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Title: Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor

Abstract

The primary goal of these studies was to localize the steroid-binding and phosphorylated sites of the glucocorticoid receptor. The synthetic steroid, dexamethasone 21-mesylate (DM) forms a covalent thioether bond via the sulfhydryl group of a cysteine residue in the receptor. To determine the covalent site of attachment of this ligand, receptors in WEHI-7 mouse thymoma cells were labeled with ({sup 3}H)DM and purified with a monoclonal antibody. The receptor was completely digested with trypsin and a single peptide covalently labeled with steroid identified by reversed-phase HPLC. This peptide was analyzed by automated Edman degradation to determine the location of the steroid-labeled residue. A similar analysis was performed on an overlapping peptide produced by Staphylococcus aureus protease digestion. Analysis of tryptic peptides from receptors labeled with both ({sup 3}H)DM and L-({sup 35}S)methionine indicated that this peptide contained methionine. These analyses, coupled with the published amino acid sequence of the receptor, identified Cysteine-644 in the steroid-binding domain of the mouse glucocorticoid receptor as the residue involved in covalent steroid-binding. A synthetic peptide representing amino acids 640-650 of the mouse receptor was prepared and analyzed to confirm the identification. These biochemical studies represent a direct demonstration of an amino acid important in receptormore » function. It has been proposed that the receptor functions through a phosphorylation-dephosphorylation cycle to explain the dependence of hormone binding capacity upon cellular ATP. The glucocorticoid receptor has been shown to be a phosphoprotein. As an initial step to identifying a role of phosphorylation in receptor action, phosphorylated sites within the functional domains of the protein were identified.« less

Authors:
Publication Date:
Research Org.:
Dartmouth Coll., Hanover, NH (USA)
OSTI Identifier:
6414871
Resource Type:
Miscellaneous
Resource Relation:
Other Information: Thesis (Ph. D.)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; GLUCOCORTICOIDS; RECEPTORS; STEROIDS; BIOCHEMICAL REACTION KINETICS; AMINO ACID SEQUENCE; CYSTEINE; FRACTIONATION; LIGANDS; LIQUID COLUMN CHROMATOGRAPHY; METHIONINE; MICE; PHOSPHORYLATION; STAPHYLOCOCCUS; SULFUR 35; TRACER TECHNIQUES; TRITIUM COMPOUNDS; TUMOR CELLS; ADRENAL HORMONES; AMINO ACIDS; ANIMAL CELLS; ANIMALS; BACTERIA; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CARBOXYLIC ACIDS; CHEMICAL REACTIONS; CHROMATOGRAPHY; CORTICOSTEROIDS; DAYS LIVING RADIOISOTOPES; DRUGS; EVEN-ODD NUCLEI; HYDROGEN COMPOUNDS; HYDROXY COMPOUNDS; ISOTOPE APPLICATIONS; ISOTOPES; KETONES; KINETICS; LIGHT NUCLEI; LIPOTROPIC FACTORS; MAMMALS; MEMBRANE PROTEINS; MICROORGANISMS; MOLECULAR STRUCTURE; NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PREGNANES; PROTEINS; RADIOISOTOPES; REACTION KINETICS; RODENTS; SEPARATION PROCESSES; SULFUR ISOTOPES; THIOLS; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Smith, L I. Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor. United States: N. p., 1989. Web.
Smith, L I. Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor. United States.
Smith, L I. Sun . "Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor". United States.
@article{osti_6414871,
title = {Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor},
author = {Smith, L I},
abstractNote = {The primary goal of these studies was to localize the steroid-binding and phosphorylated sites of the glucocorticoid receptor. The synthetic steroid, dexamethasone 21-mesylate (DM) forms a covalent thioether bond via the sulfhydryl group of a cysteine residue in the receptor. To determine the covalent site of attachment of this ligand, receptors in WEHI-7 mouse thymoma cells were labeled with ({sup 3}H)DM and purified with a monoclonal antibody. The receptor was completely digested with trypsin and a single peptide covalently labeled with steroid identified by reversed-phase HPLC. This peptide was analyzed by automated Edman degradation to determine the location of the steroid-labeled residue. A similar analysis was performed on an overlapping peptide produced by Staphylococcus aureus protease digestion. Analysis of tryptic peptides from receptors labeled with both ({sup 3}H)DM and L-({sup 35}S)methionine indicated that this peptide contained methionine. These analyses, coupled with the published amino acid sequence of the receptor, identified Cysteine-644 in the steroid-binding domain of the mouse glucocorticoid receptor as the residue involved in covalent steroid-binding. A synthetic peptide representing amino acids 640-650 of the mouse receptor was prepared and analyzed to confirm the identification. These biochemical studies represent a direct demonstration of an amino acid important in receptor function. It has been proposed that the receptor functions through a phosphorylation-dephosphorylation cycle to explain the dependence of hormone binding capacity upon cellular ATP. The glucocorticoid receptor has been shown to be a phosphoprotein. As an initial step to identifying a role of phosphorylation in receptor action, phosphorylated sites within the functional domains of the protein were identified.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1989},
month = {1}
}

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