Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor
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.
- Research Organization:
- Dartmouth Coll., Hanover, NH (USA)
- OSTI ID:
- 6414871
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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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