Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants
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Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR). We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm). Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.
KeywordsCulture Supernatant Tissue Culture Medium Spinner Flask Follicle Stimulate Hormone Receptor Hypotonic Lysis
Reproductive hormones (luteinizing hormone [LH], follicle stimulating hormone [FSH] and human chorionic gonadotropin [hCG)]) are collectively known as gonadotropins because they stimulate gonads (testes in male and ovaries in female). The hormonal activation occurs through specific ligand-receptor interactions on the surface of the target cells. The LH and hCG utilize a common receptor encoded by a single copy ~70 kb LHCGR gene, located at human chromosome 2p21 . LHCGR has 11 exons and codes for multiple alternatively spliced species (at least 6) of mRNA. Transcriptional activation to generate these mRNAs is initiated at multiple sites spanning a region more than a kilobase upstream of the first exon . Alternatively spliced mRNAs produce several truncated proteins which have the ligand binding capacity but are ineffective in transducing signals [3, 4, 5, 6, 7]. In addition to testis, ovary and placenta, various isoforms of LHCGR are expressed in uterine myometrium, vascular endothelial and smooth muscle cells, adrenals, brain, skin, lymphocytes, human sperm, macrophages and in fetal tissues [, for a review].
The earliest biochemical evidence on the existence of cell-free soluble LH receptor was the purification of an hCG-binding protein, relative molecular mass of 65K (Mr, 65K) from porcine follicular fluid and was based on gel filtration followed by affinity chromatography . A different experimental approach by West and Cooke  demonstrated the secretion of Mr 80-90K hCG-LH receptor complex into the culture media from ligand-induced rat and mouse leydig cells. Tsai-Morris et al.  first reported the production and secretion of soluble LH receptor following transient transfection of a naturally truncated variant of the receptor in COS cells. These data were independently substantiated using different combinations of in vitro expression systems [12, 13], In some studies, the lack of detection of an actively secreted soluble form of expressed LH receptor was attributed to misfolding and intra-cellular retention of the expressed protein or proteolytic degradation of the protein released into the culture media [13, 14]. Moreover, the lack of a secreted form of the receptor was overcome by co-expression of hCG with the extra-cellular domain of porcine LH receptor leading to the secretion of a mixture of free as well as hCG-receptor complex .
Notably, the natural rat Lhcgr variant first described by Tsai-Morris et al. , instead of being secreted, was retained in the ER prior to degradation . Additionally, the truncated Lhcgr, misrouted the full-length Lhcgr in ER, thereby decreasing the cell surface expression of the mature functional receptor. The role of circulating sLHCGR as in inhibitor of LH/hCG functions by ligand binding has been questioned; instead, a specific intra-cellular role of truncated receptor in regulating the cell surface expression of a full-length ligand-binding Lhcgr has been proposed . Therefore, how a complex LHCGR protein, embedded within the membrane lipid bilayer, could be secreted as soluble receptor remained unexplained.
Structurally, LHCGR is similar to thyroid and follicle stimulating hormone receptors (TSHR and FSHR respectively). The autoantibody-binding extracellular domain (ECD; N-terminal 418 amino acid residues) of TSHR when expressed in CHO cells had a high content of mannose residues, was misfolded and retained in cells. However, expression of progressively truncated carboxy- terminal domains of TSHR in stably transfected CHO cells resulted in the secretion of the TSHR ECD with an efficiency that was inversely proportional to the length of the recombinant proteins . These reports prompted us to generate C-terminally truncated recombinant variants of LHCGR and examine the expression of the secreted recombinant proteins in serum-free medium. Contrary to our expectation, we have demonstrated here that the release of soluble LHCGR protein from transfected cells as well as from placental explants was independent of the length of the receptor protein and mediated via structurally defined membrane vesicles.
