Purification of T47D human progesterone receptor and immunochemical characterization with monoclonal antibodies.
In order to obtain steroid-independent probes for human progesterone receptor (PR), the A [88-93 kilodalton (kDa)] and B (109-119 kDa) forms of PR from T47D human breast cancer cells were partially purified and used to generate a series of 14 monoclonal antibodies. Initially, unoccupied PR was isolated from cytosol extracts by steroid affinity chromatography, followed by chromatography on diethylaminoethyl Bio-Gel. The partially pure (3-15%) PR consisted of two steroid-binding components that migrated at 89 kDa and 109 kDa in reducing sodium dodecyl sulfate gels after being photoaffinity labeled with the synthetic progestin [3H]R5020. Two unique monoclonal antibodies to PR were derived from a male Lewis rat immunized with this material. One of these antibodies (JU601) was coupled to Sepharose 4B and used to purify T47D nuclear PR for additional immunizations. Highly purified (30-70%) PR migrated as 93 kDa and 119 kDa progestin-binding proteins in sodium dodecyl sulfate gels. In all, thirteen monoclonal antibodies were obtained that recognized epitopes shared by both receptor forms. One mouse immunoglobulin G (KC146) was completely specific for the larger B form. Interestingly, the epitope for this antibody was present on all PRs tested, including the B form of PR from chicken oviduct, whereas nine other antibodies recognized only human PR and the remaining four cross reacted with rabbit PR. With the exception of the JU145 and JU601 rat immunoglobulin Ms, all antibodies appeared to be completely specific for the A or B forms of PR. Each recognized the cytosol and nuclear forms of occupied as well as unoccupied PR. Although the relationship between B and A was not established, it is clear that an amino-terminal region of B is not present in A, and that a significant portion of A and B are either identical or very similar in amino acid sequence.
Greene
G L
GL
Ben May Institute, University of Chicago, Illinois 60637.
Harris
K
K
Bova
R
R
Kinders
R
R
Moore
B
B
Nolan
C
C
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D005260:Female; D006801:Humans; D011980:Receptors, Progesterone / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D011980:Receptors, Progesterone