Immunologic analysis of human breast cancer progesterone receptors. 2. Structure, phosphorylation, and processing

Journal Article · Tue Sep 22 04:00:00 EDT 1987 · Biochemistry; (United States)

OSTI ID:5599134

Wei, L L; Sheridan, P L; Krett, N L; Francis, M D; Toft, D O; Edwards, D P; Horwitz, K B

The authors have used a monoclonal antibody (MAb) directed against chick oviduct progesterone receptors (PR), that cross-reacts with human PR, to analyze PR structure and phosphorylation. This MAb, designated PR-6, interacts only with B receptors (M/sub r/ 120,000) of T47D human breast cancer cells; it has no affinity for A receptors (M/sub r/ 94,000) or for proteolytic fragments from either protein. The antibody immunoprecipitates native B receptors and was used to study the structure of native untransformed 8S and transformed 4S receptors, using sucrose density gradient analysis, photoaffinity labeling, and gel electrophoresis. The independence of A- and B-receptor complexes was confirmed by the fining that purified, transformed B receptors bind well to DNA-cellulose. Additional studies focused on the covalent modifications of receptors. The previously described shifts in apparent molecular weight of nuclear PR following R5020 treatment using in situ photoaffinity labeling. To show whether these shifts can be explained by receptor phosphorylation, untreated cells and hormone-treated cells were metabolically labeled with (/sup 32/P)orthophosphate, and the B receptors were isolated by immunoprecipitation with PR-6 and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis. In both treatment states, B receptors were labeled in vivo with /sup 32/P, thus demonstrating directly that human PR are phosphoproteins. Since B receptors were labeled in the absence of hormone and also after their in vivo transformation by hormone, they appear to be substrates for two phosphorylation reactions, one in the untransformed state and another after they are tightly bound to chromatin. The second phosphorylation may account for the mobility shift of the receptors on SDS gels. On the basis of these data a model of human PR structure and subcellular receptor dynamics is presented.

• Research Organization: Univ. of Colorado Health Sciences Center, Denver
• OSTI ID: 5599134
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:19; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMAL CELLS
ANIMALS
ANTIBODIES
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
GLANDS
HORMONES
IMMUNOLOGY
ISOTOPES
KETONES
LABELLED COMPOUNDS
LIGHT NUCLEI
MAMMALS
MAMMARY GLANDS
MAN
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
MONOCLONAL ANTIBODIES
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
PREGNANES
PRIMATES
PROGESTERONE
PROTEINS
RADIOISOTOPES
RECEPTORS
STEROID HORMONES
STEROIDS
TRITIUM COMPOUNDS
TUMOR CELLS
VERTEBRATES