Cloning and expression of a bovine cDNA for the regulatory subunit (RIIbeta) of cAMP-dependent protein kinase and cloning and characterization of the mouse RIIbeta gene

Abstract

cDNAs for RII{dollar}\beta{dollar} were cloned from a bovine brain {dollar}\lambda{dollar}gt11 library. The composite cDNA coded for a protein of 418 amino acids which was 98% homologous to the rat and human RII{dollar}\beta{dollar} proteins. The binding sites in RII{dollar}\beta{dollar} for microtubule-associated protein 2 (MAP2) and 75,000-dalton calmodulin-binding protein (P75) were localized to the NH{dollar}\sb2{dollar}-terminal 50 amino acids by using truncated RII{dollar}\beta{dollar} fusion proteins expressed in Escherichia Coli. MAP2 prevented RII from binding to P75 suggesting that the binding sites for MAP2 and P75 are located near each other or that one site on RII is binding to both proteins.;Genomic DNA clones for RII{dollar}\beta{dollar} were isolated from a mouse library in {dollar}\lambda{dollar}EMBL-3. 1.4 kb of the 5{dollar}\sp\prime{dollar}-flanking region upstream of the ATG initiation codon, exon 1 and part of intron 1 were sequenced. The 5{dollar}\sp\prime{dollar}-flanking region lacked TATA and CAAT sites but contained GC rich boxes found in constitutively expressed housekeeping genes. It contained five Sp1 sites, one each, AP1 and AP2 consensus sequences and ten copies of binding motifs for the helix-turn-helix class of transcription factors. Expression of fusion genes containing the CAT structural gene and RII{dollar}\beta{dollar} DNA (bp-1426/-49) upstream of the ATG initiation codon showed a 20 fold higher level of expression in NB2a cells than in CHO cells. Basal promoter was localized between {dollar}-{dollar}291 to {dollar}-{dollar}121 relative to ATG start codon. Deletion analysis suggested the presence of an enhancer element and inhibitory sequences. Gel shift assays revealed that several regions of the 5{dollar}\sp\prime{dollar}-flanking DNA interacted with nuclear binding factors in sequence and tissue specific manners. The defined basal promoter bound at least two factors present in both NB2a and CHO cells, one that bound Sp1 sites, one that bound to HLH motif. Our results suggest that tissue specific factors present in certain cell types are responsible for the preferential expression of RII{dollar}\beta{dollar}.