Glucocorticoid receptor expression in receptorless mutants isolated from the human leukemic cell line CEM-C7.
The molecular basis for the loss of steroid binding activity in receptorless (r-) glucocorticoid-resistant (dexr) mutants isolated from the glucocorticoid-sensitive (dexs) cell line CEM-C7 was investigated. Although there was little binding of the reversibly associating ligand [3H]dexamethasone in r- mutants, labeling with the covalent affinity ligand [3H] dexamethasone 21-mesylate revealed significant amounts of a 92 kilodalton human glucocorticoid receptor (hGR) protein. Immunoblots of hGR protein in r- and normal cells showed that r- mutants expressed approximately half the amount of immunoreactive hGR protein seen in dexs cells. Comparison of the genomic organization of the hGR genes in normal and mutant cells revealed no discernable differences in the structure, or dosage, indicating that the r- phenotype was not the result of gross deletion or rearrangement of the hGR genes. In addition, r- cells expressed the same 7 kilobase mRNA as normal cells. More importantly, the amount of hGR mRNA expressed in r- cells was never significantly less, and in some cases was greater than, that seen in normal cells, indicating that the decrease in immunoreactive hGR protein seen in r- cells is not the result of loss of hGR mRNA expression. Taken together with the known mutation rate of the hGR gene(s) in these cells, these results suggest that the hGR genes in dexs CEM-C7 cells are allelic and that dexs cells express both a normal hGR protein and one with an altered steroid binding site. Furthermore, they suggest that the r- phenotype is acquired as the result of mutation within the coding region of the originally functional allele, leading to loss of ligand binding and expression of immunoreactive product.
Harmon
J M
JM
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Elsasser
M S
MS
Eisen
L P
LP
Urda
L A
LA
Ashraf
J
J
Thompson
E B
EB
D015152:Blotting, Northern; D015139:Blotting, Southern; D005786:Gene Expression Regulation; D005838:Genotype; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D008024:Ligands; D009154:Mutation; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D014407:Tumor Cells, Cultured
D008024:Ligands; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid