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Marine Science Institute (P.T., Y.P., J.D.), University of Texas at Austin, Port Aransas, Texas 78373; and Department of Medicine (E.J.F.), Brown University School of Medicine, Providence, Rhode Island 02903
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.
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Introduction |
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and ERß, or ER-like proteins are likely candidates for the membrane ERs (mERs) mediating these estrogen actions in a variety of target cells, including endothelial, neuronal, and pituitary cells (7, 8, 9, 10, 11). However, evidence has also been obtained for the involvement of novel mERs unrelated to nuclear ERs (nERs) in nonclassical estrogen actions in several other cell types, many of which are associated with G proteins (12, 13, 14, 15, 16). Our recent discovery of a hitherto unknown family of membrane progesterone receptors (mPRs), unrelated to nuclear steroid receptors, but instead with characteristics of G protein-coupled receptor (GPCRs) (17, 18), prompted us to search for other GPCRs with characteristics of steroid membrane receptors. The orphan GPCR-like protein, GPR30, is widely distributed in neural, breast cancer, placental, heart, ovarian, prostate, hepatic, vascular epithelial, and lymphoid tissues and shows structural sequence homology to receptors for angiotensin, interleukin, and a variety of chemokines, suggesting it may be a peptide receptor (19, 20, 21, 22). However, a broad range of chemotactic peptides and angiotensins showed no binding affinity for GPR30 (20, 23). Instead, evidence was obtained for an involvement of GPR30 in estrogen-induced transactivation of epidermal growth factor receptor and adenylyl cyclase activity in SKBR3 breast cancer cells that lack nERs (24, 25, 26), suggesting GPR30 may be a novel mER. The present results demonstrate that expression of GPR30 in cells lacking ER and ERß is associated with the presence of high affinity, limited capacity, and specific 17ß-estradiol (E2) binding to their plasma membranes characteristic of mERs. Evidence is presented that GPR30 is directly coupled to a stimulatory G protein to up-regulate adenylyl cyclase activity and is a GPCR. |
Materials and Methods |
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-estradiol (E2), estrone (E1), estriol (E3), cortisol, testosterone, and progesterone (P4) and the synthetic estrogen diethylstilbestrol were purchased from Steraloids (Newport, RI). The antiestrogen tamoxifen (Tmx) and the fungal metabolite, zearalenone, were purchased from Sigma-Aldrich Corp. (St. Louis, MO). The ortho,para derivative of the pesticide ortho,para-dichlorodiphenyldichloroethylene, o,p'-DDE (DDE), was purchased from Chem Service (West Chester, PA). The synthetic antiestrogen ICI182,780 (ICI) was purchased from Tocris (Ellisville, MO). 17ß-[2,4,6,7-3H]estradiol ([3H]E2); approximately 89 Ci/mmol, was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All other chemicals, buffers, and media were purchased from Sigma-Aldrich unless noted otherwise.
Cell culture and transfections
Human SKBR3 cells and human embryonic kidney (HEK)293 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM/Ham’s F-12 medium without phenol red supplemented with 10% fetal bovine serum (FBS) and 100 µg/ml of gentamicin, with changes of medium every 1–2 d. SKBR3 cells were transiently transfected with GPR30 small interfering (si)RNA (100 nM), or nonspecific, presynthesized siRNA (control) using Lipofectamine 2000 (Life Technologies Inc., Gaithersburg, MD) at 25 C, following the manufacturer’s procedures (Dharmacon, Lafayette, CO) to interfere with GPR30 expression, and experiments were conducted 18 h later. HEK293 cells were transfected with a GPR30 construct, consisting of the full-length cDNA ligated into the pBK-CMV expression vector (25), using Lipofectamine 2000 and grown to confluence. Geneticin (500 µg/ml) was added and the geneticin-resistant cells containing the GPR30 construct were propagated to generate stable cell lines (selectively maintained with 500 µg/ml geneticin). Cells reached 80% confluence after 3 d in culture (
2 x 108 cells, 0.6 mg cell membrane protein/150 mm dish) and were replaced with fresh media containing 0 or 5% FBS 1 d before experiments.
Treatments
GPR30 expression and receptor binding were up-regulated in SKBR3 cells by incubating them for 16 h in FBS-free media with 200 nM P4 or E2 or media alone, followed by repeated washes with buffer before measurement of E2 binding. The effects of uncoupling G proteins on E2 binding affinity was investigated with membranes of transfected HEK293 cells pretreated with 0 or 25 µM GTP
-S at 25 C for 30 min. Cells were also incubated with 10 µg/ml activated cholera toxin [CTX; activated with 4 mM dithioerythritol (DTT)], inactive CTX (inactivated by boiling), or media alone for 30 min at 37 C immediately before preparation of the cell membrane for assay of E2 binding. Cells were collected with a cell scraper and washed twice with fresh media before preparation of plasma membranes. All experiments were repeated at least three times with different batches of cultured cells.
Membrane preparation and solubilization
Plasma membrane fractions of healthy human placental tissue (obtained from a live birth at Women and Infant’s Hospital, Providence, RI, following National Institutes of Health guidelines), and cells were obtained after homogenization and centrifugation procedures described previously (27, 28). Placental tissue plasma membranes were further purified by centrifuging the membrane pellet with a sucrose pad (1.2 M sucrose) at 6500 x g for 45 min (17, 29). Membranes were solubilized with 12 mM Triton X-100 in four volumes HEPES buffer (25 nM HEPES, 10 mM NaCl, 1 mM DTT) for 30 min, followed by removal of the detergent with polystyrene adsorbents (2:1 volume-weight; SM-2, Bio-Rad Laboratories, Hercules, CA) and subsequent removal of the adsorbents by filtration (G-8 filter, Fisher Scientific, Fair Lawn, NJ) before the addition of loading buffer for Western blot analyses (17).
ER binding assays
General procedures used in our laboratory for assaying saturation, association, and dissociation kinetics and steroid specificity of ligand binding to steroid membrane receptors (27, 28, 29) were used to measure [3H]E2 binding to plasma membrane preparations. For saturation analysis, one set of tubes contained a range (0.5–8.0 nM) of [3H]E2, 89 Ci/mmol) alone (total binding) and another set also contained 100-fold excess (50–800 nM) E2 competitor (nonspecific binding). For competitive binding assays, tubes contained 4 nM [3H]E2 and the steroid competitors (concentration range 1 nM to 100 µM; dissolved in 5 µl ethanol, 1% of the total volume, which does not affect [3H]E2 binding in the assay). After a 30-min incubation at 4 C with the membrane fractions, the reaction was stopped by filtration (GF/B filters, Whatman), the filters were washed and bound radioactivity measured by scintillation counting. The displacement of [3H]E2 binding by the steroid competitors was expressed as a percentage of the maximum specific binding of E2. Each assay point was run in triplicate, and the assays were repeated using different batches of cultured cells for each test chemical.