Construction of LHCGR cDNA clones
Initially, three LHCGR cDNA clones inserted into the mammalian expression vectors (30233-0, 30233-1, 30233-2) were created. A cDNA fragment encoding the N-terminal 318 amino acids of human LHCGR (CCDS1842.1; UniProtKB/Swiss-Prot P22888) was obtained by polymerase chain reaction (5'-AAGCTTATGAAACAGCGTTT-3' and 5'-GGATCCCAGGGTCTTGTTAC-3'; forward and reverse primers containing HindIII and BamHI sites respectively). The PCR amplified fragment was engineered to have a CTGCAG insertion (corresponding to Leu-Gln, LQ insertion) at nucleotide position 55-60 (amino acid residues 19-20). The idea behind this being that the LQ containing polymorphic variant in the 'signal peptide' appears to express and function efficiently (2, 4). The resultant 966bp PCR amplified fragment (30233-0) was cloned into the HindII and BamHI site of p3XFLAG-CMV-14 vector (Sigma Aldrich). The cDNA encoding the first 291 amino acids of LHCGR (30233-1) was created employing 885 bp PCR fragment (5'-AAGCTTATGAAACAGCGTTT-3' and 5'-GGATCCCTCTTTAGTGGGCA-3', forward and reverse primers respectively). The cDNA encoding the first 229 amino acid residues of LHCHR (30233-2) was obtained by PCR amplification of 699bp fragment (5'-AAGCTTATGAAACAGCGTTT-3' and 5'-GGATCCGAGGGTCTTGGGGC-3', forward and reverse primers, respectively). To generate 30233-3 (a vector containing the N-terminal 416 amino acids of LHCGR), a 300bp cDNA (5'-TACTCCTCCA-3' and 5'-GGCGATCAGC-3', forward and reverse primers, respectively) was ligated to the 3' end (corresponding to the C-terminus) of 30233-0. The codon optimized DNA sequences of each clone were verified prior to plasmid amplification and purification. Plasmid DNA was prepared using Purelink HiPure Plasmid kit (Invitrogen) and sterile filtered through a 0.22 μm filter.
Cell culture, transfection and expression of recombinant LHCGR protein in CHO-S cells
One vial of 107 frozen CHO-S cells was added to 30 ml of pre-warmed serum-free Freestyle CHO medium (Invitrogen) containing 8 mM L-glutamine without antibiotics in a 125 ml disposable polycarbonate spinner flask with vented cap (Sigma-Aldrich) and incubated at 37°C, 8% CO2 with 150 rpm stirring. Total cell count and cell viability were monitored every 24h. A cell viability of >70% was required in order to continue with the cell expansion. Cells were subcultured by seeding at a cell density of 0.3 - 0.5 × 106 viable cells/ml. The cell density was never allowed to exceed 2 × 106 cells/ml. Following expansion, cells were pooled for measurement of cell count and viability. The cell viability of expanded cells before transfection exceeded 95%. Pooled cells were used to seed fresh flasks of pre-warmed serum free medium containing 8 mM L-glutamine at a density of 1 × 106 cells/ml in a minimum of 60 ml per 125 ml spinner flask prior to DNA transfection.
75 μg (75 μl) of filter sterilized plasmid DNA was used for each transfection of 1 × 106 cells per ml in a total volume of 60 ml of cells. The sterile DNA was first diluted into Opti-Pro SFM (Invitrogen) to a total volume of 1.2 ml and mixed. In a separate tube, 75 μl of FreeStyle Max Transfection Reagent (Invitrogen) was added to Opti-Pro SFM to a total volume of 1.2 ml and mixed gently by inverting the tube. The diluted FreeSTyle MAX Transfection Reagent was added to the diluted DNA solution to obtain a total volume of 2.4 ml and mixed gently. As a negative control (no DNA and no FreeStyle MAX Reagent), 1.2 ml of Opti-Pro SFM was mixed with 1.2 ml tissue culture medium. After incubation at room temperature for 10 minutes, the DNA-FreeStyle MAX mix was added to the CHO suspension culture and the flasks were transferred to the incubator (37°C, 150 rpm, 8% CO2). Following transfection, aliquots (4ml) was removed from each transfection every 24h up to day 7 (D7). No extra tissue culture medium or passaging of cells was carried out after transfection. Both cell density and cell viability for each culture were recorded. Aliquots were collected in sterile 15 ml conical tubes and spun at 700 rpm in a Beckman J6 centrifuge using a JS-4.2A rotor for 6 minutes at room temperature. To monitor any secretion of proteins from the cells, the tissue culture medium (supernatant) was carefully removed to another sterile tube after centrifugation, leaving the last 200 μl so as not to disturb the cell pellet. Supernatents were stored at -20°C. Cell pellets were washed three times with sterile PBS (4°C) to remove residual tissue culture medium and transfection reagents, aspirated and stored at -20°C.
Placenta explant culture
This study was approved by the local ethics committee of Planned Parenthood, Fannin Health center, Houston, Texas and written consent was obtained from patients before the collection of samples. Placental tissue was obtained from patients undergoing elective termination of pregnancy with gestational age range of 9-12 wks (gestational age was determined by ultrasound measurement of biperietal diameter or crown-rump length). Immediately after collection, the placenta tissue was rinsed with sterile PBS and sequentially washed (10-12 times) to remove traces of blood. The tissue was dissected in a sterile cell culture hood to produce small pieces of placental explants (20-50 mg in weight, approximately 0.5-1.5 mm) and was resuspended in serum-free culture medium. The suspension was transferred to disposable sterile spinner flask and was cultured at constant stirring speed 150 rpm under 8% CO2 up to 48h.
