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Steroid and G Protein Binding Characteristics of the Seatrout and Human Progestin Membrane Receptor Subtypes and Their Evolutionary Origins

University of Texas Marine Science Institute (P.T., Y.P., J.D., C.T.), Port Aransas, Texas 78373; Centre for Molecular and Biomolecular Informatics (P.G., J.K., J.d.V.), Nijmegen Centre for Molecular Life Sciences, Radbout University Nijmegen, 6500 HC Nijmegen, The Netherlands; and Department of Biology (Y.Z.), East Carolina University, Greenville, North Carolina 27858

Address all correspondence and requests for reprints to: Peter Thomas, University of Texas Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373. E-mail: thomas{at}utmsi.utexas.edu.

	Abstract

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A novel progestin receptor (mPR) with seven-transmembrane domains was recently discovered in spotted seatrout and homologous genes were identified in other vertebrates. We show that cDNAs for the mPR subtypes from spotted seatrout (st-mPR) and humans (hu-mPR) encode progestin receptors that display many functional characteristics of G protein-coupled receptors. Flow cytometry and immunocytochemical staining of whole MDA-MB-231 cells stably transfected with the mPRs using antibodies directed against their N-terminal regions show the receptors are localized on the plasma membrane and suggest the N-terminal domain is extracellular. Both recombinant st-mPR and hu-mPR display high affinity (Kd 4.2–7.8 nM), limited capacity (Bmax 0.03–0.32 nM), and displaceable membrane binding specific for progestins. Progestins activate a pertussis toxin-sensitive inhibitory G protein (G_i) to down-regulate membrane-bound adenylyl cyclase activity in both st-mPR- and hu-mPR-transfected cells. Coimmunoprecipitation experiments demonstrate the receptors are directly coupled to the G_i protein. Similar to G protein-coupled receptors, dissociation of the receptor/G protein complex results in a decrease in ligand binding to the mPRs and mutation of the C-terminal, and third intracellular loop of st-mPR causes loss of ligand-dependent G protein activation. Phylogenetic analysis indicates the mPRs are members of a progesterone and adipoQ receptor (PAQR) subfamily that is only present in chordates, whereas other PAQRs also occur in invertebrates and plants. Progesterone and adipoQ receptors are related to the hemolysin3 family and have origins in the Eubacteria. Thus, mPRs arose from Eubacteria independently from members of the GPCR superfamily, which arose from Archeabacteria, suggesting convergent evolution of seven-transmembrane hormone receptors coupled to G proteins.

	Introduction

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ALTHOUGH THE IMPORTANCE of rapid (i.e. nonclassical) steroid actions initiated at the cell surface through binding to steroid membrane receptors has become more widely accepted within the past few years, details of the initial steroid-mediated events, including the identities of the steroid membrane receptors and their mechanisms of action, remain unclear and are surrounded by controversy (1, 2, 3). There is clear evidence that a variety of receptor proteins are involved in initiating these nonclassical steroid actions in different cell models, including nuclear steroid receptors or nuclear steroid receptor-like forms (1, 2, 4), receptors for other ligands that also bind steroids (2, 5), and unidentified receptors with different characteristics from those of any known receptors (2, 6). Recently, a novel cDNA was discovered in spotted seatrout ovaries that has several characteristics of the progestin membrane receptor (mPR) mediating progestin induction of oocyte maturation in this species by a nongenomic mechanism (7). The seatrout cDNA (st-mPR) encodes a 40 kDa protein, which has seven transmembrane domains, and receptor activation alters pertussis toxin-sensitive adenylyl cyclase activity, both of which suggest st-mPR is a G protein-coupled receptor (GPCR) or GPCR-like protein (7). More than 20 closely related genes have been cloned from other vertebrate species, including three mPR subtypes in humans, named , ß, and , which show high levels of expression in human reproductive, brain, and kidney tissues, respectively (8). The identification of a new class of putative steroid receptors, unrelated to nuclear steroid receptors, but instead related to GPCRs, provides a plausible explanation of how steroids can initiate rapid hormonal responses in target cells by activating receptors on the cell surface. There has been broad recognition of the potential significance of these findings (1, 9, 10) and also an extensive research effort to determine the distribution, hormonal regulation, and biological roles of the mPRs in various vertebrate models (11, 12, 13, 14, 15, 16). However, critical information is still lacking on several key features of mPRs essential for clearly establishing this proposed alternative model of steroid action and for understanding its likely evolutionary origins.

The st-mPR protein has been localized to the plasma membrane of seatrout oocytes (7), but progestin binding and activation of signal transduction pathways in the plasma membranes of cells transfected with the st-mPR and human mPRs remain to be demonstrated. To date, progestin binding has only been shown to the soluble recombinant mPR proteins produced in a bacterial expression system (7, 8). Moreover, the binding characteristics of st-mPR in the plasma membrane and its physiological role are still uncertain, because the principal teleost progestin hormones that induce oocyte maturation do not show any binding affinity for this soluble recombinant protein (7). Practically no information is currently available for the human counterpart, hu-mPR, including whether its recombinant protein is expressed in plasma membranes and binds progestins specifically, whether it transduces signals in target cells by activating G proteins, and its orientation in the plasma membrane. Several phylogenetic analyses have grouped the mPRs with adiponectin receptors as members of a progesterone and adipoQ receptor (PAQR) family (17, 18, 19), which have an opposite orientation in the plasma membrane to GPCRs with an intracellular N terminal (20), and it has been proposed that all PAQRs, including mPRs, do not activate G proteins (18). In addition, an intracellular location, rather than a plasma membrane one of mammalian mPRs, has been observed in certain eukaryotic expression systems (15, 16). Although our previous structural and functional analyses suggest st-mPR may be a GPCR, clear evidence that the receptor activates a G protein is directly coupled to it and has the characteristics of a GPCR is lacking. Finally, the phylogenetic relationship of the mPRs to GPCRs is unknown. Therefore, in the present study, we investigated the localization of the recombinant seatrout and human mPR proteins, steroid binding, and activation of second messengers in the plasma membranes of st-mPR and hu-mPR-transfected human breast cancer cells (MDA-MB-231 cells), which do not express the nuclear progestin receptor (21). Activation of G proteins, their identities, as well as direct receptor/G protein coupling were also examined after progestin treatment. A preliminary investigation of the functional domains of the st-mPR protein for G protein coupling and their similarity to GPCRs was conducted with st-mPR mutants with a truncated C-terminal and modified intracellular loop three domains. Finally, phylogenetic analyses of mPRs and other PAQRs were performed to reveal their likely origins. Collectively, the results show that the mPRs are membrane-bound specific progestin receptors that activate G proteins and function as GPCRs but have a different ancestral origin to members of the GPCR superfamily.

