Regulation of DHP receptor expression by elements in the 5'-flanking sequence.
The alpha(1)-subunit of the cardiac/vascular Ca(2+) channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca(2+) entry. It contains the binding sites for multiple classes of drugs collectively known as Ca(2+) antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac alpha(1C)-subunit gene, we have cloned a 2.3-kb fragment containing the 5'-flanking sequences and identified the alpha(1C)-subunit gene transcription start site. The rat alpha(1C)-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of alpha(1C)-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5'-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5'-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca(2+) channel alpha(1C)-subunit gene. Electrophoretic mobility shift assays identified a region of the alpha(1C)-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.
Liu
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Program in Molecular and Cellular Cardiology, Harper Hospital, Wayne State University School of Medicine, and Department of Veterans Affairs Medical Center, Detroit, Michigan 48201, USA.
Fan
Q I
QI
El-Zaru
M R
MR
Vanderpool
K
K
Hines
R N
RN
Marsh
J D
JD
D020121:5' Untranslated Regions / Q000235:genetics*; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D020746:Calcium Channels, L-Type / Q000235:genetics*; D002478:Cells, Cultured; D004586:Electrophoresis / Q000379:methods; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D012739:Gonadal Steroid Hormones / Q000494:pharmacology; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000166:cytology; D009206:Myocardium / Q000737:chemistry / Q000166:cytology; D010656:Phenylephrine / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013739:Testosterone / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D014162:Transfection
D020121:5' Untranslated Regions; D000316:Adrenergic alpha-Agonists; D000318:Adrenergic beta-Agonists; D020746:Calcium Channels, L-Type; D012739:Gonadal Steroid Hormones; D012333:RNA, Messenger; D010656:Phenylephrine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013739:Testosterone; D008156:Luciferases; D007545:Isoproterenol