Interaction of nuclear factors with the cAMP response elements of the human β(3)-adrenoceptor gene.

Four potential cyclic adenosine 3',5'-monophosphate (cAMP) response elements (CREs), each having at most two mismatches from the classical canonical sequence, have been identified in the 5'UTR of the human β(3)-adrenoceptor gene by Liggett and Schwinn (1991). Recently, three of these CREs were shown to confer responsiveness to cAMP when cloned into a CAT reporter vector (Thomas et al., 1992). In this study, in vitro gel-retardation assays have shown that recombinant human CRE binding protein-1 (CREB-1) or activating transcription factor-1 (ATF-1) can interact specifically with these four putative CREs (termed β(3)CRE2), although with different affinities. Nuclear extracts from human brown or white adipose tissue contain proteins interacting with β(3)CRE3 and β(3)CRE2. These adipose nuclear factors were shown by competition assays and the use of antibodies to differ from CREB-1 or ATF-1. The nuclear factor(s) interacting with β(3)CRE2 was found in human and rat brown and white adipose tissues, but not in the other nonadipose tissues examined, i.e., rat lung, liver, kidney, and heart, suggesting an adipose tissue-specific DNA binding or expression pattern. β(3)CRE2 is found to constitute a hexameric element that is highly homologous to the binding site for members of the nuclear hormone receptor superfamily, and a competition assay using this site has provided evidence that an adipose tissuespecific orphan member of this superfamily may bind to β(3)CRE2. Reporter gene assays have indicated that β(3)CRE2 and β(3)CRE3 slightly repress the basal level of transcription and that β(3)CRE2 confers cAMP responsiveness, whereas β(3)CRE3 does not.