Murine glucocorticoid receptors: new evidence for a discrete receptor influenced by H-2

The glucocorticoid receptor contents in the lungs of females of two congenic strains of mice, B10.A (H-2a) and B10 (H-2b), differing only in the H-2 histocompatibility region of chromosome 17, have been measured by the dextran-charcoal method and by our previously described methods of molecular sieving and ion exchange chromatography [M. Katsumata, C. Gupta, and A. S. Goldman (1985) Arch. Biochem. Biophys. 243, 385-395]. As reported, two receptors, II and IB, are demonstrable by each column chromatographic method, and 5,5-diphenylhydantoin binds to receptor IB but not to receptor II. Receptor IB cannot be detected unless molybdate is added in cytosols prepared with hypotonic buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 10 mM dithiothreitol, pH 7.35) according to S. L. Liu, J. F. Grippo, R. P. Erickson, and W. B. Pratt (1984) J. Steroid Biochem. 21, 633-637], a method which has been reported to give maximal receptor levels. Using hypotonic buffer containing 10 mM molybdate we observed a small but significantly higher content of receptor IB in B10.A mice than that in B10 mice, but no significant difference in receptor II or total receptor content. On the other hand, cytosols prepared with isotonic buffer (50 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, and 10 mM molybdate, a modification of the buffer used in our previous report) contained significantly higher levels of receptor IB and of total binding in pulmonary cytosols of B10.A as compared to those of B10. There was no difference in receptor II content. Molybdate stabilizes receptor IB in both buffers. These results explain the apparent contradiction between our results and those of Liu et al. by showing that the hypotonic buffer used by them allows for determination of maximal levels of receptor II, but permits selective destruction of receptor IB. However, the use of isotonic buffer gives maximal values of both receptors II and IB. With isotonic buffer, it is demonstrated that only the level of receptor IB is influenced by H-2-linked genes.