Western blot analysis
Solubilized membrane proteins were resolved by electrophoresis and Western blot analysis performed as described previously (17), using a GPR30 polyclonal antibody generated against a C-terminal 19-amino acid peptide fragment (24) (dilution 1:1000) in an overnight incubation. The membrane was 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 before incubation with the GPR30 antibody. The membrane was subsequently washed several times and then incubated for 1 h at room temperature with horseradish peroxidase conjugated to goat antirabbit antibody (Cell Signaling, Beverly, MA), and visualized by treatment with enhanced chemiluminescence substrate (SuperSignal, Pierce, Rockford, IL).
cAMP measurement
Plasma membranes (1.5 mg/ml) were incubated in buffer [20 mM KCl, 12 mM MgCl2, 3 mM EDTA, 2 mM ATP, 0.2 mM DTT, 10 mM creatine phosphate, 1 U creatine kinase, 1 U pyruvate kinase, and 20 mM HEPES (pH 7.5)] with or without 100 nM of the test compounds for 20 min at 25 C. A standard concentration of 100 nM was chosen for comparison of the effects of compounds with low binding affinities for the receptor, although E2 has previously been shown to be effective in SKBR3 cells at a much lower concentration, 1 nM (25). Activated CTX (10 µg/ml) was coincubated with 100 nM E2 in some experiments. The reaction was terminated by boiling the samples for 10 min. cAMP concentrations were measured in cytosolic fractions using an enzyme immunoassay kit following the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI).
[35S]GTP-S binding to cell membranes
Binding of [35S]GTP-S to plasma membranes (10 µg protein) was assayed following the procedure of Liu and Dillon (30) with few modifications. Plasma membranes were incubated with 10 µM GDP and 0.5 nM [35S]GTP-S (12,000 cpm, 1.0 Ci/mol) in 250 µl Tris buffer [100 nM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.6 mM EDTA, 0.1%BSA, and 50 mM Tris-HCl (pH 7.4)] at 25 C for 30 min in the presence of 100 nM of the test compounds. Nonspecific binding was determined by addition of 100 µM GTP-S. At the end of the incubation period, 100-µl aliquots were filtered through GF/B glass fiber filters (Whatman), followed by several washes and subsequent scintillation counting.
Immunoprecipitation of [35S]GTP-S-labeled G protein -subunits
Immunoprecipitation of the G protein -subunits coupled to [35S]GTP-S was performed as described elsewhere (30). Plasma membranes (20 µg protein) of transfected HEK293 cells were incubated with 1 µM E2 for 30 min at 25 C in 250 µl Tris buffer containing 4 nM [35S]GTP-S, 10 µM GDP, and protease inhibitor cocktail (Sigma-Aldrich). The incubation was stopped by addition of 750 µl ice-cold buffer containing 100 µM GDP and 100 µM unlabeled GTP-S. Samples were centrifuged at 20,000 x g for 15 min and the pellet resuspended in immunoprecipitation buffer containing 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 5 mM EDTA, 25 mM Tris-HCl (pH 7.4), and protease inhibitors. Specific antisera to the -subunits of G proteins (Gi, Gs, Sigma-Aldrich, dilution 1:500) were incubated with the samples for 6 h at 4 C. Protein A-Sepharose was added, and after an overnight incubation, the immunoprecipitates were collected by centrifugation (12,000 x g for 2 min) and washed in buffer (50 mM HEPES, 100 µM NaF, 50 mM sodium phosphate, 100 mM NaCl, 1% Triton X-100, and 1% SDS). The pellets were boiled in 0.5% SDS and the radioactivity in the immunoprecipitated [35S]GTP-S-labeled G protein -subunits counted.
RT-PCR of GPR30
Total RNA was extracted with Tri-reagent (Sigma-Aldrich). Reverse transcription was performed by adding 1–3 µg RNA to the 10-µl reaction mix containing 1x first-strand buffer (10 mM DTT, 0.5 mM of each deoxynucleotide triphosphate, 50 ng/µl oligo-dT primer, and 100 U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), and the mixture was incubated for 2 h at 42 C. The PCR was conducted in 30 µl PCR SuperMix (Invitrogen) consisting of 0.5 µl of the reverse transcription reaction and 0.2 µM of each of the primers. The following gene-specific primers for GPR30 were designed according to the GPR30 sequence from GenBank (accession no. BC011634): 1) sense, 5'-GGC TTT GTG GGC AAC ATC-3'; antisense, 5'-CGG AAA GAC TGC TTG CAG G-3'; 2) sense, 5'-TGG TGG TGA ACA TCA GCT TC-3'; antisense, 5'-TGA GCT TGT CCC TGA AGG TC-3'; and 3) sense, 5'-GCA GCG TCT TCT TCC TCA CC-3'; antisense, 5'-ACA GCC TGA GCT TGT CCC TG-3'. After an initial denaturation for 5 min at 94 C, the PCR was performed on the Eppendorf Mastercycler for 35 cycles with the cycling profile of 30 sec at 94 C, 30 sec at 55 C, and 2 min at 72 C followed by a 10-min extension at 72 C. The PCR (5 µl) was electrophoresed on an agarose gel (1%) containing ethidium bromide to visualize the products. For semiquantitative RT-PCR, 25 cycles of PCR were performed (linear portion of cycle/product curve).
Statistics
Linear and nonlinear regression analyses for all receptor binding assays and calculations of dissociation constant (Kd) and binding capacity were performed using GraphPad Prism for Windows (version 3.02; GraphPad Software, San Diego, CA). Student’s paired t test was used for paired comparisons and one-way ANOVA and Tukey tests for multiple comparisons (Sigma Stat, SPSS, Chicago, IL).
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). The binding was displaceable and the kinetics of association and dissociation of binding were rapid, with halftimes (t1/2) of 5.5 and 8.1 min, respectively (Fig. 1B). Competitive binding assays showed that steroid binding was specific for E2. P4, cortisol, and testosterone had no affinity for the receptor at concentrations up to 1 µM (Fig. 1C). As observed previously for some nERs and other estrogen membrane receptors, the inactive estradiol isomer, E2, failed to significantly displace E2 binding (Fig. 1D). An unexpected finding was that the other natural estrogens, E3 and E1, had very low affinities for the receptor, less than 0.1% that of E2. In contrast, ICI and Tmx were effective competitors, with relative binding affinities (RBAs) approximately 10% that of E2. Interestingly, comparatively low concentrations (0.1–1 µM) of the xenoestrogen o,p'-DDE also caused significant displacement of [3H]E2 binding. Treatment of the cells with GPR30 siRNA caused decreased expression of GPR30 mRNA that was accompanied by an 80% decrease in specific [3H]E2 membrane binding (Fig. 1E). A major immunoreactive band was detected in plasma membranes of SKBR3 cells by Western blotting, and GPR30 mRNA was detected in SKBR3 cells by RT-PCR (Fig. 1F). Immunocytochemical analysis of whole SKBR3 cells demonstrates that GPR30 protein is concentrated at the cell periphery, consistent with its function as a membrane receptor (see Supplemental Fig. 1B and the supporting text, published on The Endocrine Society’s Journals online web site at http://endo.endojournals.org). The absence of ER and ERß mRNA in SKBR3 cells was confirmed by PCR using three sets of specific primers for each receptor (see Supplemental Fig. 1A and supporting text).