	Materials and Methods

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Chemicals
The steroids progesterone, 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S), 17,20ß-dihydroxy-4-pregnen-3-one, 17-hydroxyprogesterone, 20ß-hydroxyprogesterone, cortisol, estradiol-17ß, testosterone, 11-deoxycorticosterone were purchased from Steraloids (Newport, RI). The synthetic progestin R5020 was purchased from Amersham (Piscataway, NJ). The synthetic antiprogestin RU486 was purchased from Sigma-Aldrich (St. Louis, MO). The other synthetic and natural progestins were obtained from Organon (Oss, The Netherlands). [2,4,6,7-³H]-11-deoxycortisol, activity 50 Ci/mmol was obtained from American Radiolabeled Chemicals (ARC, St. Louis, MO) and [2,4,6,7-³H]-progesterone ([³H-P4]), approximately 102 Ci/mmol, was purchased from Amersham. 20ß-hydroxysteroid dehydrogenase and all other chemicals, buffers, and media were purchased from Sigma-Aldrich unless otherwise noted.

Culture of MDA-MB-231 cells stably expressing mPRs
MDA-MB-231 cells stably transfected with the st-mPR, obtained as described previously (7), or hu-mPR (described below) were cultured in DMEM/Ham’s F-12 medium supplemented with 10% charcoal-stripped fetal bovine serum (FBS) and 100 µg/ml of gentamicin. Charcoal-stripped FBS was prepared by incubating FBS with 0.5% activated charcoal and 0.05% dextran T-70 for 30 min at 55 C. The charcoal particles then were removed by centrifugation at 4 C for 20 min at 4500 x g. The stripped serum was sterile filtered and stored in aliquots at –80 C until use. The transfected cells were selectively maintained with 500 µg/ml geneticin with changes of medium every 1–2 days. The cells reached 80% confluence after 3 days in culture. One day before the experiments, fresh medium without phenol red and supplemented with 5% charcoal-stripped FBS was added to the cell cultures. A 150-mm culture dish with a monolayer culture typically contained approximately 2 x 10⁸ cells and yielded approximately 0.6 mg of cell membrane protein. Cells were subsequently collected with a cell scraper and washed twice before experimentation.

Expression of hu-mPR and st-mPR mutants in MDA-MB-231 cells
The procedures described previously for PCR amplification of the mPR cDNA, its insertion into an expression vector, and transfection in human MDA-MB-231 breast carcinoma cells (American Type Culture Collection, Manassas, VA) (7) were followed with few modifications for stable expression of hu-mPR and transient transfection of st-mPR mutants (see supplemental Fig. 1, A and B, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The coding regions of hu-mPR and st-mPR mutants were amplified by PCR from full-length cDNA plasmid clones (2 min denaturation at 94 C; 5 PCR cycles with denaturation at 94 C for 1 min, annealing at 50 C for 1 min, and polymerization at 72 C for 2 min followed by 25 cycles under the same conditions except annealing, which was conducted at 55 C) and the PCR products were purified by electrophoresis using a low-melting agarose and a QIAquick Gel Extraction Kit as described previously (8) before ligation into a PBK-CMV expression vector (Stratagene, La Jolla, CA). The correct insertion was confirmed by DNA sequencing. Cells were transfected with hu-mPR, st-mPR, st-mPR mutant cDNAs, or vectors containing reversed mPR inserts using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) following the manufacturer’s suggestions. Transient transfection experiments with st-mPR mutants were conducted with cells grown to confluence for 2–3 d in media containing 10% charcoal-stripped FBS, whereas experiments with stably transfected hu-mPR cells were conducted after continued selection with geneticin (500 µg/ml) for several (8, 9, 10) weeks.

Confirmation of correct expression of mPR mRNAs in transfected cells
RT-PCR was performed periodically during the course of the functional studies as described previously followed by sequencing to confirm continued successful expression of the entire coding regions of st-mPR and hu-mPR in stably transfected cells (reverse transcriptase reactions without the addition of reverse transcriptase were used as controls to verify lack of genomic DNA contamination). Two overlapping DNA fragments from RT-PCR of hu-mPR and st-mPR obtained from the transfected cell lines [primers: hu-mPR 1, sense 5'-GCT CCC TGC CCA GGC CCA CA-3' (before start codon), antisense: 5'-GCC AGC AGA AAG AAG ACC AC-3'; 2, sense: 5'-TCT TTG TGG AGA CCG TGG AC-3', antisense 5'-TCC CTA CCA GAT GCC ATC CC-3' (after stop codon); st-mPR 1, sense: 5'-TAC CGT CTA CAA GTT TGC C-3' (before start codon), antisense: 5'-GTG AGC AGC AGC CAA AGC AAG-3'; 2, sense 5'-CGC CAT AGA GAA AGA GTG G-3' antisense, 5'-AGT CAC TGT CAC AAA CTT CAT T-3' (after stop codon)] were cloned into a pGEM vector with a TA cloning system (Promega, Madison, WI). The plasmids containing the mPRs were subsequently sequenced with SP6 and T7 primers from both ends. The results confirmed that the transfected mPRs were correctly transcribed.

