Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies.

Abstract

Monoclonal antibodies raised against the rat liver glucocorticoid receptor were used to investigate receptors of wild‐type and glucocorticoid‐resistant variants of mouse lymphoma cells. Two of the variant types contained receptors of ‘nuclear transfer deficient’ (nt‐) and ‘increased nuclear transfer’ (nti) phenotypes, respectively, while the third was of the ‘receptorless’ (r‐) phenotype with negligible hormone binding activity. Three monoclonal antibodies of the IgM class and one of the IgG class reacted with both wild‐type and nt‐ receptors but not with the steroid binding form of nti receptors. Some of the antibodies bound the wild‐type and nt‐ receptors more efficiently after activation at 20 degrees C. By use of an immuno‐competition assay we were able to detect cross‐reacting material in considerable amounts in extracts of nti and r‐ cell variants. This material was further characterized by gel filtration and immunoblotting. The immunoreactive material of wild‐type, nti and r‐ cells gave a major band of mol. wt. 94 000 upon SDS‐gel electrophoresis while the steroid‐binding polypeptides of wild‐type and nti receptors have mol. wts. of 94 000 and 40 000, respectively. The data show that in S49.1 mouse lymphoma cells the products of two receptor alleles can be distinguished.

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

“…These cells produced no detectable signal in the cytoplasmic dot hybridization assay or the panning assay (21); the fluorescence signal for the uninfected cells was less than that for the W7MG1 cells grown without hormone (21) and was used to establish the background level of nonspecific fluorescence. W7M326.4, a previously characterized glucocorticoid-resistant cell line (derived from W7MG1) that lacks functional glucocorticoid receptors (nt-), was previously established as a negative control for hormone responsiveness in each of the assays described above (21,22 (29) and an alkaline phosphatase immunostain (Promega Biotec).…”

Tetrameric structure of the nonactivated glucocorticoid receptor in cell extracts and intact cells

“…Since, however, other cellular material is non-specifically labeled with this reagent [19], a receptor specific purification procedure was necessary. For the wild-type receptor we used immunoaffinity chromatography with the monoclonal antibody mab 49 which recognizes a domain of the steroid-binding polypeptide that does not participate in cross-linking [6] and is missing from the nt' mutant receptor [20]. Fig.1 shows a series of SDS gels in which wild-type receptors were cross-linked with DMS for various lengths of time.…”

Glucocorticoid receptor alpha, beta and gamma expression vs in vitro glucocorticod resistance in childhood leukemia

“…8 The GRa is a 97 Kd a protein that is organized into structurally and functionally defined domains: the highly variable region that includes a transactivation domain that is required for the regulation of gene expression, the DNA-binding domain crucial for the specific interaction between the receptor and the GC-responsive elements and the carboxyl-terminus involved in hormone binding, interaction with heat-shock proteins (hsp's), nuclear translocation, receptor dimerization and transactivation. [9][10][11][12][13][14][15][16][17] In the absence of hormone, the GRa predominantly resides in the cytoplasm as a multiprotein complex consisting of the GR itself, two molecules of hsp90 and several other proteins. [18][19][20] Upon binding of a GC, the receptor complex dissociates and translocates to the nucleus.…”