Heterologous expression of human mPRalpha, mPRbeta and mPRgamma in yeast confirms their ability to function as membrane progesterone receptors

Abstract

The nuclear progesterone receptor (nPR) mediates many of the physiological effects of progesterone by regulating the expression of genes, however, progesterone also exerts non-transcriptional (non-genomic) effects that have been proposed to rely on a receptor that is distinct from nPR. Several members of the progestin and AdipoQ-Receptor (PAQR) family were recently identified as potential mediators of these non-genomic effects. Membranes from cells expressing these proteins, called mPRalpha, mPRbeta and mPRgamma, were shown to specifically bind progesterone and have G-protein coupled receptor (GPCR) characteristics, although other studies dispute these findings. To clarify the role of these mPRs in non-genomic progesterone signaling, we established an assay for PAQR functional evaluation using heterologous expression in Saccharomyces cerevisiae. Using this assay, we demonstrate unequivocally that mPRalpha, mPRbeta and mPRgamma can sense and respond to progesterone with EC(50) values that are physiologically relevant. Agonist profiles also show that mPRalpha, mPRbeta and mPRgamma are activated by ligands, such as 17alpha-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR. These results strongly suggest that these receptors may indeed function as the long-sought-after membrane progesterone receptors. Additionally, we show that two uncharacterized PAQRs, PAQR6 and PAQR9, are also capable of responding to progesterone. These mPR-like PAQRs have been renamed as mPRdelta (PAQR6) and mPRvarepsilon (PAQR9). Additional characterization of mPRgamma and mPRalpha indicates that their progesterone-dependent signaling in yeast does not require heterotrimeric G-proteins, thus calling into question the characterization of the mPRs as a novel class of G-protein coupled receptor.

Figure 1. Functional expression of human mPRα, mPRβ and mPRγ in yeast

All cells are wild type and are grown in iron-deficient LIM. Medium in panels (A) and (B) contains 0.05% galactose/1.95% raffinose and medium in panel (C) contains 2% galactose. In all boxes, the activity of the FET3 gene is monitored by measuring β-galactosidase activity produced by the FET3-lacZ reporter. (A) All PAQRs are cloned into the pYES260 vector except AdipoR2, which is cloned into pGREG536. White symbols show the effect of progesterone on FET3 in cells carrying either empty expression vector or vectors that express mPRα, mPRβ or mPRγ. Grey symbols show the effect of adiponectin on FET3 in cells carrying either empty expression vector or vectors that express AdipoR1 or AdipoR2. (B) The effect of various steroids on the FET3 gene cells expressing either mPRγ or mPRα from the pYES260 plasmid. (C) The effect of various steroids on FET3 in cells expressing mPRα from the pRS316 plasmid. When data points overlap or are very close to overlapping, combined symbols are used.

Figure 2. Steroid specificities for mPRγ and mPRα

In all cases, FET3 expression is measured using the FET3-lacZ reporter. All PAQR are cloned into the pGREG536 vector. All cells are wild type and are grown in iron-deficient LIM containing 0.05% galactose/1.95% raffinose. (A) The dose response of FET3 in cells expressing mPRγ plasmid to progesterone either alone or in the presence of 10 µM β-estradiol (E), 10 µM testosterone (test) or 100 nM RU-486 (RU). (B) Dose response of FET3 to either progesterone (P) or RU-486 (RU) in cells carrying either empty expression vector or vectors that express mPRγ or mPRα. (C) Dose response of FET3 to RU-486 in cells carrying either empty expression vector or a vector that expresses mPRγ. Cells are either treated with RU-486 alone or in the presence of 100 nM progesterone (P). When data points overlap or are very close to overlapping, combined symbols are used.

