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.
Project description:Three members of the progestin and adipoQ receptor (PAQR) family, PAQR-7, PAQR-8, and PAQR-5 [membrane progesterone (P4) receptor (PR) (mPR)?, mPR?, and mPR?], function as plasma mPRs coupled to G proteins in mammalian cells, but the characteristics of two other members, PAQR6 and PAQR9 (mPR? and mPR?), remain unclear, because they have only been investigated in yeast expression systems. Here, we show that recombinant human mPR? and mPR? expressed in MDA-MB-231 breast cancer cells display specific, saturable, high-affinity [(3)H]-P4 binding on the plasma membranes of transfected cells with equilibrium dissociation constants (K(d)s) of 2.71 and 2.85 nm, respectively, and low affinity for R5020, characteristics typical of mPRs. P4 treatment increased cAMP production as well as [(35)S]-guanosine 5'-triphosphate (GTP)?S binding to transfected cell membranes, which was immunoprecipitated with a stimulatory G protein antibody, suggesting both mPR? and mPR? activate a stimulatory G protein (Gs), unlike other mPRs, which activate an inhibitory G protein (Gi). All five mPR mRNAs were detected in different regions of the human brain, but mPR? showed greatest expression in many regions, including the forebrain, hypothalamus, amygdala, corpus callosum, and spinal cord, whereas mPR? was abundant in the pituitary gland and hypothalamus. Allopregnanolone and other neurosteroids bound to mPR? and other mPRs and acted as agonists, activating second messengers and decreased starvation-induced cell death and apoptosis in mPR?-transfected cells and in hippocampal neuronal cells at low nanomolar concentrations. The results suggest that mPR? and mPR? function as mPRs coupled to G proteins and are potential intermediaries of nonclassical antiapoptotic actions of neurosteroids in the central nervous system.
Project description:Recent results showing that the binding characteristics of 33 steroids for human membrane progesterone receptor alpha (hu-mPRalpha) differ from those for the nuclear progesterone receptor (nPR) suggest that hu-mPRalpha-specific agonists can be identified for investigating its physiological functions. The binding affinities of an additional 21 steroids for hu-mPRalpha were determined to explore the structure-activity relationships in more detail and to identify potent, specific mPRalpha agonists. Four synthetic progesterone derivatives with methyl or methylene groups on positions 18 or 19, 18a-methylprogesterone (18-CH(3)P4, Org OE 64-0), 13-ethenyl-18-norprogesterone (18-CH(2)P4, Org 33663-0), 19a-methylprogesterone (19-CH(3)P4, Org OD 13-0) and 10-ethenyl-19-norprogesterone (19-CH(2)P4, Org OD 02-0), showed similar or higher affinities than progesterone for hu-mPRalpha and displayed mPRalpha agonist activities in G-protein and MAP kinase activation assays. All four steroids also bound to the nPR in cytosolic fractions of MCF-7 cells. However, two compounds, 19-CH(2)P4 and 19-CH(3)P4, showed no nPR agonist activity in a nPR reporter assay and therefore are selective mPRalpha agonists suitable for physiological investigations. The structure-binding relationships of the combined series of 54 steroids for hu-mPRalpha deviated strikingly from those of a published set of 60 3-keto or 3-desoxy steroids for nPR. Close correlations were observed between the receptor binding affinities of the steroids and their physicochemical properties calculated by comparative molecular field analysis (CoMFA) for both hu-mPRalpha and nPR. A comparison of the CoMFA field graphs for the two receptors revealed several differences in the structural features required for binding to hu-mPRalpha and nPR which could be exploited to develop additional mPR-specific ligands.
Project description:The progestin and AdipoQ receptor (PAQR) family of proteins comprises three distinct structural classes, each with seemingly different agonist specificities. For example, Class I receptors, like the human adiponectin receptors (AdipoR1 and AdipoR2), sense proteins with a particular three-dimensional fold, while Class II receptors are nonclassical membrane receptors for the steroid hormone progesterone. Using a previously developed heterologous expression system to study PAQR receptor activity, we demonstrate that human PAQRs from all three classes are antagonized by both 1(S),2(R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, a ceramidase inhibitor, and TNFalpha, a homologue of adiponectin that functions antagonistically to both adiponectin and progesterone in human cells.