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) or ER or ERß mRNA (see Supplemental Fig. 2 and supporting text) as shown by RT-PCR and Western blot analysis. Therefore, we investigated whether they acquire the ability to specifically bind E2 upon transfection with a cDNA encoding GPR30. Expression levels of GPR30 mRNA and protein in the transfected HEK293 cells and membranes (Fig. 2A) were similar to those in SKBR3 cells (data not shown). Significant amounts of specific estrogen binding were detected in plasma membranes of cells transfected with GPR30, whereas negligible specific binding was detected in the plasma membranes of untransfected cells (Fig. 2B). Saturation analysis showed high-affinity (Kd 3.3 nM), saturable (Bmax 100 pM) specific [3H]E2 binding, and the Scatchard plot indicated a single binding site (Fig. 2C). The kinetics of association/dissociation of [3H]E2 binding to the recombinant protein produced in HEK393 cells were rapid with halftime (t
sec) of 1.3 and 4.9 min (Fig. 2D). Competitive binding studies showed that binding was specific for E2 and certain estrogens. E1, E3, diethylstilbestrol, and nonestrogenic compounds failed to significantly displace [3H]E2 at concentrations up to 10 µM, whereas Tmx, ICI, o,p'-DDE, and the mycotoxin estrogenic compound, zearalenone, displayed significant binding affinity (Fig. 2, E and F). The binding affinities of most of the compounds for the recombinant GPR30 protein in plasma membranes from transfected HEK293 cells were similar to their affinities for the wild-type GPR30, with the exception of the antiestrogens ICI and Tmx, which displayed somewhat lower binding affinities. Immunocytochemical analysis showed that GPR30 was detected only on the plasma membranes of transfected whole cells (see Supplemental Fig. 2 and supporting text).
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-S binding to membranes from HEK293 cells transfected with GPR30 but not to mem-branes of untransfected cells (Fig. 3A), whereas E2 treatment was ineffective (Thomas, P., Y. Pang, and J. Dong, unpublished observations). Immunoprecipitation of the membrane-bound [35S]GTP-S with specific G protein -subunit antibodies showed that the majority of the GTP-S is bound to the s-subunit on E2 treatment (Fig. 3B), which suggests the receptor activates a stimulatory G protein (Gs). Treatment with 100 nM E2 caused a similar increase in specific [35S]GTP-S binding to membranes of SKBR3 cells expressing wild-type GPR30, whereas E1 and E3, which display low binding affinities for the mER in this cell line, failed to activate the G proteins (Fig. 3C). Two other compounds with higher RBAs for the mER, Tmx and DDE, also significantly increased [35S]GTP-S binding to SKBR3 cell membranes, suggesting they mimic the actions of E2 on this nonclassical signaling pathway. Adenylyl cyclase activity, measured as an increase in cAMP content, was significantly increased in transfected HEK293 cells after 15 min treatment with 100 nM E2 and ICI but not in untransfected cells (Fig. 3D), in agreement with previous findings in SKBR3 cells (26). Moreover, the estrogen-induced increase in cAMP concentrations was blocked by prior treatment with activated CTX (Fig. 3E), which is consistent with coupling of GPR30 to a stimulatory G protein and activation of this pathway. E1 and E3 did not alter cAMP production, but other compounds with higher RBAs for the mER in transfected cells, Tmx, ICI, and o,p'-DDE significantly increased cAMP (Fig. 3F).
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-S (Fig. 4, A and B), which causes uncoupling of G proteins from their receptors. Plasma membranes of SKBR3 cells cultured for 2 d in FBS-free media had low amounts of specific E2 binding. Pretreatment of the cells with 200 nM P4 or E2 for 16 h in FBS-free media caused dramatic 9- and 2.5-fold increases in receptor binding, respectively, which paralleled the increases in GPR30 mRNA and protein expression (Fig. 4, C and D). The hormonal treatments did not alter ER and ERß mRNA expression levels, which remained undetectable by RT-PCR analysis. However, a transcript for the nuclear P4 receptor (nPR-A) was detected by RT-PCR (see Supplemental Fig. 3
and supporting text). Saturation analysis and Scatchard plotting also showed the presence of a high-affinity (Kd 6.3 nM) limited capacity (Bmax 1.4 nM) single E2 binding site on human placental plasma membranes (Fig. 4E). Immunocytochemistry and Western blots showed specific GPR30 staining of human placenta synctiotrophoblast and other cell types and a 38-kDa protein band, respectively (Fig. 4F).
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Discussion |
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and ERß mRNAs and proteins (Supplemental Fig. 1) (24, 32, 33, 34, 35) but expressing GPR30 (24) show high-affinity, limited capacity, displaceable, specific binding to E2 suggests the presence of a previously unknown ER in these cells. The orphan GPCR-like protein, GPR30, is a candidate for a novel ER because it is expressed and is a requisite signaling intermediary in estrogen-dependent activation of adenylyl cyclase and epidermal growth factor receptor (EGFR) in SKBR3 in breast cancer cells that lack known ERs (Supplemental Fig. 1) (24, 25, 26). Evidence in support of this hypothesis was obtained from experiments showing that both P4-induced increases and siRNA-induced decreases in GPR30 expression in SKBR3 cells were accompanied by parallel changes in specific E2 binding. Moreover, the observation that a tissue that expresses GPR30, human placenta (19), also shows similar high-affinity E2 binding in its plasma membranes suggests GPR30 is a functional mER in human tissues. Direct evidence that GPR30 binds estrogens and has the signal transduction characteristics of a mER was obtained from the studies with transfected HEK293 cells, which lack both nuclear ER and ERß (Supplemental Fig. 2) (36). Untransfected HEK293 cells showed negligible E2 binding and E2 activation of G proteins in their membrane fractions. However, plasma membranes of cells transfected with GPR30 displayed specific E2 binding almost identical with that of the SKBR3 cells and characteristic of mERs identified previously (20, 21, 22, 23, 24, 25).
The steroid binding characteristics of the recombinant GPR30, like those of the wild-type receptor, fulfill all the criteria for its designation as a mER. Both forms of GPR30 display high affinity and saturable E2 binding with Kds of approximately 3.0 nM, similar to the affinities of other mERs (28). E2 consistently occupies a single binding site in cell and tissue membrane preparations as shown in the Scatchard plots. Moreover, E2 readily dissociates from the binding site, a critical feature of steroid receptors. The kinetics of association and dissociation were rapid, with t1/2 less than 10 min, which is characteristic of membrane steroid receptors (27, 29). In addition, it was demonstrated that GPR30 can act as a mER in transfected cells to transduce the signals of estrogenic compounds with high RBAs for the receptor, resulting in activation of a stimulatory G protein and up-regulation of adenylyl cyclase activity, whereas E3 and E1, which have low RBAs for the receptor, were inactive. Finally, the decrease in mER binding in transfected cell membranes observed after treatment with agents causing uncoupling of G proteins from GPCRs, GTP-S, and CTX (30, 37), indicates the mER is directly coupled to G protein and is a GPCR, consistent with its identity as GPR30. To our knowledge this is the first report of a protein structurally unrelated to nuclear ERs that has the characteristics of an ER. The discovery of this novel mER provides a plausible mechanism by which estrogens can initiate rapid steroid actions at the cell surface and act in certain nER-negative target cells. The existence of this mER-mediated signaling pathway also explains some of the pleiotropic actions of estrogens in breast and other estrogen target tissues.
