Phosphorylated sites within the functional domains of the @ 100-kDa steroid-binding subunit of glucocorticoid receptors

Journal Article · Tue May 16 04:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00436a055· OSTI ID:5434686

Smith, L I; Mendel, D B; Bodwell, J E; Munck, A ^[1]

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1. Dartmouth Medical School, Hanover, NH (USA)

The steroid-binding subunit of the glucocorticoid receptor is known to be a {approximately} 100-kDa phosphoprotein composed of an immunogenic, DNA-binding, and steroid-binding domain. To identify the domains that contain these phosphorylated sites, the authors have analyzed the phosphate content of selected proteolytic fragments of the {approximately} 100-kDa steroid-binding protein from nonactivated and activated receptors. The {approximately} 100-kDa steroid-binding protein from WEHI-7 cells grown in the presence of ({sup 32}P)orthophosphate was covalently labeled with ({sup 3}H)dexamethasone 21-mesylate, purified with the BuGR2 monoclonal antibody, digested with chymotrypsin or trypsin, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin digestion of this protein yields a {approximately} 45-kDa fragment containing both the steroid-binding and DNA-binding domains, which contained both {sup 32}P and {sup 3}H. The {sup 32}P/{sup 3}H ratio of each fragment provides a measure of phosphate content per steroid-binding site and indicated that each fragment has approximately 30% of the phosphate content of the intact protein. This is sufficient to account for one of the three receptor phosphoryl groups. To determine more directly the phosphate content of the DNA-binding domain of the {approximately} 100-kDa protein, they isolated a {approximately} 16-kDa tryptic fragment from cytosol of WEHI-7 cells grown in the presence of ({sup 32}P)orthophosphate and ({sup 35}S)cysteine. Comparison of the {sup 32}P/{sup 35}S ratio of this fragment to that of the intact protein indicated that it contains less than 0.2 mol of phosphoserine/mol of protein.

• OSTI ID: 5434686
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:10; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADRENAL HORMONES
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CORTICOSTEROIDS
CYSTEINE
DAYS LIVING RADIOISOTOPES
DEXAMETHASONE
EVEN-ODD NUCLEI
GLUCOCORTICOIDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPES
KETONES
LABELLING
LIGHT NUCLEI
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
PREGNANES
PROTEINS
PURIFICATION
RADIOISOTOPES
RECEPTORS
STEROIDS
SULFUR 35
SULFUR ISOTOPES
THIOLS
TRITIUM COMPOUNDS