The role glucocorticoids (GCs) as lymphocytolytic agents is the primary basis for using these hormones in the clinical management of leukemias. Although lack of glucocorticoid receptor (GR) precludes any clinical response to GCs, a high level of intracellular GR (iGR) does not guarantee therapeutic response; therefore, the mechanism of the lytic response is not well understood and elucidation of the function of the mGR subpopulation is important. I previously demonstrated a correlation of lymphocytolysis with the presence of plasma membrane-resident GR (mGR) on immuno-separated GC-sensitive S-49 mouse T lymphoma cells. Using our monoclonal antibody to the rodent GR (BUGR) for Western analysis of purified membrane extracts, we demonstrated that the molecular size of some of these mGR forms (85,00-145,00 KD) were significantly larger than the iGR form (94,000 KD). Repeated immuno-selection of these cells for mGR produced highly enriched (mGR++ & GC lysis sensitive), or very depleted (mGR-- & GC lysis resistant) S-49 cells. We not propose to utilize mGR++ cells to [predict which protein domain of the mouse GR is responsible for hormone-mediated cell killing and what role this domain may have in directing the protein to the plasma membrane. We will purify and characterize mGR with respect to physicochemical, steroid- and DNA- binding, molecular size, susceptibility to proteolysis, post- translational modifications, and functional properties. Western blot analysis of translation products and purified preparations revealed several high M/r size mGR forms expressed in mGR++ cells only, and not in mGR-- cells. Screenings of a mGR++ cDNA library revealed: 1) one or two base substitutions; and 2) four alternatively spliced RNA transcripts; two of which were novel. One of these transcripts was expressed in mGR++ cells only, but not in mGR-less cells. Using a hybrid selection technique, will isolate the unique full-length GR mRNA and mGR++ cells, study the bases for M/r variations between mGR and iGR by in vitro translation experiments, clone full-length cDNAs, and then transfect these constructs into Xenopus oocytes and mGR-less cells to study subcellular localization of mGR and function in lymphocytolysis. Achieving these goals will help to define the mechanisms of GC-mediated lymphocytolysis, and ultimately allow us to use immunoanalysis to select patients likely to respond to therapeutic GCs.
| Powell, C E; Watson, C S; Gametchu, B (1999) Immunoaffinity isolation of native membrane glucocorticoid receptor from S-49++ lymphoma cells: biochemical characterization and interaction with Hsp 70 and Hsp 90. Endocrine 10:271-80 |
| Chen, F; Watson, C S; Gametchu, B (1999) Association of the glucocorticoid receptor alternatively-spliced transcript 1A with the presence of the high molecular weight membrane glucocorticoid receptor in mouse lymphoma cells. J Cell Biochem 74:430-46 |
| Chen, F; Watson, C S; Gametchu, B (1999) Multiple glucocorticoid receptor transcripts in membrane glucocorticoid receptor-enriched S-49 mouse lymphoma cells. J Cell Biochem 74:418-29 |