The identification of a novel mER that activates a Gs indicates that estrogen and antiestrogen signaling in human breast cancer is more complex than previously recognized. Several characteristics of the receptor have important implications for the development of the disease. Estrogens can activate pathways involved in proliferative responses, such as MAPK via EGFR transactivation and c-fos expression, in nER-negative breast cancer cells via GPR30 (24, 38). Recent studies show that Gs-coupled GPCRs, in addition to Gq protein-coupled ones, can stimulate EGFR transactivation (39). GPR30 transactivates the EGFR by release of heparan-bound epidermal growth factor from the cell surface by a Gß-Src-Shc signaling pathway (26). Gs-coupled receptors can signal to Src and Shc via ß-arrestin scaffolds (40), and this could provide an alternative mechanism by which they transactivate EGFR. Estrogen also attenuates the EGFR-to-ERK signaling axis by cAMP-dependent signaling (25) via GPR30, indicating an additional role of this novel receptor in regulating EGF action. Interestingly, GPR30 is abundantly expressed in human primary breast carcinomas and breast cancer cells lines that are nER positive but shows no or minimal expression in ER-negative breast cancer tissues and cells (19).
The observation that GPR30 is up-regulated by P4 confirms the results of a previous study (41) and raises the possibility of coordinate hormonal control by P4 of GPR30 and the nERs in cells expressing both receptors. The nuclear P4 receptor is a potential mediator of the P4 up-regulation of GPR30 in SKBR3 cells, whereas estrogen is presumably acting via a nER-independent mechanism, possibly through GPR30 and activation of adenylyl cyclase (42), although additional experiments will be required to confirm this. Environmental contaminants that are weak nER agonists (xenoestrogens), such as polychlorinated biphenyls (PCBs) and the DDT derivative, o,p'-DDE, have also been implicated in tumorigenesis in breast and other estrogen target tissues, presumably via nER activation (43, 44). The finding that o,p'-DDE is an agonist for GPR30 receptor activity demonstrates that xenoestrogens can also activate this alternative estrogen signaling pathway in breast cancer cells, as has been shown for mERs in other tissues (14, 27). Interestingly, the RBA of o,p'-DDE binding to GPR30 and nERs are similar (45), possibly indicating a similar susceptibility of these two estrogen signaling pathways to interference by this xenoestrogen. Interference with nontraditional steroid actions by xenoestrogen binding to steroid membrane receptors has previously demonstrated for the mPR on fish gametes (46). The present results extend this novel mechanism of endocrine disruption to a second GPCR-like steroid receptor and suggest that interactions of xenoestrogens with ligand binding sites is a shared feature of both nuclear and GPCR-like steroid receptors.
The results also have profound implications for the treatment of breast cancer. Patients treated for ER-positive breast cancers are frequently administered the antiestrogen Tmx to prevent reoccurrence of tumor growth (47). However, our results show that Tmx and the nER antagonist ICI have opposite actions on the alternative mER-mediated pathway, acting as estrogen agonists by binding to GPR30 and activating G proteins. The agonist activity of the pure ER antagonist ICI was expected because we found it has a high RBA for GPR30 and it has previously been shown to mimic estrogen actions initiated at the cell membrane in a broad range of targets, including SKBR3 cells (25, 28). The identification of GPR30 as a mER facilitates investigations on its role in the physiology and pathology of breast, prostate, placenta, ovarian, neural, and vascular tissues and also provides a potential target for therapeutic intervention.
The discovery of a second class of ERs unrelated to nERs provides an entirely new model to explore the structural requirements for estrogen binding and activation of receptor proteins. The marked differences in the RBAs of some estrogens to GPR30 and their affinities to ER and ERß and a third distinct ER subtype in fishes was expected, considering the lack of structural similarity between GPR30 and the nERs (48, 49). Initial binding studies with a limited number of nER ligands suggests GPR30 has a higher specificity for E2 binding than the nERs; all the other estrogens tested had RBAs of 10% or lower for the membrane receptor. Interestingly, the presence of other functional groups on the D ring of the steroid molecule in the vicinity of the 17 position or alteration of the 17ß-OH configuration dramatically decreases binding to GPR30, E1, E3, and E2 having RBAs less than 1% that of E2, whereas these changes result in relatively modest decreases in binding affinity to the nERs (48, 49). In contrast, alteration of the four carbon ring structure to produce Tmx or addition of a large side chain at the 7 position to produce ICI caused only minor decreases in RBA to GPR30, similar to that observed with some nERs (48, 49).
Although the identities of mERs remain uncertain and a topic of intense debate, there is a growing body of evidence indicating a role for nER or ER-like proteins in many tissues showing rapid, cell surface-mediated estrogen actions (7, 8, 9, 10, 11, 12). The absence of GPR30 in several well-characterized cell models of rapid, nongenomic estrogen actions, sheep endothelial cells and rat pituitary and hypothalamic cells (Supplemental Fig. 3) (8, 9, 31), suggests that not all these estrogen actions are mediated via GPR30 and that at least two classes of mERs are present in vertebrates. The physiological significance of the presence of both types of mERs in certain cell types, such as MCF-7 cells is unclear (38, 50). However, the Kd of E2 binding to membranes of SKBR3 and HEK293 cells expressing GPR30 in the present study ranged from 2.7 to 3.3 nM, 10-fold higher than that reported for membranes of Chinese hamster ovary cells transfected with ER (7) and may be indicative of a higher threshold concentration for activation of GPR30-dependent signaling pathways by estrogens.
GPR30 and the mPRs we discovered previously represent two distinct classes of GPCRs with no sequence homology and few apparent structural similarities. Thus, there is no indication that these mERs and mPRs arose from a common ancestor, unlike members of the nuclear steroid receptor superfamily (51). The C-terminal domain of GPR30 is longer than that of the mPRs (47 vs. 12 amino acids), the DRY sequence involved in signal transduction in intracellular loop 2 is absent in the mPRs, whereas the length of the second extracellular loop in GPR30 (10–20 amino acids) is shorter than that of the mPRs (50 amino acids). On the other hand, both receptors have seven-transmembrane domains, N-terminal glycosylation sites, and two conserved cysteines in the first two extracellular loops, which can form disulfide bonds to help stabilize the structure, basic features of GPCRs (19, 20). In addition, both receptors have large N-terminal extracellular domains, 57–75 amino acids long, that could possibly be involved in ligand binding. The discovery of two apparently unrelated families of GPCR-like membrane steroid receptors raises interesting evolutionary questions regarding their origins, such as whether the ancestral proteins were receptors for nonsteroidal ligands that subsequently acquired new functions (neofunctionalization) to bind and transduce specific steroid signals and, if so, whether the receptors have retained their responses to these nonsteroidal ligands. Information on the tissue distribution, regulation, and ligand specificity of these receptors should provide insights into the evolution and functions of this new class of steroid receptors.
