Determination of conformational changes in the progesterone receptor using ELISA-like assays.
The conformation of proteins often influences their functional activity. The effect of progesterone receptor ligands on the C-terminal conformation of the progesterone receptor affects the recruitment of transcriptional cofactors. These conformations can be studied by differential sensitivity to proteolytic cleavage or immunoprecipitation with a conformation-specific antibody. This study describes an ELISA-like method using conformation-specific antibodies to the C-terminal or an area adjacent to the DNA binding region. Progesterone receptor ligands are shown to influence how the progesterone receptor interacts with these antibodies in a concentration dependent manner. This method allows for quick determination of the potency of agonists as well as mechanistic studies of antagonism. The conformation inducing activity of several standard agonist and antagonist compounds were compared to their binding affinity and ability to induce alkaline phosphatase in T47D cells. This method is useful for screening compounds for functional activity at the progesterone receptor and demonstrates that J867 induces an antagonist conformation of the progesterone receptor similar to the antagonist RU486.
Pullen
Mark A
MA
Department of Urogenital Biology, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, Mail Code UW2521, King of Prussia, PA 19406, USA. Mark.A.Pullen@gsk.com
Laping
Nicholas
N
Edwards
Richard
R
Bray
Jeffrey
J
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D015735:Mifepristone / Q000494:pharmacology; D010091:Oximes / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D011980:Receptors, Progesterone / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014599:Uterus
D004963:Estrenes; D010091:Oximes; D011980:Receptors, Progesterone; D015735:Mifepristone; C488516:asoprisnil