Monoclonal and polyclonal antibodies to human progesterone receptor peptide-(533-547) recognize a specific site in unactivated (8S) and activated (4S) progesterone receptor and distinguish between intact and proteolyzed receptors.
We have synthesized three peptides with amino acid sequences corresponding to amino acids 533-547, 597-611, and 765-779 of the human progesterone receptor (hPR). These peptides were conjugated to keyhole limpet hemocyanin and injected into mice and rabbits to develop antibodies to hPR. Antibodies to the undenatured form of PR were elicited only by the peptide with amino acid sequence 533-547. Fusion of SP2/0 myeloma cells with spleen cells from mice immunized with this peptide produced several active clones. Rabbit sera from immunized animals produced one antiserum that reacted with the undenatured form of PR. One monoclonal antibody (PR-AT 4.14) and one antiserum (PR-AT533) raised against peptide-(533-547) were characterized. Binding of these antibodies to the undenatured form of PR was demonstrated by analysis of the antibody-receptor complexes on sucrose density gradients and by immunoprecipitation techniques. Binding of PR to the antibodies was inhibited by excess peptide. The antibodies did not react with estrogen, glucocorticoid, or androgen receptors, but recognized PR from human breast cancer as well as calf, rabbit, mouse, and rat uteri, indicating that this epitope was conserved among these species. Based on sucrose density gradient analysis of PR prepared and labeled in the presence of proteolysis inhibitors and sodium molybdate, the antibodies bound to a site on the intact undenatured PR, but failed to bind to partially degraded steroid-binding form of the receptor, suggesting that the antibody-binding domain is at or near a site sensitive to proteolysis.
Traish
A M
AM
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
Wotiz
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001666:Binding Sites, Antibody; D001943:Breast Neoplasms / Q000032:analysis; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000032:analysis; D005260:Female; D006433:Hemocyanins / Q000276:immunology; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011980:Receptors, Progesterone / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D013045:Species Specificity
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000941:Antigens; D010446:Peptide Fragments; D011980:Receptors, Progesterone; D006433:Hemocyanins; D010447:Peptide Hydrolases; C032808:keyhole-limpet hemocyanin