Protein extraction, Western blots and quantitative analysis of protein expression
Protein extraction from cultured cells was carried out using hypotonic buffer (10 mM Tris-HCl, pH7.5 containing 25 mM EDTA, 25 mM, NaF, 1 mM Na3VO4, and EDTA-free protease inhibitor mix [Sigma-Aldrich]). Detergent extraction of cell culture pellets following hypotonic lysis was carried out with lysis buffer (25 mM Hepes pH 7.5, 150 mM NaCl, 1% igepal CA-630 [Sigma-Aldrich], 10 mM MgCl2, 1 mM EDTA, 25 mM NaF, 1 mM Na3VO4, and EDTA-free protease inhibitor mix [Sigma-Aldrich]. This sequential extraction method was employed to obtain a predominantly cytosolic extraction (via hypotonic lysis) and a predominantly membrane extraction (hypotonic followed by detergent lysis). Extraction of placental villous tissue was performed using either T-PER reagent alone [Perbio, Helsinborg, Sweden] or T-Per containing 25 mM bicine buffer pH 7.6 [Thermo Fisher Scientific].
Western blot analysis was as described [17, 18]. Primary and secondary antibodies used were as follows: anti-human LHCGR mouse monoclonal antibody [LHR-29, ATCC clone CRL2685]; anti-FLAG monoclonal antibody [Sigma]; anti -β Actin, clone AC-15 [Sigma] and goat anti-mouse IgG (H+L) HRP-conjugated [Chemicon International Inc., CA, USA]; mouse control IgG [Sigma]. The antibody concentrations used for Western blots were: LHR-29 diluted to 1 μg/ml; anti-FLAG at 1:1000 dilution and anti-mouse IgG-HRP at 1:10,000 in the incubation medium. The epitope which the LHR29 monoclonal antibody recognizes has been mapped to a region between amino acids 227 to 289 of the LHCGR ECD (unpublished data).
Five times concentrated N-Glycanase Reaction Buffer (100 mM sodium phosphate pH 7.5, 0.1% sodium azide); Denaturation solution (2% SDS, 1 M β-mercaptoethanol); Detergent Solution (15% NP-40 solution); Endo H and PNGase F (glycerol-free) were obtained from Prozyme [San Leandro, California, USA]. According to vendor's protocol, both Endo H and PNGase F were added to a final concentration 50 mU/ml and 50 U/ml, respectively. Samples were incubated at 30°C for 16 h and reactions were terminated by adding SDS-PAGE loading buffer.
Microvesicle purification from culture supernatants
The placenta explant cultures were collected at 24h and 48h. The explant tissues and the cell debris were first pelleted by centrifugation at 3000 × g for 15 min. The supernatant was re-centrifuged at 14000 × g for 15 min and two-thirds of the clear supernatant from the top of each tube was transferred to new tubes prior to the purification of microvesicles by two alternative methods; ultrafiltration  or ultracentrifugation [20, 21]. For ultrafiltration, the vivaspin nanomembrane concentrators [Vivasin 500 Sartorius Inc., Goettingen, Germany] were washed twice with 600 μl PBS (with centrifugation at 300 × g) for 5 min to remove glycerol and other preservatives. Subsequently, 600 μl of the clear culture supernatant was centrifuged in each concentrator column at 6000 × g for 10 min at 4°C. This process was repeated and the combined retanate was then mixed with EDTA-free protease inhibitor mix (Sigma Aldrich) and stored at -20°C. Alternatively the culture supernatant was centrifuged at 10000 × g [L8-70M ultracentrifuge, Beckman Coulter, Inc. CA; 70.1 Ti rotor] for 1h and the pellet was resuspended in PBS containing EDTA-free protease inhibitor mix ]Sigma-Aldrich] before storage at -20°C.
Staining of microvesicles and immune-electron microscopy
For negative staining, grids were incubated at room temperature for 10 min with 25 μl of 2% uranyl acetate and examined by electron microscopy [Jeol 1200EX TEM operating at an acceleration voltage of 80 kV]. For immuno-labelling, 10 μl of microvesicles suspension was dropped onto a 200 mesh nickel Formvar/Carbon coated grid [Agar Scientific-S162N] and incubated for 40 min in a humid box at room temperature. A drop of 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH7.2 was added to each grid. Following PFA, grids were treated sequentially with 0.15% glycine, 1% BSA in phosphate buffer followed by primary antibodies (anti-LHCGR, LHR29 or anti-FLAG monoclonal antibodies or mouse IgG, diluted 1:10 in phosphate buffer with 1% BSA) for 30 min at room temperature. Unbound primary antibodies were removed by rinsing with 0.1% BSA in phosphate buffer four times, 2 min each time. The grids were labelled with secondary antibody conjugated with 10 nm colloidal gold [Clone M2, Sigma-Aldrich] for 1h at room temperature in a humid box and then washed multiple times with phosphate buffer before fixation using 1% glutaraldehyde in 0.1 M phosphate buffer for 5 min at room temperature. Following removal of excess glutaraldehyde, grids were washed six times with distilled water for 1 min each time prior to negative staining with 2% aqueous uranyl acetate for 1 min. All experiments were done in duplicate. Samples were examined in a Jeol 1200EX transmission electron microscope and images were captured and stored.
