The lymphoid cell killing effect of glucocorticoids is the fundamental basis for using these hormones in the treatment of leukemias and lymphomas. An imperfect correlation exists between glucococoid receptor (GR) concentration and clinical response. Although absence of GR precludes any response, many patients with high numbers of intracellular receptor (iGR) fail to respond to therapy. While the mechanism of this lytic response is not understood, my recent work has demonstrated the presence of a membrane-bound GR (mGR) on the surface of glucocorticoid- sensitive S-49 mouse T-lymphoma cells. I used immunoadsorption to obtain S-49 cell populations enriched (100%) and depleted (38%) for this mGR. The mGR+ cells showed complete glucocorticoid induced cytolysis, while the mGR-deficient population was only partially (58%) sensitive, suggesting a role for mGR in the cytolytic process. Gel electrophoresis, specific immunoblot, and autoradiographic analysis revealed multiple large size proteins on the plasma membrane of mGR+ cells. I now propose to use a combination of steroid, DNA-cellulose, and immunoaffinity chromatographies with HPLC to purify the mGR to homogeneity. I will then study its physicochemical properties, functional domains, and structural and functional relationship to the classical intracellular iGR. Experiments designed to explain the larger size of mGR over iGR will include study of possible post- translational modifications and comparison of immunoreactive in vitro translation products from mGR+ and mGR- S-049 cells. If these initial studies suggest a different primary structure for the mGR, its primary sequence will be established by amino acid and cDNA sequence analysis, and the origin of the different mRNA will be investigated. To study the cytolytic role of mGR, I will fuse lysis-deficient mGR- cells with mGR-containing vesicles or cell ghosts, followed by tests for glucocorticoid sensitivity. Finally, I hope to develop a clinically useful immunocytochemical method for rapid quantitation of mGR so as to identify lymphoma patients likely to benefit from steroid treatment.
| Gametchu, B; Watson, C S; Shih, C C et al. (1991) Studies on the arrangement of glucocorticoid receptors in the plasma membrane of S-49 lymphoma cells. Steroids 56:411-9 |
| Gametchu, B; Watson, C S; Pasko, D (1991) Size and steroid-binding characterization of membrane-associated glucocorticoid receptor in S-49 lymphoma cells. Steroids 56:402-10 |
