UV cross-linking/immunoprecipitation assay: glucocorticoid receptor-adrenergic receptor gene sequence interaction.
The rat beta 1-adrenergic receptor (beta 1-AR) gene contains glucocorticoid response element (GRE) half-sites at positions -2767 and -945. In electrophoretic mobility shift assay (EMSA) experiments, neither beta 1-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 beta 1-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 beta 1-AR GRE half-sites. This represents one of the first reports demonstrating the utility of a UV cross-linking/immunoprecipitation assay in the detection of mammalian GR interaction with beta 1-AR sequences, is consistent with the lack of specific DNA-GR protein complexes observed in EMSA experiments using oligonucleotide probes containing the beta 1-AR GRE half-sites, and provides evidence that mammalian GR interaction occurs at complex rate beta 1-AR gene sequences.
Bai
Ying
Y
Oregon Health & Science University, Portland, OR, USA.
Kirigiti
Philbert
P
Li
Xiaorong
X
Li
Biao
B
Tian
Li
L
Ma
Mark Y J
MY
Machida
Curtis A
CA
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002522:Chlorocebus aethiops; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests / Q000379:methods*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011941:Receptors, Adrenergic / Q000737:chemistry / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D011941:Receptors, Adrenergic; D018342:Receptors, Adrenergic, beta-1; D011965:Receptors, Glucocorticoid