Abstract
The rat angiotensin II type 1a receptor (AT1a-R) gene is expressed in a cell-specific manner. We demonstrated that the negative regulatory element (NRE) between -489 and -331 is active in PC12 cells (Murasawa, S., Matsubara, H., Urakami, M., and Inada, M. (1993) J. Biol. Chem. 268, 26996-27003). Gel retardation assays confirmed that PC12 cells have a trans-acting factor bound to the NRE. By means of a DNase I footprint assay we identified the core of the NRE as an (A+T)-rich sequence (TAATCTTTTATTTTA) located at nucleotides -456 to -442. Oligonucleotides corresponding to the NRE core sequence bound to nuclear protein. Site-directed mutagenesis at nucleotides -451 to -448 eliminated the specific protein/DNA binding and restored expression of the AT1a-R in transient transfection assays (2.7-fold increase). The NRE did not negatively affect the thymidine kinase promoter. No homology was found with known NREs, suggesting that this is a novel NRE. Southwestern blotting revealed a 53-kDa, specific binding protein in PC12 cells and the rat brain, but not in the liver, spleen, adrenal gland, and kidney. These findings demonstrate that the NRE of the rat AT1a-R is an (A+T)-rich sequence located at nucleotides -456 to -442 and the 53-kDa protein is a specific binding protein, and suggest that this protein may be a trans-acting factor which determines the neuron-specific down-regulation of the AT1a-R gene.
Highlights
Angiotensin II has multiple physiological effects in the cardiovascular, endocrine, and nervous systems that are initiated by binding to specific receptors located on the plasma membrane [1]
The negative cis-regulatory element (NRE) was located within 159 nucleotides from Ϫ489 to Ϫ331 and the core sequence has not been mapped in detail
Gel Retardation Analysis of Negative Cis-regulatory Element—Our previous study demonstrated that three positive cis-regulatory elements and one strong NRE are present in the 980-bp 5Ј-flanking region of the rat Angiotensin II type 1a receptor (AT1a-R) gene [7]
Summary
Angiotensin II has multiple physiological effects in the cardiovascular, endocrine, and nervous systems that are initiated by binding to specific receptors located on the plasma membrane [1]. We characterized one negative and three positive cis-regulatory elements in the 5Ј-flanking region of the rat AT1a-R gene [7]. The negative cis-regulatory element (NRE) was located between Ϫ489 and Ϫ331 and inhibited the promoter activity of the 590-bp 5Ј-flanking region by a factor of 10. The trans-acting factor that binds to the element was present in PC12, not in vascular smooth muscle and glial cells This suggested that the trans-acting factor is a major determinant which regulates the expression of the rat AT1a-R gene in PC12 cells. Southwestern blotting revealed that a nuclear protein of about 53 kDa bound to the NRE in PC12 cells and the rat brain, but not in vascular smooth muscle cells, glial cells, kidney, spleen, adrenal gland, and liver
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