Membrane preparation and solubilization
Plasma membrane fractions of transfected MDA-MB-231 cells were obtained following procedures described previously (7, 22) with few modifications. The cell suspension was washed once with assay buffer and then sonicated for 15 sec followed by a 1000 x g centrifugation for 7 min to remove any nuclear and heavy mitochondrial material (nuclear fraction). The resulting supernatant was centrifuged at 20,000 x g for 20 min to obtain the plasma membrane fraction. The remaining supernatant was centrifuged at 100,000 x g for 60 min to obtain the microsomal and the cytosolic fractions. The plasma membrane was further purified for some studies by centrifuging the membrane pellet one to three times with a sucrose pad (1.2 M sucrose) at 6500 x g for 45 min (7).

mPR binding assays
The general procedures for measuring binding of radioactive steroid ligand to plasma membranes (22, 23) was used to measure binding of [2,4,6,7-³H]-17,20ß,21-trihydroxy-4-pregnen-3-one ([³H]-20ß-S,41.9 Ci/mmol) to the recombinant st-mPR and [2,4,6,7-³H]-progesterone ([³H]-P4,102.1 Ci/mmol) to the recombinant hu-mPR in the presence or absence of steroid competitors. [³H]-20ß-S was converted from [³H]-11deoxycortisol as described in (24). One set of tubes contained radiolabeled progestin alone (total binding); another set also contained nonradiolabeled progestin at concentrations 60- to 100-fold greater than the Kd of the receptor (450–750 nM) to measure nonspecific binding (NSB). For competition assays, a third set of tubes contained the radiolabeled progestins and 4–6 different concentrations of the steroid (range 1–10 µM) competitors (dissolved in 1–5 µl ethanol, which does not affect ligand binding in the receptor assays). After a 30-min incubation at 4 C with the membrane fractions, the reaction was stopped by filtration (Whatman GF/B filters, presoaked in assay buffer). The filters were washed twice with 25 ml assay buffer and bound radioactivity was measured by scintillation counting. The displacement of the radiolabeled steroid binding by the steroid competitors was expressed as a percentage of the maximum specific binding of the steroid for its receptor. Progestin binding to membranes pretreated with nonradiolabeled GTPS (25–50 µM) or activated and inactive pertussis toxin (0.5 µg/ml) was performed as described previously (25). The same filtration assay protocol was used to measure specific [³H]-P4 binding to microsomal and nuclear fractions of MDA cells transfected with hu-mPR (>65% of the proteins in these subcellular fractions are retained on the glass-fiber filters), whereas dextran-coated charcoal was used to separate bound from free [³H]-P4 in a soluble radioreceptor assay for cytosolic fractions as described previously (7).

Western blot analysis and immunocytochemistry
Polyclonal antibodies generated by a commercial vendor (Sigma-Genosys, Woodlands, TX) in rabbits against six injections of synthetic 15-mer peptides derived from the N-terminal domains of st-mPR (YRQPDQSWRYYFLTL) and hu-mPR (TVDRAEVPPLFWKPC) and a 11-mer peptide from C-terminal domain of hu-mPR (RPIYEPLHTHW) conjugated to keyhole limpet hemocyanin were used for immunodetection of the mPRs (see supplemental Fig. 1, A and B). Plasma membrane fractions were resuspended and solubilized in gel loading buffer containing sodium dodecyl sulfate (SDS) for electrophoresis. Ten micrograms of solubilized plasma membrane proteins were resolved in 12% SDS-polyacrylamide gel electrophoresis gels and transferred to a nitrocellulose membrane for Western blot analysis. The membranes were incubated for 1 h with blocking solution (5% nonfat milk in TBST buffer: 50 mM Tris/100 mM NaCl/0.1% Tween 20, pH 7.4) before incubation with the mPR antibodies overnight at 4 C. The specificity of the immunoreactions was evaluated by blocking them with the peptide antigens (2–3 ng/µl mPR antiserum diluted 1:20 in PBS buffer and preincubated at room temperature with the antibodies for 1.5–2 h). The next day, the membranes were washed with TBST buffer and incubated for 1 h at room temperature with horseradish peroxidase conjugated to goat antirabbit antibody (Cell Signaling, Beverly, MA). The blots were washed three times for 15 min with TBST buffer and treated with enhanced chemiluminescence (Pierce) and exposed to x-ray film.

Transfected cells were grown on coverslips for immunocytochemical analysis. The cells were rinsed twice with PBS and fixed with 2% paraformaldehyde and 0.25% glutaraldehyde in PBS for 15 min at 4 C. Cells were permeabilized by adding 0.5% Triton X-100 to the fixative. The cells were then rinsed briefly with PBS, incubated with 13 mM NaBH₄ in PBS for 10 min at 4 C to reduce autofluorescence, followed by three 5-min washes in PBS. The cells were subsequently blocked in 2% BSA in PBS for 1.5 h at 4 C followed by three 5-min washes in PBS. The cells were then incubated with the st-mPR or hu-mPR primary antibodies (dilution 1:1000) described previously in 2% BSA overnight at 4 C followed by three 5-min washes in PBS. Antisera were preabsorbed with peptide (0.02 mg peptide/1 ml antibody) overnight at 4 C for peptide block controls. The cells were then incubated with AlexaFluor 488 goat antirabbit secondary antibody (Molecular Probes, Carlsbad, CA; dilution 1:2000) followed by three 5-min washes in PBS. The coverslips were wet-mounted to slides using 80% glycerol in PBS and the presence of fluorescent-labeled mPR proteins in the cells visualized using a Nikon C1 confocal microscope.