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Footnotes |
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First Published Online November 11, 2004
Abbreviations: Bmax, Maximal binding capacity; CTX, cholera toxin; DDE, o,p'-DDE; DTT, dithioerythritol; E1, estrone; E2, 17ß-estradiol; E2, 17-estradiol; E3, estriol; EGFR, epidermal growth factor receptor; ER, estrogen receptor; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; GPR30, orphan GPCR-like protein; Gs, stimulatory G protein; HEK, human embryonic kidney; ICI, ICI182,780; Kd, dissociation constant; mER, membrane estrogen receptor; mPR, membrane progestin receptor; nER, nuclear ER; P4, progesterone; RBA, relative binding affinity; SDS, sodium dodecyl sulfate; si, small interfering; t1/2, halftime; Tmx, tamoxifen.
Received August 16, 2004.
Accepted for publication November 4, 2004.
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References |
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 G. G J Hazell, S. T Yao, J. A Roper, E. R Prossnitz, A.-M. O'Carroll, and S. J Lolait Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues J. Endocrinol., August 1, 2009; 202(2): 223 - 236. [Abstract] [Full Text] [PDF]
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 S. H. Lindsey, J. A. Cohen, K. B. Brosnihan, P. E. Gallagher, and M. C. Chappell Chronic Treatment with the G Protein-Coupled Receptor 30 Agonist G-1 Decreases Blood Pressure in Ovariectomized mRen2.Lewis Rats Endocrinology, August 1, 2009; 150(8): 3753 - 3758. [Abstract] [Full Text] [PDF]
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 E. Swedenborg, J. Ruegg, S. Makela, and I. Pongratz Endocrine disruptive chemicals: mechanisms of action and involvement in metabolic disorders J. Mol. Endocrinol., July 1, 2009; 43(1): 1 - 10. [Abstract] [Full Text] [PDF]
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N. Tagawa, R. Yuda, S. Kubota, M. Wakabayashi, Y. Yamaguchi, D. Kiyonaga, N. Mori, E. Minamitani, H. Masuzaki, and Y. Kobayashi 17{beta}-Estradiol inhibits 11{beta}-hydroxysteroid dehydrogenase type 1 activity in rodent adipocytes J. Endocrinol., July 1, 2009; 202(1): 131 - 139. [Abstract] [Full Text] [PDF]
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J. A. Quinn, C. T. Graeber, A. R. Frackelton Jr., M. Kim, J. E. Schwarzbauer, and E. J. Filardo Coordinate Regulation of Estrogen-Mediated Fibronectin Matrix Assembly and Epidermal Growth Factor Receptor Transactivation by the G Protein-Coupled Receptor, GPR30 Mol. Endocrinol., July 1, 2009; 23(7): 1052 - 1064. [Abstract] [Full Text] [PDF]
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 B. C. Lin, M. Suzawa, R. D. Blind, S. C. Tobias, S. E. Bulun, T. S. Scanlan, and H. A. Ingraham Stimulating the GPR30 Estrogen Receptor with a Novel Tamoxifen Analogue Activates SF-1 and Promotes Endometrial Cell Proliferation Cancer Res., July 1, 2009; 69(13): 5415 - 5423. [Abstract] [Full Text] [PDF]
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 C. Ohlsson and L. Vandenput The role of estrogens for male bone health Eur. J. Endocrinol., June 1, 2009; 160(6): 883 - 889. [Abstract] [Full Text] [PDF]
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 Y. Zhang, H. Zhao, S. Asztalos, M. Chisamore, Y. Sitabkhan, and D. A. Tonetti Estradiol-Induced Regression in T47D:A18/PKC{alpha} Tumors Requires the Estrogen Receptor and Interaction with the Extracellular Matrix Mol. Cancer Res., April 1, 2009; 7(4): 498 - 510. [Abstract] [Full Text] [PDF]
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J. Isensee, L. Meoli, V. Zazzu, C. Nabzdyk, H. Witt, D. Soewarto, K. Effertz, H. Fuchs, V. Gailus-Durner, D. Busch, et al. Expression Pattern of G Protein-Coupled Receptor 30 in LacZ Reporter Mice Endocrinology, April 1, 2009; 150(4): 1722 - 1730. [Abstract] [Full Text] [PDF]
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S. D. Noel, K. L. Keen, D. I. Baumann, E. J. Filardo, and E. Terasawa Involvement of G Protein-Coupled Receptor 30 (GPR30) in Rapid Action of Estrogen in Primate LHRH Neurons Mol. Endocrinol., March 1, 2009; 23(3): 349 - 359. [Abstract] [Full Text] [PDF]
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J. Kuo, O. R. Hariri, G. Bondar, J. Ogi, and P. Micevych Membrane Estrogen Receptor-{alpha} Interacts with Metabotropic Glutamate Receptor Type 1a to Mobilize Intracellular Calcium in Hypothalamic Astrocytes Endocrinology, March 1, 2009; 150(3): 1369 - 1376. [Abstract] [Full Text] [PDF]
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 S. H. Windahl, N. Andersson, A. S. Chagin, U. E. A. Martensson, H. Carlsten, B. Olde, C. Swanson, S. Moverare-Skrtic, L. Savendahl, M. K. Lagerquist, et al. The role of the G protein-coupled receptor GPR30 in the effects of estrogen in ovariectomized mice Am J Physiol Endocrinol Metab, March 1, 2009; 296(3): E490 - E496. [Abstract] [Full Text] [PDF]
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U. E. A. Martensson, S. A. Salehi, S. Windahl, M. F. Gomez, K. Sward, J. Daszkiewicz-Nilsson, A. Wendt, N. Andersson, P. Hellstrand, P.-O. Grande, et al. Deletion of the G Protein-Coupled Receptor 30 Impairs Glucose Tolerance, Reduces Bone Growth, Increases Blood Pressure, and Eliminates Estradiol-Stimulated Insulin Release in Female Mice Endocrinology, February 1, 2009; 150(2): 687 - 698. [Abstract] [Full Text] [PDF]
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S. P. Yun, M. Y. Lee, J. M. Ryu, C. H. Song, and H. J. Han Role of HIF-1{alpha} and VEGF in human mesenchymal stem cell proliferation by 17{beta}-estradiol: involvement of PKC, PI3K/Akt, and MAPKs Am J Physiol Cell Physiol, February 1, 2009; 296(2): C317 - C326. [Abstract] [Full Text] [PDF]
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 B. Kleuser, D. Malek, R. Gust, H. H. Pertz, and H. Potteck 17-{beta}-Estradiol Inhibits Transforming Growth Factor-{beta} Signaling and Function in Breast Cancer Cells via Activation of Extracellular Signal-Regulated Kinase through the G Protein-Coupled Receptor 30 Mol. Pharmacol., December 1, 2008; 74(6): 1533 - 1543. [Abstract] [Full Text] [PDF]
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 S. M. Carroll, J. T. Bridgham, and J. W. Thornton Evolution of Hormone Signaling in Elasmobranchs by Exploitation of Promiscuous Receptors Mol. Biol. Evol., December 1, 2008; 25(12): 2643 - 2652. [Abstract] [Full Text] [PDF]
