DNA ELEMENTS AND PROTEIN FACTORS INVOLVED IN THE TRANSCRIPTION OF THEBETA(2)-ADRENERGIC RECEPTOR GENE IN RAT-LIVER - THE NEGATIVE REGULATORY ROLE OF C EBP-ALPHA/

Primer extension and RNase protection analyses of the rat beta(2)-adre nergic receptor (beta(2)AR) gene identify two transcription start poin ts at -64 and -220 nt, respectively. Transient transfections of putati ve promoter/pCAT constructs into DDT1 MF-2 cells indicate that fragmen ts -36 to -100 (PI) and -186 to -312 (P2) are sufficient to promote tr anscription, whereas -911 to -1122 contains a negative regulatory elem ent(s). RNase protection analysis of the 3' untranslated region (3'-UT R) indicates the presence of two transcripts with 3'-UTR of 111 and 60 4 nt exclusive of the poly(A(+)) tails. Northern blots of beta(2)AR mR NA using full-length and partial cDNA probes indicate that a major 2.2 kb and a minor 1.6 kb species arise from the use of alternative promo ters as well as different polyadenylation signals. DNase I footprintin g and DNA mobility shift assays (DMSA) using rat liver nuclear extract s identify a number of transcription factors binding to sequence eleme nts within or upstream from P1 and P2, including Spl, CRE, CPI, AP-2, NF-I, NF-kappa B, and C/EBP. Supershift assays using antibodies agains t C/EBP alpha and C/EBP beta and mutational analyses indicate that the protein binding to the C/EBP consensus recognition site at -925 to -9 33 is C/EBP alpha. The activity of promoter/CAT constructs containing the C/EBP recognition site is significantly decreased by cotransfectio n of C/EBP alpha but not C/EBP beta into either DDT1 MF-2 cells or pri mary rat hepatocytes. Partial hepatectomy causes a transient decrease in C/EBP alpha, as measured by DMSA, and an increase in beta(2)AR mRNA levels and rate of transcription in the remnant liver. Thus, derepres sion via C/EBP alpha is likely involved in the up-regulation of beta(2 )AR in the regenerating rat liver.