Adenylyl cyclase activity in transfected MDA-MB-231 cells
The production of cAMP by isolated plasma membranes over 30 min at 25 C was measured as an estimation of adenylyl cyclase activity in response to treatment with progestins (20–100 nM) in the presence or absence of activated (30 min incubation with 50 mM DTT) or heat-inactivated (15 min incubation at 100 C) pertussis toxin (0.5 µg/ml). cAMP concentrations in the membranes were measured by enzyme immunoassay using a kit following the manufacturer’s instructions (Biomedical Technologies Inc., Stoughton, MA).

[³⁵S]GTP-S binding to cell membranes
Activation of G proteins was determined by measuring an increase in specific binding of [³⁵S]GTP-S to plasma membranes as described previously (6, 23). Membranes (10 µg protein) were incubated for 30 min at 25 C in the presence of 20 nM progestins with 10 µM GDP and 0.5 nM [³⁵S]GTP-S (12,000 cpm, 1 Ci/mol; Amersham) in the absence (total binding) or presence of 100 µM GTP-S (nonspecific binding). Bound [³⁵S]GTP-S was separated from free by filtering the incubation mixture through Whatman GF/B glass fiber filters followed by several washes.

Immunoprecipitation of [³⁵S]GTP-S-labeled G protein -subunits
Immunoprecipitation of the G protein -subunits was performed as described previously (23, 26). Plasma membranes (20 µg protein) of the transfected cells were incubated for 30 min at 25 C in 250 µl buffer containing 4 nM [³⁵S]GTP-S, 10 µM GDP, and protease inhibitors with 1 µM progestin and stopped by the addition of ice-cold buffer containing 100 µM GDP and 100 µM unlabeled GTP-S. The samples were subsequently centrifuged and the pellet resuspended in immunoprecipitation buffer containing Triton X-100, SDS, and protease inhibitors. Specific antisera to the -subunits of G proteins (G_i, G_o, and G_s, 1:300; Santa Cruz Biotechnology, Santa Cruz, CA) were added to the mixture and incubated at 4 C with gentle shaking for 6 h. Protein A-Sepharose beads were added and after an overnight incubation the immunoprecipitates were collected by centrifugation, washed, boiled in SDS, and the radioactivity in the immunoprecipitated [³⁵S]GTP-S-labeled G protein -subunits counted.

Coimmunoprecipitation of G protein i-subunit with mPRs
Transfected cells were treated with 100 nM progestin (20ß-S for recombinant st-mPR and progesterone for hu-mPR) for 10 min or untreated (controls) followed by two washes with PBS at 4 C. Triethanolamine buffer (50 mM triethanolamine, 25 mM KCl, 5 mM MgCl₂, 0.25 M sucrose, 0.1% protease inhibitor cocktail; Sigma-Aldrich; pH 7.5) was added and the cells were frozen at –80 C until analyzed. Plasma membranes were prepared as described previously and resuspended in immunoprecipitation buffer (0.1 mM EDTA, 1% Triton X-100 in Ca²⁺- and Mg²⁺-free PBS, pH 7.5) to a final volume of 300 µl (2 mg/ml membrane protein). The membrane suspension was incubated overnight at 4 C with 1:100 of goat anti-Gi and -Go antibody and control goat IgG (Santa Cruz Biotechnology, Inc.). Plasma membranes were then incubated for an additional 2 h at 4 C with 20 µl protein-A agarose beads (Santa Cruz Biotechnology, Inc.) added in the immunoprecipitation buffer. Beads were washed twice with 1 ml immunoprecipitation buffer, and immunoprecipitates were eluted by boiling for 10 min in SDS sample buffer. Samples were run on a 10% Tris-glycine SDS-polyacrylamide gel, and proteins were transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in a buffer of 50 mM Tris, 100 mM NaCl, and 0.1% Tween 20 (pH 7.4) for 1 h, and then incubated at 4 C overnight with the st-mPR or hu-mPR antibodies (1:2500). The membrane was then washed three times with Tris-buffered saline and then incubated for 1 h at room temperature with horseradish peroxidase conjugated goat antirabbit (hu-mPR) or mouse (st-mPR) antibodies (Cell Signaling), and visualized by treatment with enhanced chemiluminescence substrate (SuperSignal kit; Pierce, Rockford, IL).

Flow cytometry
Cells were carefully removed from the culture plates with a scraper and washed several times in PBS followed by low speed centrifugation to remove any cellular debris and damaged cells. Before conducting flow cytometry, the integrity of the cell membranes and their impermeability was confirmed by incubating them with clathrin antibody. Cells were either pretreated with 90% methanol for 15 min on ice and then blocked in the 1% BSA in PBS (permeabilized group) or directly incubated in blocking solution (nonpermeabilized group). N-terminal and C-terminal hu-mPR antibodies (1:1000) were added to the cell suspension in blocking solution and incubated at room temperature for 1 h followed by two washes in PBS. Alexa Fluor 488 goat antirabbit IgG antibody (Alexa 488; Molecular Probes) in blocking solution was added to the cell suspension and incubated for 30 min at room temperature in the dark followed by two washes with blocking solution. The cells were resuspended in 500 µl PBS and analyzed within 24 h on a flow cytometer (Becton and Dickinson FACSCallbur). Data were analyzed with CellQuest Pro software (BD Biosciences, San Jose, CA).