Project description:Adaptive responses in fungi result from the interaction of membrane receptors and extracellular ligands. Many different classes of receptors have been described in eukaryotic cells. Recently a new family of receptors classified as belonging to the progesterone-adiponectin receptor (PAQR) family has been identified. These receptors have the seven transmembrane domains characteristic of G-protein coupled receptors, but their activity has not been associated directly to G proteins. They share sequence similarity to the eubacterial hemolysin III proteins.A new receptor, SsPAQR1 (Sporothrix schenckii progesterone-adiponectinQ receptor1), was identified as interacting with Sporothrix schenckii G protein alpha subunit SSG-2 in a yeast two-hybrid assay. The receptor was identified as a member of the PAQR family. The cDNA sequence revealed a predicted ORF of 1542 bp encoding a 514 amino acids protein with a calculated molecular weight of 57.8 kDa. Protein domain analysis of SsPAQR1 showed the 7 transmembrane domains (TM) characteristic of G protein coupled receptors and the presence of the distinctive motifs that characterize PAQRs. A yeast-based assay specific for PAQRs identified progesterone as the agonist. S. schenckii yeast cells exposed to progesterone (0.50 mM) showed an increase in intracellular levels of 3', 5' cyclic adenosine monophosphate (cAMP) within the first min of incubation with the hormone. Different progesterone concentrations were tested for their effect on the growth of the fungus. Cultures incubated at 35°C did not grow at concentrations of progesterone of 0.05 mM or higher. Cultures incubated at 25°C grew at all concentrations tested (0.01 mM-0.50 mM) with growth decreasing gradually with the increase in progesterone concentration.This work describes a receptor associated with a G protein alpha subunit in S. schenckii belonging to the PAQR family. Progesterone was identified as the ligand. Exposure to progesterone increased the levels of cAMP in fungal yeast cells within the first min of incubation suggesting the connection of this receptor to the cAMP signalling pathway. Progesterone inhibited the growth of both the yeast and mycelium forms of the fungus, with the yeast form being the most affected by the hormone.
Project description:BACKGROUND:Nuclear progesterone receptor (nPR) is an evolutionary innovation in vertebrates that mediates genomic responses to progesterone. Vertebrates also respond to progesterone via membrane progesterone receptors (mPRs) or membrane associated progesterone receptors (MAPRs) through rapid nongenomic mechanisms. Lampreys are extant agnathan vertebrates, residing at the evolutionary juncture where vertebrates diverged from invertebrates. A survey of the progesterone receptor (PR) gene sequences in lamprey genomes would inform PR gene evolutionary events during the transition from invertebrates to vertebrates. RESULTS:In this study, we annotated sequences of one nPR, four mPR (?, ?, ? and ?) and four MAPR genes from genomes of two lamprey species (Petromyzon marinus and Lethenteron japonicum). To infer the origin and evolutionary history of PR genes, we constructed phylogenetic trees of PR homologous sequences across representative species of metazoans. Phylogenetic analyses revealed that the mPR? gene first appeared in non-bilaterians, and the mPR? gene likely arose from a duplication of mPR?. On the other hand, the mPR? gene gave rise to the mPR? and ? genes much later in the vertebrate lineage. In addition, the mPR? gene first appeared in cartilaginous fishes, likely derived from duplication of mPR? after the agnathan-gnathostome divergence. All known MAPR genes were present in the lamprey genomes. Progesterone receptor membrane component 1 (PGRMC1), neudesin and neuferricin genes probably evolved in parallel in non-bilaterians, whereas two copies of PGRMC genes probably derived from duplication of ancestral PGRMC1 sequence and appeared before the speciation of lampreys. CONCLUSIONS:Non-classical mPR and MAPR genes first evolved in non-bilaterians and classical nPR genes evolved later in basal vertebrates. Sequence repertoires for membrane progesterone receptor genes in vertebrates likely originated from an ancestral metazoan sequence and expanded via several duplication events.
Project description:Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPRalpha, mPRbeta and mPRgamma) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPRgamma. The mPRalpha mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPRbeta mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPRgamma and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPRalpha and mPRbeta in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period.
Project description:Steroid hormones induce changes in gene expression by binding to intracellular receptors that then translocate to the nucleus. Steroids have also been shown to rapidly modify cell function by binding to surface membrane receptors. We identified a candidate steroid membrane receptor, the progestin and adipoQ receptor (PAQR) 10, a member of the PAQR family, in a screen for genes differentially expressed in mouse pancreatic beta-cells. PAQR10 gene expression was tissue restricted compared with other PAQRs. In the mouse embryonic pancreas, PAQR10 expression mirrored development of the endocrine lineage, with PAQR10 protein expression confined to endocrine islet-duct structures in the late embryo and neonate. In the adult mouse pancreas, PAQR10 was expressed exclusively in islet cells except for its reappearance in ducts of maternal islets during pregnancy. PAQR10 has a predicted molecular mass of 29 kDa, comprises seven transmembrane domains, and, like other PAQRs, is predicted to have an intracellular N-terminus and an extracellular C-terminus. In silico analysis indicated that three members of the PAQR family, PAQRs 9, 10, and 11, have a candidate mitochondrial localization signal (MLS) at the N-terminus. We showed that PAQR10 has a functional N-terminal MLS and that the native protein localizes to mitochondria. PAQR10 is structurally related to some bacterial hemolysins, pore-forming virulence factors that target mitochondria and regulate apoptosis. We propose that PAQR10 may act at the level of the mitochondrion to regulate pancreatic endocrine cell development/survival.
