Unusual Properties
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History of the GeneSwitch System

Unusual Properties of Truncated Progesterone Receptors

The GeneSwitch regulator protein is based on the unusual properties of a mutant progesterone receptor that was identified by a yeast (Saccharomyces cerevisiae) screening system (Vegeto et al., 1992). A library of mutations in the ligand binding domain of the human progesterone receptor (form B) was generated by error-prone polymerase chain reaction (PCR) using low fidelity conditions. The resulting library was transformed into a yeast strain that contained a progesterone-dependent reporter gene (b-Galactosidase linked to a promoter with a progesterone response element (PRE)). One colony, called UP-1, displayed an unusual phenotype: b-Gal expression was induced in response to the progesterone antagonist, MFP, but not in response to the natural agonist, progesterone. The level of b-Gal induction achieved by the UP-1 mutant in response to the antiprogestin was of the same order of magnitude of that achieved by the wild type receptor in response to progesterone. Thus, the mutant receptor exhibited a nearly complete reversal of specificity. Ligand binding analysis revealed that the wild type and UP-1 mutant receptors had similar affinities (3-4 nM) for MFP. However, the UP-1 mutant receptor had lost the ability to bind progesterone (specific binding was reduced by at least 50-fold). DNA sequence analysis revealed that the UP-1 phenotype was due to a single nucleotide deletion that introduced a frame shift, which caused 54 amino acids of the authentic progesterone receptor to be truncated and 12 novel amino acids to be added to the C-terminus. Thus, the C-terminus of the progesterone receptor ligand binding domain plays a critical role in determining how the receptor responds to different ligands.


The wild type human progesterone receptor (hPR) is 933 amino acids in length. To further define the sequences responsible for the unusual phenotype, stop codons were introduced into the wild type progesterone receptor, resulting in C-terminal truncations of 54 amino acids (hPR-B879) or 42 amino acids (hPR-B891). Both of these truncated receptors had the same phenotype as the UP-1 mutant receptor – they demonstrated the ability to bind and to be activated by progesterone receptor antagonists (MFP, Org31806, Org31376), but not by progesterone receptor agonists (progesterone, R5020).