Phylogenetic analyses
The majority of the sequences used to construct the phylogeny of the PAQR/hemolysin 3 (HLY3) family was derived from the SMART database (27) corresponding to the Pfam model HLY3 (291 sequences) including all species. The Hidden Markow Model was used to screen for additional PAQR protein sequences in the complete eukaryotic genome sequences using HMMSearch (version 2.3.2c; part of HMMer package by S. Eddy, Janelia Farm Research Campus, Howard Hughes Medical Institute Laboratory). These complete proteomes were obtained from the Ensembl database (www.ensembl.org). The Ciona intestinalis protein sequences were obtained from the Joint Genome institute (http://genome.jgi-psf.org/Cioin2/Cioin2.home.html). The total set of protein sequences (397) was then aligned using ClustalW1.83 (28). The alignment was manually curated to remove redundancy (fragments, duplicates, chimaeras). All the remaining sequences (281) were aligned again and used for construction of a phylogeny using the Neighbor-Joining method and subsequently visualized using Treeview 1.6 (29).

	Results

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Steroid binding characteristics of recombinant seatrout and human mPRs
Expression of st-mPR and hu-mPR mRNAs in MDA-MB-231 cells and proteins in the plasma membranes was confirmed after stable transfection of their cDNAs (Figs. 1A and 2A). Weak endogenous expression of hu-mPR mRNA was also detected in untransfected MDA-MB-231 cells after 35 cycles of RT-PCR (Fig. 2A). Plasma membranes of st-mPR-transfected cells showed a 6.5-fold increase in specific 20ß-S binding compared with untransfected cells in single-point receptor binding assays (P < 0.001, Fig. 1B). Saturation analysis and Scatchard plotting indicated the presence of a high-affinity (Kd 7.58 ± 0.93 nM, n = 3), limited-capacity (Bmax 0.026 nM), saturable, single, specific binding site for 20ß-S (Fig. 1C). Dissociation of [³H]-20ß-S from the receptor was demonstrated in the presence of excess unlabeled steroid (Fig. 1D). Moreover, the rates of dissociation and association were rapid and reached 50% binding within 5 and 2 min, respectively (Fig. 1D). Competitive binding assays revealed that the major seatrout progestin hormone, 20ß-S, and the tetrapod hormone, progesterone, bound with high affinity to the receptor with IC₅₀ values of 47.5 nM and 185 nM, respectively (Fig. 1, E and F). Receptor binding was specific for progestins. Testosterone had a 28-fold lower affinity than 20ß-S (IC₅₀: 1374 nM), whereas 200-fold higher concentrations of estradiol-17ß and cortisol (10 µM) than the IC₅₀ of 20ß-S did not cause any displacement of [³H]-20ß-S from the receptor. Interestingly, the synthetic progestin R5020 and antiprogestin RU486, which have relatively high binding affinities for the mammalian nuclear progesterone receptor, failed to bind to the recombinant seatrout mPR at concentrations up to 10 µM (Fig. 1F).

View larger version (41K): [in this window] [in a new window]   FIG. 1. Progestin binding to plasma membranes of MDA-MB-231 cells stably transfected with st-mPR. A, Detection of st-mPR protein in transfected (Tr-231) cell membranes by Western blot analysis and st-mPR mRNA in cells by RT-PCR [(+)RT]. 231, Untransfected cells; OV, seatrout ovarian membranes; M, molecular weight protein standards; (-)RT, lacking reverse transcriptase; peptide blocked, blocked with peptide antigen. B, Specific [3H]-20ß-S binding to transfected cell membranes in a single point assay (see key in A) (n = 6,*, P < 0.05, Student’s t test). C, Representative saturation curve and Scatchard plot of specific [3H]-20ß-S binding to plasma membranes of transfected cells. D, Time course of association (Assoc) and dissociation (Dissoc) of [3H]-20ß-S binding. E and F, Competition curves for steroid (E) and progestin (F) binding expressed as a percentage of maximum specific 20ß-S binding. E2, Estradiol-17ß; T, testosterone; P4, progesterone; cort, cortisol; 17,20ß-P, 17,20ß-dihydroxy-4-pregnen-3-one; RU486, mifepristone; R5020, promegestone.

View larger version (42K): [in this window] [in a new window]   FIG. 2. Progestin binding to plasma membranes of MDA-MB-231 cells stably transfected with hu-mPR (see Fig. 1 for experimental details and key). A, Detection of hu-mPR protein and mRNA. B, Specific [3H]-progesterone (P4) binding in a single point assay. C, Representative saturation curve and Scatchard plot of specific [3H]-P4 binding (n = 5). D, Time course of association and dissociation of [3H]-P4 binding. E and F, Competition curves for steroid (E) and progestin (F) binding expressed as a percentage of maximum specific P4 binding. Norp, Norprogesterone; Nandr, nandrolone; Ethist, ethisterone; Nore, norethisterone; Norg, norgestrel.

View this table: [in this window] [in a new window]   TABLE 1. Rank order of binding affinities of natural and synthetic steroids to plasma membranes prepared from MDA-231 cells transfected with hu-mPR

