Analysis of the alpha2C-adrenergic receptor gene promoter and its cell- type-specific activity

JS Saulnier-Blache, Q Yang, JD Sherlock and SM Lanier

Department of Pharmacology, Medical University of South Carolina, Charleston 29425, USA.

As an initial approach to define the regulatory elements and transcriptional factors that account for cell-restricted expression of the alpha2c-adrenergic receptor (AR) gene, we isolated and characterized the receptor gene and identified regions of the gene conferring cell-specific expression. A 4300-nucleotide (nt) fragment of the 5'-flanking region of the rat alpha2c-AR gene was isolated from a genomic library. The genomic sequence contained the uninterrupted sequence of the 5'-untranslated region of a previously isolated alpha2c- AR cDNA clone indicating the lack of introns in the 5' gene segment. RNase protection assays and/or RNA blot analysis indicated the expression of alpha2c-AR mRNA in rat brain but not in kidney or liver, which is consistent with the major expression of this gene in neuronal tissue. The 5' gene segment was used to identify sites of transcriptional initiation and promoter activity by RNase protection assays and transient transfection of reporter gene constructs. With the use of RNA probes progressively upstream of the translational start site, RNase protection assays with rat brain total RNA indicated multiple sites of transcriptional initiation within a approximately 70- nt span (-660 to -730 nt 5' to the translational start codon). The zone of transcriptional initiation was part of a larger GC-rich area of the 5' gene segment that is a characteristic of genes initiating transcripts at multiple sites. The promoter activity of this zone of transcriptional initiation and the influence of gene segments 5' to this area were addressed using chloramphenicol acetyl transferase reporter gene constructs. Transient transfection of reporter gene constructs indicated that a 96-nt gene fragment (-699/-603 relative to the translational start codon) was sufficient to direct transcription in the neuroblastoma X glioma hybrid cell line NG108-15, a cell line expressing the endogenous alpha2c-AR. Promoter activity was not observed in constructs lacking the zone of transcriptional initiation. The promoter segment was inactive when introduced into the rat glioma cell line C6B4, the rat submandibular cell line RSMT-A5, and the rat pancreatic beta cell line RIN-5AH, all of which do not express the endogenous alpha2c-AR gene. Upon incubation with nuclear extracts, a 129-nt fragment encompassing the promoter exhibited a gel mobility shift pattern that was specific for cells expressing the receptor protein and involved a nuclear protein that recognized a Sp1 oligonucleotide. These data indicate that a 96-nt gene promoter segment of the alpha2c-AR gene functions in a cell-type-specific manner.

Volume 50, Issue 6, pp. 1432-1442, 12/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				T. D. O'Connell, D. G. Rokosh, and P. C. Simpson Cloning and Characterization of the Mouse alpha 1C/A-Adrenergic Receptor Gene and Analysis of an alpha 1C Promoter in Cardiac Myocytes: Role of an MCAT Element That Binds Transcriptional Enhancer Factor-1 (TEF-1) Mol. Pharmacol., April 16, 2001; 59(5): 1225 - 1234. [Abstract] [Full Text]

				Q. Yang, P. J. McDermott, E. Duzic, C. W. A. Pleij, J. D. Sherlock, and S. M. Lanier The 3'-Untranslated Region of the alpha 2C-Adrenergic Receptor mRNA Impedes Translation of the Receptor Message J. Biol. Chem., June 13, 1997; 272(24): 15466 - 15473. [Abstract] [Full Text] [PDF]

				N. Pizzinat, A. Takesono, and S. M. Lanier Identification of a Truncated Form of the G-protein Regulator AGS3 in Heart That Lacks the Tetratricopeptide Repeat Domains J. Biol. Chem., May 11, 2001; 276(20): 16601 - 16610. [Abstract] [Full Text] [PDF]