full transcriptional activity. Mutational analysis, antibody supershift, and electrophoretic mobility shift assay competition experiments indicated that element A binds the transcription factor Sp1, element B binds protein(s) present only in nuclear extracts from SK-N-MC cells and brown adipose tissue, and element C binds protein(s) present in both SK-N-MC and HeLa cells. In addition, element C exhibits characteristics of an S1 nuclease-hypersensitive site. These data indicate that cell-specific positive cis-regulatory elements located 6.5 kb upstream from the translation start site may play an important role in transcriptional regulation of the human beta(3)-adrenergic receptor. These data also suggest that brown adipose tissue-specific transcription factor(s) may be involved in the tissue-specific expression of the beta(3)-adrenergic receptor gene.

Expression of the Human β3-Adrenergic Receptor Gene in SK-N-MC Cells Is Under the Control of a Distal Enhancer