View larger version (43K): [in this window] [in a new window]   FIG. 3. Activation of G proteins and second messengers in membranes of cells transfected with st-mPR and hu-mPR. A, Effects of 15-min treatment with 100 nM 20ß-S on cAMP production by membranes of st-mPR-transfected cells (Tr-231) with or without 30-min pretreatment with activated (aPTX) or heat-inactivated pertussis toxin (iPTX, 0.5 µg/ml). 231-untransfected cells. *, P < 0.05, n = 4. B, Effects of 10-min treatment with 20 nM progesterone (P4), 20 nM cortisol (Cort), or vehicle (Veh) on cAMP production by membranes from cells transfected with hu-mPR. C, Effects of treatment with 20 nM 20ß-S or 20 nM R5020 on specific [35S]GTP-S binding to membranes of cells transfected with st-mPR. *, P < 0.05 compared with vehicle treatment (Veh), n = 4. D, Effects of treatment with 20 nM progesterone, 20 nM cortisol (Cort), or 20 nM R5020 on specific [35S]GTP-S binding to membranes of cells transfected with hu-mPR. *, P < 0.05 compared with vehicle treatment (Veh). E, Immunoprecipitation of [35S]GTP-S bound to G protein -subunits activated on 1 µM 20ß-S treatment of st-mPR-transfected cell membranes with specific Gs (anti-Gs) and Gi (anti-Gi) G protein antibodies or control rabbit serum (rabbit ser). *, P < 0.05 (n = 4) compared with vehicle control. F, Immunoprecipitation of [35S]GTP-S bound to G protein -subunits activated upon 1 µM progesterone treatment of hu-mPR-transfected cell membranes with specific antibodies described in E. *, P < 0.05 (n = 4) compared with vehicle control.

G protein coupling to seatrout and human mPRs

View larger version (36K): [in this window] [in a new window]   FIG. 4. Coupling of seatrout and human mPR proteins to G proteins. A and B, Coimmunoprecipitation of st-mPR (A) and hu-mPR (B) coupled to G protein -subunits with specific G protein antibodies followed by immunodetection of the mPRs by Western blot analysis. Pretreated for 10 min with 100 nM 20ß-S or progesterone. C and D, Effects of 30-min pretreatment with 0.5 µg/ml activated pertussis toxin (aPTX) or inactivated pertussis toxin (iPTX) on specific [3H]-20ß-S binding to membranes of st-mPR-transfected cells (C) or on specific [3H]-progesterone binding to membranes of hu-mPR-transfected cells (D). *, P < 0.05, n = 4. E and F, Effects of 30-min pretreatment with 25 µM GTPS on specific [3H]-20ß-S binding to membranes of st-mPR-transfected cells (E) or pretreatment with 25 or 50 µM GTPS on specific [3H]-P4 binding to membranes of hu-mPR transfected cells (F). *, P < 0.05, n = 4.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Localization, orientation, and functional domains of the mPRs. A and B, Immunocytochemical localization of st-mPR (Aa) and hu-mPR (Ba) on external surface of plasma membranes of transfected cells with specific N-terminal directed antibodies. Ab and Bb, Blocked with peptide antigens; Ac and Bc, lack of immunoreaction with nontransfected cells; Bd, lack of immunoreaction with nonpermeabilized cell using C-terminal antibody. C and D, Flow cytometry of cells transfected with hu-mPR using N-terminal (C) and C-terminal (D) antibodies. C, 231, Background fluorescence on nontransfected cells in the presence mPR N-terminal antibody; Tr-231 Non-perm., immunodetection of the hu-mPR N-terminal on the surface of nonpermeabilized transfected cells by flow cytometry using the N-terminal antibody. D, Tr-231 Non-perm., fluorescence of nonpermeabilized transfected cells using the C-terminal antibody; Tr-231 Perm., immunodetection of the hu-mPR C-terminal in permeabilized transfected cells by flow cytometry using a C-terminal directed antibody. E, Specific binding of [3H]-P4 to subcellular fractions of cells transfected with hu-mPR. m, Plasma membranes; m-sp, plasma membranes further purified with a sucrose pad; ms, microsomes; nu, nuclear fraction; cyt, cytosolic fraction; #, dextran-coated charcoal used to separate bound from free. F and G, Mutational analysis of st-mPR functional domains. F, Effects of C-terminal truncation (Mu1) and amino acid substitution in the third intracellular loop (Mu3) on specific [35S]GTPS binding to cell membranes in response to 20ß-S treatment. G, Effects of C-terminal truncation (mutant 1) on specific [3H]-20ß-S binding to cell membranes.

View larger version (53K): [in this window] [in a new window]   FIG. 6. Schematic representation of the presence of PAQR family members 1–11 among the eukaryotes. The phylogram on the left depicts the evolutionary relationships between the different species used in this study. The table on the right shows the presence of a particular PAQR family member by a dot. A double dot indicates a duplication event. *, Missing data, incomplete genome sequence; ¶, gene duplication; ¥, possible redundancy; low-quality sequence shows divergence in phylogenetic tree. The results show that the mPR, mPRß, and mPR genes first appeared in the Euteleostomi.

View larger version (42K): [in this window] [in a new window]   FIG. 7. Schematic depiction of the parallel convergent evolution of the PAQR family and GPCR superfamily. The bacteriorhodopsins are strictly found in Archaebacteria and constitute the origin of the GPCR superfamily, whereas the HLY3 genes are strictly found in Eubacteria.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The high binding specificity of the recombinant st-mPR produced in the mammalian expression system for 20ß-S is consistent with its identity as the major progestin hormone regulating maturation of oocytes and motility of sperm in this species and with previous receptor binding results with seatrout ovarian and sperm membranes (22, 31). The lack of 20ß-S binding to the soluble recombinant st-mPR protein produced in Escherichia coli observed in our earlier study is probably due to protein modification deficiencies in this prokaryotic expression system (7). In general, however, the steroid binding characteristics of recombinant st-mPR and hu-mPR produced in MDA-MB-231 cell membranes are remarkably similar to those of the recombinant soluble receptors produced in the prokaryotic system (8). The more than 100-fold lower binding affinity of 20ß-S and the other teleost progestin, 17,20ß-P, for hu-mPR (RBAs <1%) is noteworthy and warrants investigation, because all the other steroids tested have similar affinities for the two mPRs. For example, the nuclear receptor agonist R5020 and antagonist RU486 have very low affinities for both mPRs, whereas testosterone, which shows low affinities for nuclear PRs, has RBAs of 10% and 22% for st-mPR and hu-mPR, respectively. The finding that the steroid specificities of the recombinant seatrout and human mPRs differ markedly from those of their nuclear progestin receptor counterparts was anticipated because there is no homology in any regions of the mPRs to the ligand binding domain of the nuclear progesterone receptor. Competitive binding assays with more than 30 steroidal compounds revealed different effects of substituting various functional groups on the progesterone nucleus on their binding affinities to hu-mPR compared with previously published results with human nPR (Refs. 33 and 34 and Table 1). For example, removal of carbon 19 (substitution of a methyl group for hydrogen: norprogesterone, also see R5020 and Organon 2058) decreases binding affinity to mPR (Table 1) but modestly increases it for the nPR (33, 34). However, removal of the side chain and addition of a hydroxyl at C17 (i.e. testosterone, dihydrotestosterone, nandrolone), which practically eliminated binding to the human nPR, only reduced binding with mPR to 22–7% that of P4 (33, 34). Finally, the hu-mPR also does not recognize C19 steroids (androgens) with a substitution of a hydrogen on C17 with an ethinyl group (i.e. ethisterone, norethisterone, and norgestrel), whereas these steroids have relatively high affinities for the nPR (33, 35). These marked differences in binding affinities suggest selective mPR modulators, in addition to nPR ones such as R5020, can be developed to independently explore progestin actions in target tissues mediated by each of these two receptor systems.

