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

Journal Article · Tue May 16 00:00:00 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), Vol. 28:10; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

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