Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors
Introduction
The classic paradigm for progesterone-mediated signal transduction involves diffusion of the steroid hormone into cells and binding to soluble intracellular progesterone receptors [1]. This type of signal transduction is often referred to as genomic signaling because this receptor, also called the nuclear progesterone receptor (nPR), doubles as a DNA binding transcription factor and, as such, modulates gene transcription in response to progesterone. It has long been recognized, however, that many of the physiological effects of progesterone occur far too rapidly to require gene transcription and often occur in cells that are transcriptionally silent, such as sperm [2]. Moreover, the receptor(s) responsible for mediating the non-genomic effects of progesterone have a markedly different agonist profiles from nPR [3], suggesting that it is a distinct receptor. These facts have led researchers to propose the existence of an integral membrane progesterone receptor (mPR) that binds progesterone at the cell surface and rapidly generates intracellular second messengers [4]. This mechanism for progesterone response has been called non-genomic signaling because it does not absolutely require transcription to proceed, although there is no reason to suspect that non-genomic mechanisms do not also regulate transcription [5]. The existence of mPR is a matter of intense debate. Indeed, it has been argued that there is no need to invoke the existence of a distinct mPR because the nPR itself is capable of mediating rapid non-genomic effects by binding to and regulating the activity of other signaling proteins [6], [7], [8], [9]. Nevertheless, several candidate mPRs have emerged and, while it is clear that some of these candidates bind progesterone, it is not clear how, or even if, they function as receptors to mediate the non-genomic effects of progesterone.
Recently, a series of seminal papers were published identifying three integral membrane proteins from fish that not only bind progesterone, but also seem to mediate many of its rapid non-genomic effects [10], [11]. These three proteins, renamed mPRα, mPRβ and mPRγ, are conserved in vertebrates and belong to a newly characterized family of proteins that includes the yeast osmotin receptor (Izh2p) [12] and human adiponectin receptors (AdipoR) [13]. This family is known as the progestin- and AdipoQ-Receptor (PAQR) family [14].
A series of follow-up studies produced compelling evidence that mPRα, mPRβ and mPRγ function as membrane progesterone receptors. However, as a recent review in this journal demonstrates [15], the field remains embroiled in controversy. The primary reason for this is the fact that a recent study was unable to reproduce the original results [16], [17]. Another significant source of contention is the assertion that these receptors function as a new class of G-protein-coupled receptor (GPCR) [18], [19], [10], [11] despite the fact that no other class of PAQR seems to couple to G-proteins and that members of the PAQR family of receptors bear only superficial similarity to GPCRs.
Further investigation is needed before mPRα, mPRβ and mPRγ can be universally accepted as membrane progesterone receptors. One side of this equation involves the study of the physiological roles of these proteins in vivo. To date, no studies have demonstrated an unequivocal role for these proteins in the physiology of progesterone. Part of the reason for this is the overabundance of progesterone-binding proteins and putative progesterone receptors in vertebrates that can potentially confound data analysis. Not only do vertebrate cells possess classical nuclear progesterone receptors, they also possess the other members of the nuclear hormone receptor family for which progesterone may function as either an agonist or antagonist [20], [21]. Vertebrate cells also contain a variety of other progesterone-binding proteins that have been proposed to function as mPRs [22], [23]. Progesterone has even been shown to function as an allosteric regulator of a variety of enzymes and receptors [24], [25], [26]. Thus, it is difficult to attribute progesterone-dependent effects solely to one protein. This line of research is beyond the scope of this study and, ultimately, the discovery of the true physiological roles of mPRα, mPRβ and mPRγ may require the development of mouse knockout strains.
Herein, we address another important aspect of characterizing mPRα, mPRβ and mPRγ—their biochemistry. To date, the biochemical characterization of mPRα, mPRβ and mPRγ has involved their expression in either vertebrate cell culture or E. coli [19], [10], [11]. Expression in vertebrate cell culture has been fruitful, however, conflicting results were obtained by different groups [17], [19]. Moreover, this approach is limited by the aforementioned overabundance of progesterone-binding proteins in vertebrate cells that make data interpretation difficult. On the other hand, E. coli do not contain known progesterone receptors and heterologous expression in this organism has been successfully used to confirm progesterone binding to isolated mPRs embedded in intact E. coli membranes [10], [11]. However, while expression in E. coli is certainly useful for studying progesterone binding to these receptors, it cannot yet be used to investigate functionality, since E. coli it is not known if PAQR receptors function the same in this organism as they do in eukaryotes.