Identification of the ligand binding pocket by site-directed mutagenesis will be required for definitive proof that the mPRs directly bind progestins and are not merely components of a multiple-protein receptor complex. However, all the results obtained to date with the mPRs are consistent with our previous suggestion that they are true steroid membrane receptors (7). The finding that prokaryotic as well as eukaryotic expression systems produce recombinant st-mPRs and hu-mPRs with the binding characteristics of steroid membrane receptors suggests that the ability to bind progestins is an intrinsic property of these proteins and is not dependent on their association with other proteins present only in eukaryotic cells (7, 8). The demonstration that transfection of MDA cells with the various mPRs confers progestin binding specificity to the cell membrane preparations also suggests a direct role for these proteins in ligand binding. Membranes from cells transfected with st-mPR display a high binding affinity for 20ß-S, the principal progestin in this species, whereas those from hu-mPR-transfected cells have low affinity for this steroid but show highest affinity for progesterone, the major progestin hormone in mammals. In addition, MDA cells transfected with zebrafish mPR show the highest binding affinity for 17,20ß-P, the likely progestin hormone in this species (36). The characteristics of G protein activation and ligand binding to the mPRs on G protein uncoupling are also typical of seven-transmembrane hormone receptors. For example, dissociation of G proteins from GPCRs results in a decrease in ligand binding affinity of the receptors directly coupled to them. The finding that dissociation of the mPR-G protein complex by pretreatment with GTP-S and pertussis toxin causes a decline in progestin binding is further evidence that the progestin binding site is located on the mPR protein.

There is now evidence that representatives of all three mPR subtypes described in the original papers, , ß, and (7, 8), corresponding to PAQR types 7, 8, and 5 (17), display high-affinity, specific and limited-capacity binding to progestins characteristic of steroid membrane receptors. Sequence and phylogenetic analyses reveal that another PAQR type, PAQR 6, is closely related to the mPRs (supplemental Fig. 2) and therefore is a candidate as an additional member of this novel group of membrane progestin receptors. A notable feature of this progestin receptor PAQR subfamily (PAQR 5–9) is that representatives have only been identified in vertebrates, including teleosts and tetrapods, suggesting that they arose at the base of the vertebrate lineage. PAQR 9 could also possibly belong to this subgroup of progestin receptors because it is primarily found in the Chordata (Fig. 6) and is clustered with the mPRs (supplemental Fig. 3). In contrast, all other members of PAQR family, including the adiponectin (PAQR 1, 2) and MMD receptors (PAQR 10, 11), have both invertebrate and vertebrate representatives, indicating they have a more ancient evolutionary origin (Fig. 6). The high similarity of PAQR 10 and 11 with the HLY3 proteins in bacteria suggests that this is the archetypical PAQR in eukaryotes, although the phylogenetic analysis suggests that the ADR2 and PAQR 3 genes appeared first in prokaryotes. The present results with seatrout and human mPRs indicate that, at least for the PAQR 7 subtype, their ability to bind progestins arose early in vertebrate evolution, before the divergence of the teleost and tetrapod lineages, at least 200 million years ago (37). Members of this progestin PAQR subfamily have not been identified in the urochordate Ciona genome, which appear to lack many of the key steroidogenic enzymes (steroid dehydrogenases and cytochrome P450s) and enzymes that metabolize steroids (38, 39). The parallels between the evolution of the progestin membrane receptor subfamily, PAQR 5–8, and that of nuclear steroid receptors are striking. Duplication of nuclear steroid receptors from an ancestral estrogen receptor-like gene in vertebrates also occurred early in vertebrate evolution, coincident with the advent of the jawed vertebrates, to give rise to nuclear progesterone receptors and multiple estrogen receptors and later to give rise to androgen, glucocorticoid, and mineralocorticoid receptors (39, 40, 41). Thus, phylogenetic analyses indicate that functional mPRs probably arose around the same time as nuclear progestin receptors (nPRs), coincident with the appearance of critical steroidogenic enzymes and the production of significant quantities of progestins and other steroids in early vertebrates. The appearance of both mPRs and nPRs in early vertebrates likely reflects an intimate and complimentary relationship between the two receptor systems, both of which may be required for a coordinated or varied response of target cells in the reproductive system to progestin hormones. The first evidence of such an intimate relationship between these two progestin receptor signaling pathways has recently been obtained in human myometrial cells, where progesterone was shown to regulate transactivation of nPR and coactivator function through mPR- and mPRß-mediated pathways (26). Complex interactions between these two types of progesterone receptors are expected in some breast cancer cell lines (e.g., MCF-7), which show progesterone activation of nonclassical pathways through both mPRs and nPRs in addition to classical genomic responses through the nPR (4, 42). Information on the differing roles of mPRs and nPRs in nonclassical signaling in these cells may provide insights into why alternative signaling pathways mediated by two different classes of progesterone receptors evolved in vertebrates. Structural analyses of the nuclear steroid receptor ligand binding domains in a wide variety of chordates indicate that the ancestral receptor initially recognized the estrogens produced in vertebrate gonads associated with reproductive activity. It has been suggested that nPRs evolved subsequently to respond to progesterone, a steroid intermediate in estrogen production, by a process called ligand exploitation (40). A similar analytical approach may provide valuable insights into the evolution of mPRs from the ancestral PAQR once information on the structures of the ligand binding domains of mPRs and other members of the PAQR family becomes available.