Project description:The PAQR family of proteins comprises an intriguing group of newly discovered receptors. Although the agonist is known for 5 of the 11 human PAQRs, most are considered "orphan" receptors. We developed a yeast-based assay system for PAQR receptor activity that can be used to identify agonists for PAQRs of unknown function. Using this system, we found that the proteinaceous hormone adiponectin functions as an agonist of PAQR3, a previously uncharacterized member of this family. This is not surprising given that PAQR3 is most closely related to PAQR1 (AdipoR1) and PAQR2 (AdipoR2), which also sense adiponectin. The identification of adiponectin as an agonist for PAQR3 is of considerable clinical relevance because adiponectin suppresses the proliferation of tumor cells and it has been reported that PAQR3 suppresses tumorigenesis. Thus, the interaction between PAQR3 and adiponectin may help explain the antiproliferative properties of adiponectin.
Project description:A previous investigation showed that the endometrium normalized in women with endometrial hyperplasia after three months treatment with high dose levonorgestrel IUS (intrauterine system)  . The effect was maintained even if immunohistochemical analyses of the endometrium showed that nuclear progesterone receptors (nPRs) were completely downregulated. These observations indicated that some type of non-genomic effect existed . We conducted new investigations of endometrial hyperplasia, now with 6 months low dose levonorgestrel IUS treatment. Again, the growth disturbances were reversed with normalization of the endometrium [3,4]. In the context of these studies, RT-qPCR analyses of the endometrium were performed before and after treatment, to determine expression of nuclear progesterone receptors (nPRA+B and nPRB), membrane progesterone receptors (mPR, ?-, ?- and ?-subtypes) and progesterone receptor membrane components (PGRMC1and PGRMC2). The human cervical cell line (C-4 I)  with no detectable nPRs [6,7] , was included in the investigation as biological control .The gene expression of nPRs, mPRs and PGRMCs was determined in the logarithmic growth phase. Tissue and cellular mRNA was determined with RT-qPCR and used as a surrogate marker for receptor (protein) expression. The present data are connected to the related article entitled "Expression of nuclear progesterone receptors (nPRs), membrane progesterone receptors (mPRs) and progesterone receptor membrane components (PGRMCs) in the human endometrium after 6 months levonorgestrel low dose intrauterine therapy" .
Project description:<h4>Introduction</h4>Basal phenotype breast cancers (BPBC) are often associated with apparent epithelial to mesenchymal transition (EMT). The role of progesterone (P4) in regulating EMT of BPBC has not been reported.<h4>Methods</h4>The EMT relevant biology was investigated in vitro using human BPBC cell models (MDA-MB468 and MDA-MB231) with P4, PR agonist (RU486), and PR antagonist (R5020) treatments. The essential role of membrane progesterone receptor alpha (mPRalpha) in the P4-regulated EMT was demonstrated by knocking down the endogenous gene and/or stably transfecting exogenous mPRalpha gene in the BPBC cell models.<h4>Results</h4>The expression of snail and down-stream EMT proteins such as occludin, fibronectin, and E-cadherin was significantly regulated by P4 incubation, which was accompanied by cell morphological reversion from mesenchymal to epithelial phenotypes. In searching for the cell mediator of P4' action in the MDA-MB468 (MB468) cells, it was found that mPRalpha but not the nuclear PR has an essential role in the P4 mediated EMT inhibition. Knocking down the expression of mPRalpha with specific siRNA blocked the P4's effects on expression of the EMT proteins. In another BPBC cell line--MDA-MB231 (MB231), which is mPRalpha negative by Western blotting--P4 treatment did not alter cell proliferation and EMT protein expressions. Introduction of the exogenous mPRalpha cDNA into these cells caused cell proliferation, but not EMT, to become responsive to P4 treatment. In further studies, it was found that activation of the PI3K/Akt pathway is necessary for the P4-induced EMT reversion. To define the potential inter-mediate steps between mPRalpha and PI3K, we demonstrated that mPRalpha, caveolin-1 (Cav-1), and epidermal growth factor receptor (EGFR) are colocalized in the membrane of caveolar vesicle and the P4-repressed EMT in MB468 cells can be blocked by EGFR inhibitor (AG1478) and PI3K inhibitor (wortmannin).<h4>Conclusions</h4>Our data suggest that the signaling cascade of P4 induced mesenchymal repression is mediated through mPRalpha and other caveolae bound signaling molecules namely Cav-1, EGFR, and PI3K. This novel finding may have great impact on fully understanding the pathogenesis of BPBC and provide an essential clue for developing a targeted therapeutic strategy for treatment of BPBC.