TRANSCRIPTION OF THE RAT GLUCAGON GENE BY THE CYCLIC-AMP RESPONSE ELEMENT-BINDING PROTEIN CREB IS MODULATED BY ADJACENT CREB-ASSOCIATED PROTEINS

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-brom o-cAMP and protein kinase A (PKA) in a glucagon-producing hamster isle t cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer a nd that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CR EB and cAMP-dependent PKA-stimulated transcriptional activity in trans ient transfection experiments but does not affect the binding of adjac ent CR-EB-associated proteins. Progressive deletions and clustered poi nt mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kD a bind to each of the 5' and 3' TCATT sites. Formation of additional p rotein-DNA complexes containing 45- and 20-kDa proteins depends upon t he integrity of both TCATT sequences. Deletion or point mutation of th e TCATT motif located on the 3' side of the CRE octamer results in enh anced transcriptional responses to PKA, suggesting that the CREB-assoc iated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE b y serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.