IDENTIFICATION AND CHARACTERIZATION OF THE GC-RICH AND CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE (CAMP)-INDUCIBLE PROMOTER OF THE TYPE-II-BETA CAMP-DEPENDENT PROTEIN-KINASE REGULATORY SUBUNIT GENE

A rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II-beta regulatory subunit (RII-beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Band-shifting and DNase-I footprinting experiments using this region of the RII-beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII-beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII-beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.