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S. Xu, Y. Cheng, J. R. Keast, and P. B. Osborne 17{beta}-Estradiol Activates Estrogen Receptor {beta}-Signalling and Inhibits Transient Receptor Potential Vanilloid Receptor 1 Activation by Capsaicin in Adult Rat Nociceptor Neurons Endocrinology, November 1, 2008; 149(11): 5540 - 5548. [Abstract] [Full Text] [PDF]
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 Y. Gui, X.-L. Zheng, J. Zheng, and M. P. Walsh Inhibition of rat aortic smooth muscle contraction by 2-methoxyestradiol Am J Physiol Heart Circ Physiol, November 1, 2008; 295(5): H1935 - H1942. [Abstract] [Full Text] [PDF]
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R. Sirianni, A. Chimento, C. Ruggiero, A. De Luca, R. Lappano, S. Ando, M. Maggiolini, and V. Pezzi The Novel Estrogen Receptor, G Protein-Coupled Receptor 30, Mediates the Proliferative Effects Induced by 17{beta}-Estradiol on Mouse Spermatogonial GC-1 Cell Line Endocrinology, October 1, 2008; 149(10): 5043 - 5051. [Abstract] [Full Text] [PDF]
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 S. N. Sarkar, R.-Q. Huang, S. M. Logan, K. D. Yi, G. H. Dillon, and J. W. Simpkins Estrogens directly potentiate neuronal L-type Ca2+ channels PNAS, September 30, 2008; 105(39): 15148 - 15153. [Abstract] [Full Text] [PDF]
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C. Wang, E. R. Prossnitz, and S. K. Roy G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary Endocrinology, September 1, 2008; 149(9): 4452 - 4461. [Abstract] [Full Text] [PDF]
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J. Teng, Z.-Y. Wang, E. R. Prossnitz, and D. E. Bjorling The G Protein-Coupled Receptor GPR30 Inhibits Human Urothelial Cell Proliferation Endocrinology, August 1, 2008; 149(8): 4024 - 4034. [Abstract] [Full Text] [PDF]
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L. Albanito, D. Sisci, S. Aquila, E. Brunelli, A. Vivacqua, A. Madeo, R. Lappano, D. P. Pandey, D. Picard, L. Mauro, et al. Epidermal Growth Factor Induces G Protein-Coupled Receptor 30 Expression in Estrogen Receptor-Negative Breast Cancer Cells Endocrinology, August 1, 2008; 149(8): 3799 - 3808. [Abstract] [Full Text] [PDF]
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Y. Pang, J. Dong, and P. Thomas Estrogen Signaling Characteristics of Atlantic Croaker G Protein-Coupled Receptor 30 (GPR30) and Evidence It Is Involved in Maintenance of Oocyte Meiotic Arrest Endocrinology, July 1, 2008; 149(7): 3410 - 3426. [Abstract] [Full Text] [PDF]
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 R. Raju and I. H. Chaudry Sex Steroids/Receptor Antagonist: Their Use as Adjuncts After Trauma-Hemorrhage for Improving Immune/Cardiovascular Responses and for Decreasing Mortality from Subsequent Sepsis Anesth. Analg., July 1, 2008; 107(1): 159 - 166. [Abstract] [Full Text] [PDF]
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 V. M. Miller and S. P. Duckles Vascular Actions of Estrogens: Functional Implications Pharmacol. Rev., June 1, 2008; 60(2): 210 - 241. [Abstract] [Full Text] [PDF]
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T. J Heino, A. S Chagin, and L. Savendahl The novel estrogen receptor G-protein-coupled receptor 30 is expressed in human bone J. Endocrinol., May 1, 2008; 197(2): R1 - R6. [Abstract] [Full Text] [PDF]
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H. Abe, K. L. Keen, and E. Terasawa Rapid Action of Estrogens on Intracellular Calcium Oscillations in Primate Luteinizing Hormone-Releasing Hormone-1 Neurons Endocrinology, March 1, 2008; 149(3): 1155 - 1162. [Abstract] [Full Text] [PDF]
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A. Bouskine, M. Nebout, B. Mograbi, F. Brucker-Davis, C. Roger, and P. Fenichel Estrogens Promote Human Testicular Germ Cell Cancer through a Membrane-Mediated Activation of Extracellular Regulated Kinase and Protein Kinase A Endocrinology, February 1, 2008; 149(2): 565 - 573. [Abstract] [Full Text] [PDF]
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 V. Bourdeau, J. Deschenes, D. Laperriere, M. Aid, J. H. White, and S. Mader Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells Nucleic Acids Res., January 17, 2008; 36(1): 76 - 93. [Abstract] [Full Text] [PDF]
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C Dosiou, A E Hamilton, Y Pang, M T Overgaard, S Tulac, J Dong, P Thomas, and L C Giudice Expression of membrane progesterone receptors on human T lymphocytes and Jurkat cells and activation of G-proteins by progesterone J. Endocrinol., January 1, 2008; 196(1): 67 - 77. [Abstract] [Full Text] [PDF]
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 M. M. McCARTHY Estradiol and the Developing Brain Physiol Rev, January 1, 2008; 88(1): 91 - 134. [Abstract] [Full Text] [PDF]
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 A. S. Chagin and L. Savendahl GPR30 Estrogen Receptor Expression in the Growth Plate Declines as Puberty Progresses J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4873 - 4877. [Abstract] [Full Text] [PDF]
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H. Sakamoto, K.-i. Matsuda, K. Hosokawa, M. Nishi, J. F. Morris, E. R. Prossnitz, and M. Kawata Expression of G Protein-Coupled Receptor-30, a G Protein-Coupled Membrane Estrogen Receptor, in Oxytocin Neurons of the Rat Paraventricular and Supraoptic Nuclei Endocrinology, December 1, 2007; 148(12): 5842 - 5850. [Abstract] [Full Text] [PDF]
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 K. S. Monkkonen, R. Aflatoonian, K.-F. Lee, W. S.B. Yeung, S.-W. Tsao, J. T. Laitinen, and A. Fazeli Hormonal regulation of G{alpha}i2 and mPR{alpha} in immortalized human oviductal cell line OE-E6/E7 Mol. Hum. Reprod., December 1, 2007; 13(12): 845 - 851. [Abstract] [Full Text] [PDF]
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M. Liu, N. Yanagihara, Y. Toyohira, M. Tsutsui, S. Ueno, and Y. Shinohara Dual Effects of Daidzein, a Soy Isoflavone, on Catecholamine Synthesis and Secretion in Cultured Bovine Adrenal Medullary Cells Endocrinology, November 1, 2007; 148(11): 5348 - 5354. [Abstract] [Full Text] [PDF]
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C. Wang, E. R. Prossnitz, and S. K. Roy Expression of G Protein-Coupled Receptor 30 in the Hamster Ovary: Differential Regulation by Gonadotropins and Steroid Hormones Endocrinology, October 1, 2007; 148(10): 4853 - 4864. [Abstract] [Full Text] [PDF]
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 R. J. Pietras and D. C. Marquez-Garban Membrane-Associated Estrogen Receptor Signaling Pathways in Human Cancers Clin. Cancer Res., August 15, 2007; 13(16): 4672 - 4676. [Full Text] [PDF]
