Differential binding of mutant glucocorticoid receptors to the glucocorticoid response element of the tyrosine aminotransferase gene.
Glucocorticoid receptors (GCRs) in sublines of the mouse P1798 lymphosarcoma that are sensitive (S) or resistant (R) to glucocorticoid-induced cell lysis were examined for their ability to bind to a single glucocorticoid responsive element (GRE). Mobility shift assays detected two specific complexes that were identical in both S and R cellular extracts. Antibodies against the GCR N-terminus supershifted complexes, suggesting that the 97 kDa wild-type GCR (WT-GCR) in S cells, and the variant, 97 kDa non-steroid-binding GCR (NSB-GCR) in R cells were components of both complexes. Sephacryl S300 gel filtration column fractions containing the WT-GCR and NSB-GCR formed complexes with the GRE, while fractions containing a second GCR variant in R cells, the 45 kDa steroid-binding truncated GCR (TR-GCR), did not. Southwestern blotting detected a GRE-binding, 97 kDa protein band in both S and R extracts. A 45 kDa band was not detected. UV crosslinking of protein to DNA revealed protein in the range of 92-120 kDa crosslinked to the GRE in both S and R extracts. No crosslinking was detected at 45 kDa. Strong interaction of the NSB-GCR with GREs and lack of binding of the TR-GCR to single GREs illustrate a complex receptor system in the P1798 lymphosarcoma.
Rowan
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BG
Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
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D000180:Acrylic Resins; D000818:Animals; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D004586:Electrophoresis / Q000379:methods; D005938:Glucocorticoids / Q000494:pharmacology; D008228:Lymphoma, Non-Hodgkin / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014444:Tyrosine Transaminase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014466:Ultraviolet Rays
D000180:Acrylic Resins; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D014444:Tyrosine Transaminase