The results of the present study also demonstrate that both the seatrout and human recombinant mPR proteins activate inhibitory G proteins (G_i), are coupled to them and share many other critical features of GPCRs. Clear evidence was obtained that mPRs, like GPCRs, are localized and function as seven-transmembrane receptors in the plasma membranes of cells. However, the mPRs have also been identified in other cellular compartments in fish oocytes and in certain eukaryotic expression systems (7, 15, 16). Moreover, the mPRs are similar to GPCRs in that they likely can occur as dimers as well a monomers (43), because an 80-kDa band is also frequently observed on Western blots, the intensity of which varies with the membrane solubilization conditions used (unpublished observation). G protein activation, detected with GPCRs by an increase in specific [³⁵S]GTP-S binding to the cell membranes (44), was also demonstrated after progestin treatment of mPR-transfected cells. The demonstration that progestins activate the inhibitory G protein, G_i, and down-regulate pertussis toxin-sensitive adenylyl cyclase activity in the plasma membranes of MDA-MB-231 cells transfected with receptors indicates the mPRs function as GPCRs. The activation of an inhibitory G protein by the mPRs is also consistent with our recent findings on the signaling pathways activated by 20ß-S during meiotic maturation of seatrout oocytes (25) as well as those activated by progesterone through the wild-type hu-mPR in human myometrial cells (26). A unique characteristic of GPCRs is that treatment with nonhydrolyzable forms of guanine nucleotides such as GTP-S, and for G_i/o-protein coupled receptors with pertussis toxin, cause uncoupling of G proteins from the receptors (45, 46), resulting in a decrease in their ligand binding affinities (47, 48). These treatments caused similar decreases in ligand binding to st-mPR and hu-mPR. Thus, these results further support the conclusions of the coimmunoprecipitation studies that the mPRs are directly coupled to G proteins and are not acting through intermediaries. The results of the mutational analyses indicate that two functional domains of GPCRs critical for G protein activation/coupling, the C-terminal domain and the third intracellular loop (49), are also important for activation of G proteins through st-mPR. The demonstration that the C-terminal of st-mPR is involved in activation of G proteins suggests it is intracellular in agreement with the model we proposed earlier for the orientation of the receptor in the plasma membrane (8). This orientation is also consistent with the results of the flow cytometry studies with cells transfected with hu-mPR and probed with N-terminal and C-terminal antibodies. Therefore, several lines of evidence suggest the mPRs have the same orientation in the plasma membrane as GPCRs.

Although the mPRs function by activating G proteins and have many characteristics of GPCRs, extensive phylogenetic analysis reveals that they are unrelated to members of the GPCR superfamily, in agreement with our previous conclusions (50), which were subsequently confirmed by Tang et al. (18). The mPRs belong to the large eukaryotic family of PAQRs, which in turn is related to the prokaryotic HLY3 family, suggesting they have a common bacterial ancestor. Members of the HLY3 family have only been identified in Eubacteria, consisting of Gram-positive and -negative bacteria, and are not present in Archaebacteria (18, 50). The results clearly indicate that the eukaryotic PAQR family descended from ancestral HLY3-like proteins in Eubacteria and therefore their bacterial origin differs from that of the GPCR superfamily, which descended from Bacteriorhodopsin, which is only found in the Archaebacteria (51, 52). This would explain the structural similarity but the low degree of sequence similarity between GPCRs and PAQRs. Activation of G proteins by PAQRs has only been demonstrated to date for mPR and mPRß (PAQR 7, 8) and predicted to be absent in adiponectin receptors (20), suggesting that this receptor function was acquired early in vertebrate evolution. However, additional studies on G protein activation with other PAQRs will be required to confirm this hypothesis. In conclusion, the mPRs are coupled to G proteins, activate them on ligand binding, and have many characteristics of GPCRs. However, the mPRs and members of the GPCR superfamily are unrelated, indicating that hormone signaling through seven-transmembrane receptors coupled to G proteins arose independently more than once during vertebrate evolution, for long thought to be a unique characteristic of the GPCR superfamily.

	Footnotes

 
This work was supported by Organon and National Institutes of Health Grants ESO4214 and ESO12961.

Author Disclosure Summary: All of the authors have nothing to disclose.

First Published Online November 9, 2006

Abbreviations: GPCR, G protein-coupled receptor; HLY3, hemolysin 3; hu-mPR, human membrane progestin receptor ; MMD, monocyte to macrophage differentiation protein; mPR, membrane progestin receptor; nPR, nuclear progestin receptor; st-mPR, spotted seatrout membrane progestin receptor ; PAQR, progesterone and adipoQ receptors; RBA, relative binding affinity.

Received July 20, 2006.

Accepted for publication October 26, 2006.

	References

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