Expression of Functional Chicken Oviduct Progesterone Receptors in Yeast (Saccharomyces Cerevisiae)

P Mak,D P Mcdonnell,N L Weigel,W T Schrader,B W O'Malley

doi:10.1016/s0021-9258(20)88228-3

P Mak, D P Mcdonnell + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(20)88228-3

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Abstract

The cDNAs encoding full-length chicken oviduct progesterone receptor B (PRB) and a truncated receptor (C1C2) lacking the amino-terminal domain were expressed in yeast (Saccharomyces cerevisiae) using a ubiquitin fusion system. The expression of the fusion protein is under the control of a copper-responsive yeast metallothionein promoter, and the fusion protein is subsequently cleaved by the yeast host enzyme to produce receptor protein. Western immunoblot analyses of yeast extracts containing full-length PRB revealed a polypeptide co-migrating with authentic chicken oviduct PRB. Using a polyclonal antibody (907) directed against the "hinge" region of the authentic chicken progesterone receptor, a 42-kDa polypeptide was detected by Western analysis in yeast extracts containing C1C2 receptors. Standard hormone binding assays indicated that these receptors produced in yeast cells exhibited steroid binding affinity and specificity characteristic of the authentic chicken progesterone receptor. To test for progesterone receptor-mediated activation of transcription in yeast, reporter plasmids were constructed to transform yeast cells expressing PRB or C1C2 receptors. The reporter gene contained two copies of a progesterone response element upstream of the yeast proximal CYC1 promoter fused to the beta-galactosidase gene of Escherichia coli. The induction of beta-galactosidase activity by PRB and C1C2 was strictly dependent on specific ligand and the presence of a progesterone response element. However, overproduced C1C2 receptors had an adverse effect on the transcription of the lacZ gene. It was found that when overproduced C1C2 was activated by progesterone, an inhibitory effect on normal yeast cell growth was evident. These observations suggest that C1C2 is a potent trans-acting factor in yeast and that the amino-terminal domain of the chicken progesterone receptor may play a role in selective modulation of target gene activation.

Highlights

The cDNAs encodingfull-lengthchickenoviduct with the promoters of a variety of steroid-regulated genes progesterone receptor B (PRB) anda truncated recep- have been identified [5]
Our laboratory habseen studying the structure and function expressed in yeast (Saccharomyces cerevisiae) using aof the chicken progesterone receptor
The expression of the fusion posed of two hormone-binding forms, PRA(7’ 2 kDa) and PRB

Summary

RESULTS

Aftercopperinduction,yeastextracts from YEpPPwere analyzed by Western immunoblots. As shown in Fig. 3, a immunoblot assays, the receptor is approximately 0.1% of the polypeptide which co-migrates with authentic PRB was de- total cellular protein. Tracts from YEpPl revealed a polypeptide with a molecular The steroid binding affinityof the expressed receptors was mass of 42 kDa (Fig. 3) when probed with polyclonal antibody determined by saturation analysis. There was a 10,000-12,000- ther resolved into two binding components when plotted by fold increase of receptor hormone binding activity after copper the method of Rosenthal [22] with K d values of 0.9 and 6.0 induction. As shown in YEpPl without copper induction due to thelow sensitivity of Fig. 5 (A and B ) , a 100-fold excess of either progesterone or polyclonal antibody 907, hormone binding assays indicated R5020 could compete effectively (80-go%), whereas cortisol, that there was aconsistently low basal level of receptors triamcinolone acetonide, RU486, and estradiol were poor comexpressed (80-120 fmol/mg) under these conditions. The values represent the average of two to three separate experiments

Molar Excess Competitors

DISCUSSION

Expression of COhPivcrikdoeugncetsRteercoenpetors