Thus, a resolution to the debate about whether mPRα, mPRβ and mPRγ can sense and respond to progesterone requires a system with two fundamental properties. First, such a system must be devoid of known progesterone-binding/sensing proteins so that the mPRs can be studied in isolation. Second, this system must have an intact signaling apparatus that allows for monitoring signal transduction in response to progesterone. Herein we report the development of such a system that entails the heterologous expression of mPRα, mPRβ and mPRγ in the yeast Saccharomyces cerevisiae. This choice of model systems is advantageous for several reasons. First, yeast is a eukaryotic system for which simple yet powerful genetic tools exist. Second, yeast possesses receptors in the PAQR family suggesting that the machinery required to read second messengers produced by these proteins is present [27]. Third, S. cerevisiae neither makes nor uses progesterone. In fact, a recent publication showed that massive doses of progesterone (1 mM) did little more than weakly induce the general stress–response [28]. The low background biological activity of progesterone in this system makes it an ideal model for the study of individual progesterone receptors in a living cell. Indeed, S. cerevisiae has already been successfully adapted for the biochemical characterization of nuclear progesterone receptors [29].
We recently published a study showing that the yeast osmotin receptor (Izh2p) controls a signaling pathway in S. cerevisiae that negatively regulates the expression of a gene called FET3 [30]. The physiological importance of this regulation is neither clear nor is it actually important in the context of this investigation. What is important is that we also showed that activation of human adiponectin receptors, when heterologously expressed in yeast, had the same effect on FET3 expression. The regulation of the same pathway by fungal and human PAQRs suggests that PAQRs from diverse sources generate similar second messengers when expressed in yeast. The identity of this second messenger and the mechanism of signal transduction for PAQRs in yeast are still under investigation and these topics are beyond the scope of this study.
Herein, we demonstrate that the expression of the FET3 gene can be used as a general reporter of the functionality of human PAQR receptors. Using this reporter system to study the mPRs, we made several important findings. First and foremost, we demonstrated mPRα, mPRβ and mPRγ repress FET3 in response to progesterone while other PAQR receptors do not. This confirms beyond reasonable doubt that these proteins can sense and respond to progesterone. Moreover, the ED50 values for the activation of the mPRs by progesterone demonstrate that the mPRs are most responsive to progesterone at physiologically relevant concentrations. Second, we show that this assay can be used to probe the agonist profiles for these receptors. Not only do these receptors have specificities that are distinct from nPR, they are activated by ligands, such as 17α-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR [3]. We also demonstrate that mifepristone, an important antagonist of nuclear progesterone receptors, actually serves as a weak agonist of the mPRs, a fact that is consistent with what is known about non-genomic progesterone signaling [31]. Third, we show that human PAQR6 and PAQR9, both uncharacterized members of the PAQR family likely function as additional vertebrate mPRs. Accordingly, we renamed them mPRδ and mPRɛ, respectively. Finally, we demonstrate that the ability of these proteins to sense and respond to progesterone requires neither human nor yeast Gα-proteins making it unlikely that the mPRs function as GPCRs in the classical sense.
Section snippets
Yeast strains
Wild type BY4742 (Mat α), BY4741 (Mat a) and gpa2Δ (Mat α, BY4742 background) mutant yeast strains were obtained from Euroscarf (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/). gpa1Δ mutants are inviable due to constitutive growth arrest caused by the hyperactivation of the Ste4p/Ste118p Gβγ subunit in the absence of the Gpa1p Gα subunit. Viability of gpa1Δ mutant strains can be restored by concomitant deletion of the Ste7p MAP kinase that functions downstream of Ste4p/Ste18p. The gpa1Δste7
Heterologous expression of human mPRs in yeast
When grown in iron-deficient medium, the FET3 gene is induced to facilitate the uptake of exogenous iron. We previously demonstrated that the yeast osmotin receptor (Izh2p) activated a pathway that resulted in the constitutive repression of FET3 in iron-deficient conditions [30]. Control experiments demonstrated that this effect was not an artifact of the FET3-lacZ reporter and that the expression of FET3 was, indeed, regulated by Izh2p. We also demonstrated that the human adiponectin receptors
Discussion
The definitive classification of mPRα, mPRβ and mPRγ as membrane progesterone receptors is still a matter of debate. The recent publication of conflicting data that directly challenged the role of mPRα, mPRβ and mPRγ in progesterone signaling [17] has reinforced the controversy. To address whether or not the mPRs are, indeed, membrane progesterone receptors, we expressed the mPRs in the tractable eukaryotic model organism, S. cerevisiae. The obvious reason for this choice is that yeast do not
Acknowledgements
Funding for this study was provided by the National Institutes of Health (5 R21 DK074812-02 to TJL) and by the University of Florida, Department of Chemistry.
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- 1
Current address: Department of Neurobiology, SBR-14, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States.
- 2
Current address: Boston Museum of Science, Science Park, Boston, MA 02114, United States.