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 H. Ish, T. Tsurugizawa, M. Ogiue-Ikeda, M. Asashima, H. Mukai, G. Murakami, Y. Hojo, T. Kimoto, and S. Kawato Local Production of Sex Hormones and Their Modulation of Hippocampal Synaptic Plasticity Neuroscientist, August 1, 2007; 13(4): 323 - 334. [Abstract] [PDF]
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 M. Barton, M. R. Meyer, and E. Haas Hormone Replacement Therapy and Atherosclerosis in Postmenopausal Women: Does Aging Limit Therapeutic Benefits? Arterioscler Thromb Vasc Biol, August 1, 2007; 27(8): 1669 - 1672. [Full Text] [PDF]
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A. Morales, M. Gonzalez, R. Marin, M. Diaz, and R. Alonso Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells J. Endocrinol., July 1, 2007; 194(1): 193 - 200. [Abstract] [Full Text] [PDF]
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E. Filardo, J. Quinn, Y. Pang, C. Graeber, S. Shaw, J. Dong, and P. Thomas Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane Endocrinology, July 1, 2007; 148(7): 3236 - 3245. [Abstract] [Full Text] [PDF]
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 E. E. Connor, M. J. Meyer, R. W. Li, M. E. Van Amburgh, Y. R. Boisclair, and A. V. Capuco Regulation of Gene Expression in the Bovine Mammary Gland by Ovarian Steroids J Dairy Sci, June 1, 2007; 90(13_suppl): E55 - E65. [Abstract] [Full Text] [PDF]
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 E. Haas, M. R. Meyer, U. Schurr, I. Bhattacharya, R. Minotti, H. H. Nguyen, A. Heigl, M. Lachat, M. Genoni, and M. Barton Differential Effects of 17{beta}-Estradiol on Function and Expression of Estrogen Receptor {alpha}, Estrogen Receptor {beta}, and GPR30 in Arteries and Veins of Patients With Atherosclerosis Hypertension, June 1, 2007; 49(6): 1358 - 1363. [Abstract] [Full Text] [PDF]
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E. Brailoiu, S. L Dun, G C. Brailoiu, K. Mizuo, L. A Sklar, T. I Oprea, E. R Prossnitz, and N. J Dun Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system J. Endocrinol., May 1, 2007; 193(2): 311 - 321. [Abstract] [Full Text] [PDF]
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 Y.-C. Hsieh, H.-P. Yu, M. Frink, T. Suzuki, M. A. Choudhry, M. G. Schwacha, and I. H. Chaudry G Protein-Coupled Receptor 30-Dependent Protein Kinase A Pathway Is Critical in Nongenomic Effects of Estrogen in Attenuating Liver Injury after Trauma-Hemorrhage Am. J. Pathol., April 1, 2007; 170(4): 1210 - 1218. [Abstract] [Full Text] [PDF]
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D. W. Good Nongenomic Actions of Aldosterone on the Renal Tubule Hypertension, April 1, 2007; 49(4): 728 - 739. [Full Text] [PDF]
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L. Josefsberg Ben-Yehoshua, A. L. Lewellyn, P. Thomas, and J. L. Maller The Role of Xenopus Membrane Progesterone Receptor {beta} in Mediating the Effect of Progesterone on Oocyte Maturation Mol. Endocrinol., March 1, 2007; 21(3): 664 - 673. [Abstract] [Full Text] [PDF]
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T. Jover-Mengual, R. S. Zukin, and A. M. Etgen MAPK Signaling Is Critical to Estradiol Protection of CA1 Neurons in Global Ischemia Endocrinology, March 1, 2007; 148(3): 1131 - 1143. [Abstract] [Full Text] [PDF]
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L. Albanito, A. Madeo, R. Lappano, A. Vivacqua, V. Rago, A. Carpino, T. I. Oprea, E. R. Prossnitz, A. M. Musti, S. Ando, et al. G Protein-Coupled Receptor 30 (GPR30) Mediates Gene Expression Changes and Growth Response to 17{beta}-Estradiol and Selective GPR30 Ligand G-1 in Ovarian Cancer Cells Cancer Res., February 15, 2007; 67(4): 1859 - 1866. [Abstract] [Full Text] [PDF]
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R. J. Denver Endocannabinoids Link Rapid, Membrane-Mediated Corticosteroid Actions to Behavior Endocrinology, February 1, 2007; 148(2): 490 - 492. [Full Text] [PDF]
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N. Vasudevan and D. W. Pfaff Membrane-Initiated Actions of Estrogens in Neuroendocrinology: Emerging Principles Endocr. Rev., February 1, 2007; 28(1): 1 - 19. [Abstract] [Full Text] [PDF]
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P. Thomas, Y. Pang, J. Dong, P. Groenen, J. Kelder, J. de Vlieg, Y. Zhu, and C. Tubbs Steroid and G Protein Binding Characteristics of the Seatrout and Human Progestin Membrane Receptor {alpha} Subtypes and Their Evolutionary Origins Endocrinology, February 1, 2007; 148(2): 705 - 718. [Abstract] [Full Text] [PDF]
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 R. Kapoor, D. Nimmagadda, and J. J. Sheng Cellular Localization Studies on Human Estrogen Sulfotransferase SULT1E1 in Human Embryonic Kidney 293 Cells Drug Metab. Dispos., January 1, 2007; 35(1): 17 - 20. [Abstract] [Full Text] [PDF]
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 L. Zhao, K. O'Neill, and R. D. Brinton Estrogenic Agonist Activity of ICI 182,780 (Faslodex) in Hippocampal Neurons: Implications for Basic Science Understanding of Estrogen Signaling and Development of Estrogen Modulators with a Dual Therapeutic Profile J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1124 - 1132. [Abstract] [Full Text] [PDF]
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K. Moriarty, K. H. Kim, and J. R. Bender Estrogen Receptor-Mediated Rapid Signaling Endocrinology, December 1, 2006; 147(12): 5557 - 5563. [Abstract] [Full Text] [PDF]
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J. G. Tasker, S. Di, and R. Malcher-Lopes Rapid Glucocorticoid Signaling via Membrane-Associated Receptors Endocrinology, December 1, 2006; 147(12): 5549 - 5556. [Abstract] [Full Text] [PDF]
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E. J. Filardo, C. T. Graeber, J. A. Quinn, M. B. Resnick, D. Giri, R. A. DeLellis, M. M. Steinhoff, and E. Sabo Distribution of GPR30, a Seven Membrane-Spanning Estrogen Receptor, in Primary Breast Cancer and its Association with Clinicopathologic Determinants of Tumor Progression. Clin. Cancer Res., November 1, 2006; 12(21): 6359 - 6366. [Abstract] [Full Text] [PDF]
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A. Vivacqua, D. Bonofiglio, L. Albanito, A. Madeo, V. Rago, A. Carpino, A. M. Musti, D. Picard, S. Ando, and M. Maggiolini 17beta-Estradiol, Genistein, and 4-Hydroxytamoxifen Induce the Proliferation of Thyroid Cancer Cells through the G Protein-Coupled Receptor GPR30 Mol. Pharmacol., October 1, 2006; 70(4): 1414 - 1423. [Abstract] [Full Text] [PDF]
