UV cross-linking/immunoprecipitation assay: Glucocorticoid receptor-adrenergic receptor gene sequence interaction

The rat β₁-adrenergic receptor (β₁-AR) gene contains glucocorticoid response element (GRE) half-sites at positions -2767 and -945. In electrophoretic mobility shift assay (EMSA) experiments, neither β₁-AR GRE half-site recognized glucocorticoid receptors (GRs) obtained from baculovirus high-level expression systems or from mammalian cells. We have developed a sensitive UV cross-linking/immunoprecipitation assay, using a 524-bp fragment containing the prototypical GRE obtained from the rat tyrosine aminotransferase promoter sequence and using antibodies recognizing mammalian GR. Using this assay, we provide evidence that rat β₁-AR gene sequences recognize mammalian GRs expressed in mouse 3T3 cells and that the site of GR interaction does not appear to specifically contain the β₁-AR GRE half-sites. This represents one of the first reports demonstrating the utility of a UV cross-linking/immzmoprecipitation assay in the detection of mammalian GR interaction with β₁-AR sequences, is consistent with the lack of specific DNA-GR protein complexes observed in EMSA experiments using oligonuticleotide probes containing the β₁-AR GRE half-sites, and provides evidence that mammalian GR interaction occurs at complex rat β₁-AR gene sequences.