Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

F. C. Dalman, E. R. Sanchez, A. L Y Lin, F. Perini, W. B. Pratt

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGr anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E. Thompson, E.B., Gametchu, B., Harrison, R.W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding. DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGr epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.

Original languageEnglish (US)
Pages (from-to)12259-12267
Number of pages9
JournalJournal of Biological Chemistry
Volume263
Issue number25
StatePublished - 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Dalman, F. C., Sanchez, E. R., Lin, A. L. Y., Perini, F., & Pratt, W. B. (1988). Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor. Journal of Biological Chemistry, 263(25), 12259-12267.