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S. Ray, X. Hou, H.-E. Zhou, H. Wang, and S. K. Das Bip Is a Molecular Link between the Phase I and Phase II Estrogenic Responses in Uterus Mol. Endocrinol., August 1, 2006; 20(8): 1825 - 1837. [Abstract] [Full Text] [PDF]
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T. Simoncini, C. Scorticati, P. Mannella, A. Fadiel, M. S. Giretti, X.-D. Fu, C. Baldacci, S. Garibaldi, A. Caruso, L. Fornari, et al. Estrogen Receptor {alpha} Interacts with G{alpha}13 to Drive Actin Remodeling and Endothelial Cell Migration via the RhoA/Rho Kinase/Moesin Pathway Mol. Endocrinol., August 1, 2006; 20(8): 1756 - 1771. [Abstract] [Full Text] [PDF]
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J. Keay, J. T. Bridgham, and J. W. Thornton The Octopus vulgaris Estrogen Receptor Is a Constitutive Transcriptional Activator: Evolutionary and Functional Implications Endocrinology, August 1, 2006; 147(8): 3861 - 3869. [Abstract] [Full Text] [PDF]
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E. Karteris, S. Zervou, Y. Pang, J. Dong, E. W. Hillhouse, H. S. Randeva, and P. Thomas Progesterone Signaling in Human Myometrium through Two Novel Membrane G Protein-Coupled Receptors: Potential Role in Functional Progesterone Withdrawal at Term Mol. Endocrinol., July 1, 2006; 20(7): 1519 - 1534. [Abstract] [Full Text] [PDF]
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R. X.-D. Song and R. J. Santen Membrane Initiated Estrogen Signaling in Breast Cancer Biol Reprod, July 1, 2006; 75(1): 9 - 16. [Abstract] [Full Text] [PDF]
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M. R. Meyer, E. Haas, and M. Barton Gender Differences of Cardiovascular Disease: New Perspectives for Estrogen Receptor Signaling Hypertension, June 1, 2006; 47(6): 1019 - 1026. [Full Text] [PDF]
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W. Yan, P. L Ryan, F. F Bartol, and C. A Bagnell Uterotrophic effects of relaxin related to age and estrogen receptor activation in neonatal pigs. Reproduction, May 1, 2006; 131(5): 943 - 950. [Abstract] [Full Text] [PDF]
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H. J. Han, J. S. Heo, and Y. J. Lee Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1067 - C1075. [Abstract] [Full Text] [PDF]
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A. Vivacqua, D. Bonofiglio, A. G. Recchia, A. M. Musti, D. Picard, S. Ando, and M. Maggiolini The G Protein-Coupled Receptor GPR30 Mediates the Proliferative Effects Induced by 17{beta}-Estradiol and Hydroxytamoxifen in Endometrial Cancer Cells Mol. Endocrinol., March 1, 2006; 20(3): 631 - 646. [Abstract] [Full Text] [PDF]
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W. R. Harrington, S. H. Kim, C. C. Funk, Z. Madak-Erdogan, R. Schiff, J. A. Katzenellenbogen, and B. S. Katzenellenbogen Estrogen Dendrimer Conjugates that Preferentially Activate Extranuclear, Nongenomic Versus Genomic Pathways of Estrogen Action Mol. Endocrinol., March 1, 2006; 20(3): 491 - 502. [Abstract] [Full Text] [PDF]
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Y. Wu, W. G Foster, and E. V Younglai Rapid effects of pesticides on human granulosa-lutein cells Reproduction, February 1, 2006; 131(2): 299 - 310. [Abstract] [Full Text] [PDF]
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M. M. Khan, M. Hadman, C. Wakade, L. M. De Sevilla, K. M. Dhandapani, V. B. Mahesh, R. K. Vadlamudi, and D. W. Brann Cloning, Expression, and Localization of MNAR/PELP1 in Rodent Brain: Colocalization in Estrogen Receptor-{alpha}- But Not in Gonadotropin-Releasing Hormone-Positive Neurons Endocrinology, December 1, 2005; 146(12): 5215 - 5227. [Abstract] [Full Text] [PDF]
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 M. N. Chernova, D. H. Vandorpe, J. S. Clark, J. I. Williams, M. A. Zasloff, L. Jiang, and S. L. Alper Apparent receptor-mediated activation of Ca2+-dependent conductive Cl- transport by shark-derived polyaminosterols Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1644 - R1658. [Abstract] [Full Text] [PDF]
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P. J. Fuller and M. J. Young Mechanisms of Mineralocorticoid Action Hypertension, December 1, 2005; 46(6): 1227 - 1235. [Abstract] [Full Text] [PDF]
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 T. M. D'Eon, S. C. Souza, M. Aronovitz, M. S. Obin, S. K. Fried, and A. S. Greenberg Estrogen Regulation of Adiposity and Fuel Partitioning: EVIDENCE OF GENOMIC AND NON-GENOMIC REGULATION OF LIPOGENIC AND OXIDATIVE PATHWAYS J. Biol. Chem., October 28, 2005; 280(43): 35983 - 35991. [Abstract] [Full Text] [PDF]
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 E.V. Younglai, Y.J. Wu, T.K. Kwan, and C.-Y. Kwan Non-genomic action of estradiol and progesterone on cytosolic calcium concentrations in primary cultures of human granulosa-lutein cells Hum. Reprod., September 1, 2005; 20(9): 2383 - 2390. [Abstract] [Full Text] [PDF]
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 J. G. Tasker, S. Di, and R. Malcher-Lopes Rapid Central Corticosteroid Effects: Evidence for Membrane Glucocorticoid Receptors in the Brain Integr. Comp. Biol., August 1, 2005; 45(4): 665 - 671. [Abstract] [Full Text] [PDF]
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E. R. Levin Integration of the Extranuclear and Nuclear Actions of Estrogen Mol. Endocrinol., August 1, 2005; 19(8): 1951 - 1959. [Abstract] [Full Text] [PDF]
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M. Wehling Rapid Effects of Aldosterone: Relevant in Cardiac Ischemia? Hypertension, July 1, 2005; 46(1): 27 - 28. [Full Text] [PDF]
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 A. Hasbi, B. F. O'Dowd, and S. R. George A G Protein-Coupled Receptor For Estrogen: The End Of The Search? Mol. Interv., June 1, 2005; 5(3): 158 - 161. [Abstract] [Full Text] [PDF]
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R. R. Magness, T. M. Phernetton, T. C. Gibson, and D.-b. Chen Uterine blood flow responses to ICI 182 780 in ovariectomized oestradiol-17{beta}-treated, intact follicular and pregnant sheep J. Physiol., May 15, 2005; 565(1): 71 - 83. [Abstract] [Full Text] [PDF]
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 C. M. Revankar, D. F. Cimino, L. A. Sklar, J. B. Arterburn, and E. R. Prossnitz A Transmembrane Intracellular Estrogen Receptor Mediates Rapid Cell Signaling Science, March 11, 2005; 307(5715): 1625 - 1630. [Abstract] [Full Text] [PDF]
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, P < 0.05, one-way ANOVA).