Figure 3. Multiple sequence alignment of human Class II PAQRs

This alignment was originally performed using ClustalX but was modified manually afterwards. TM and loop regions (L) are numbered. Predicted TMs are also boxed. Black shading shows amino acids that are highly, but not universally, conserved in the entire PAQR family. Grey shading indicates amino acids that are conserved in all vertebrate members of the mPRγ/PAQR6 clade, although only human sequences are shown. Circled amino acids indicate the positions of intron/exon boundaries in the pre-mRNA. Arrow indicates the location of truncations in the mPRγ and mPRα proteins discussed in Figure 7.

Figure 4. Identification of additional membrane progesterone receptors

In all panels except (C) cells were grown in medium containing 0.05% galactose/1.95% raffinose and FET3 activity is measured using the FET3-lacZ construct as a reporter. All PAQRs are cloned into the pYES260 vector except AdipoR2, which is cloned into pGREG536. (A) Response FET3 in cells expressing all 11 human PAQR proteins to 10 µM progesterone. (B) Response of FET3 to various steroids in wild type cells expressing either PAQR6 (white symbols) or PAQR9 (grey symbols).

Figure 5. Topological analysis of Class I and Class II PAQRs. (A–D)

Hydropathy plots for the individual proteins analyzed in Figure 3 were generated and aligned (dotted lines). An average hydropathy plot for all members of each clade were generated (solid lines). The core PAQR motif is shaded in grey and the predicted TMs are numbered. All vertebrate members of the AdipoR1 and AdipoR2 clade (A), the mPRγ/PAQR6 clade (B), the mPRα/mPRβ clade (C) and the PAQR9 homologs (D). (E) Predicted topology of the core PAQR motif with the locations of the three highly conserved motifs shaded in black in Figure 4. The predicted eighth TM in the mPRs is shown with a dotted line. (F) The structure of the dual topology reporter tag. (G) DTR-tagged PAQR6 and mPRα expressed in the pJK90 plasmid repress FET3-lacZ in response to 100 nM progesterone in cells grown in medium containing 0.05% galactose/1.95% raffinose. (H) Rescue of the histidine auxotrophy of the STY50 strain by DTR-tagged PAQR6 and mPRα on plates containing histidinol. Plasmids expressing untagged versions of PAQR6 and mPRa are shown as negative controls. A plasmid expressing DTR-tagged Ost4p is shown as a positive control. (I) Membrane extracts from cells expressing DTR-tagged PAQR6 or mPRα are either left untreated (−) or treated with the endoglycosidase EndoH (+) and subsequently run on protein gels and transferred to nitrocellulose membranes for Western blot. anti-HA antibodies were used to detect the DTR tag. (left) Coomassie stained gel of EndoH treated RNase B under the same conditions as those used to treat the cell membrane extracts is shown on the right as a positive control.

Figure 6. Truncation mutations and G-protein signaling

In all cases, FET3 expression is measured using the FET3-lacZ reporter. (A) Full length and truncated mPRγ and mPRα were expressed in wild type cells grown in 2% galactose from the pGREG536 vector. The location of the C-terminal truncations is shown in Figure 4. All proteins possess an N-terminal 7x-HA tag. Proteins were detected by Western blot using an anti-HA antibody. (B) The dose response of FET3 to progesterone in cells expressing the full length or truncated mPRγ and mPRα and grown in 0.05% galactose/1.95% raffinose. (C) The ability of mPRα and mPRγ to respond to progesterone and repress FET3 is not impaired in either gpa2Δ or gpa1Δ cells (gpa1Δ cells also lack the STE7 gene, see text) (D) Overexpression of the Ste2p GPCR using the GAL1 promoter does not repress FET3 in wild type cells (mat a) grown in 2% galactose. Activation of overexpressed Ste2p via the addition of 1 µM of its agonist, α-factor, also has no effect on FET3 under these conditions. (C) Expression of constitutively active alleles of Gpa1p (Gpa1p^Q323L) and Gpa2p (Gpa2p^Q300L) from the GAL1 promoter had no effect on FET3 in cells grown in 2% galactose.