Densitometry and data analysis
Densitometry of autoradiograms and data analysis were carried out as described [17, 18]. In the majority of cases the analyses used β-actin as an internal control for computing the experimental values. However, in some experiments (see 'Results' below), where the β-actin failed to reflect the yield of LHCGR in the supernatant fraction, a Mr 63K membrane protein, stained using Coomassie blue following Western blot, was used as an internal standard to measure the corresponding LHCGR protein. A value for the level of significance (P-value) was calculated using the Poisson statistic. P < 0.05 was considered significant.
Expression of LHCGR recombinant proteins in CHO cell suspension culture
In order to examine the expression of recombinant LHCGR expression, samples were aliquoted from each transfection including corresponding control cultures at 24-hr intervals for up to seven days (D7). To distinguish the cytoplasmic soluble LHCGR (sLHCGR) from the cell membrane-associated receptor protein, the cells were first lysed in hypotonic buffer. Following hypotonic extraction, the cell pellet was subject to equal volume of detergent (Triton) lysis to recover the membrane-bound LHCGR recombinants. The cumulative expression at each time point for each recombinant was expressed as sum of the products in hypotonic and detergent lysates.
Secretion of sLHCGR into the culture medium
The amount of secreted recombinant LHCGR-318, LHCGR-291 and LHCGR-229 at D4 (Figure 3b-d and 3e), was 26.9%, 37.4% and 24.2% respectively (Figure 3f), compared to their highest cumulative cellular expression on D1 or D2 (Figure 2). Additionally, the amount of LHCGR-318, LHCGR-291 and LHCGR-229 in the culture supernatant at D4 was 7-, 11.5- and 4-fold, respectively, when compared with corresponding cellular expression on D4 (Figure 2 and Figure 3e and 3f). Similar results were obtained where secretion of LHCGR-418 into the medium was maximum on D4 (data not shown). The cellular expression of the recombinants was highest on D2 and subsequently declined. And yet, the amount of protein secreted on D4-D6 was significantly higher than the total protein produced by the cell (intracellular) on D4-D6 (Figure 3f). This paradoxical observation could be explained assuming that the cellular microvesicles accumulated up to D3, were abruptly released into the media between D3 and D4. Once released, the cellular production of the recombinant protein was almost shut down and the microvesicle-bound LHCGR proteins underwent gradual proteolytic degradation on D5 and D6 before disappearing on D7. Interestingly, the cell density being high on D3 and D4, began declining afterwards (Figure 1b).
Secreted LHCGR proteins are glycosylated and folded
Selective release of proteins into the culture medium
The release of recombinant LHCGR is mediated via microvesicles
The amount of total recombinant LHCGR on D4 and D5 in the supernatant was 4-12 fold higher than the corresponding total cellular proteins (Figure 3d-f), suggesting that the majority of protein synthesised on Days 1-3 was secreted on Days 4 and 5. The observation of specific secretion led us to hypothesise that the secretion might be active, involving sub-cellular organelles. To test this hypothesis, a three step centrifugation procedure was adopted whereby the highly clarified culture supernatant was subjected to ultracentrifugation in order to pellet any subcellular particles. As an alternative method, clarified culture supernatant was also concentrated by ultrafiltration (see Materials and Methods). Western blot analysis of the concentrated supernatant and the pellet following ultracentrifugation showed that only the pellet contained the LHCGR. Standard microscopy revealed subcellular particles with no detectable intact cells in the resuspended pellet (data not shown). Electron microscopy on the material from the pellet following ultracentrifugation revealed distinct subcellular particles. Based on the size of these particles (50-200 nm), the sLHCGR recombinants appear to have been released as components of these microvesicles (Figure 5d).
Microvesicle-mediated release of sLHCGR from human placenta explants at early pregnancy
Not all microvesicles from the placenta contain LHCGR
In this manuscript, we demonstrate that the secretion of soluble LHCGR from transfected cells as well as from placental tissues is independent of the size of the receptor and is mediated via microvesicles. Since microvesicles are also released from mock-transfected cells and only a portion of placental explant microvesicles stain positive for LHCGR, the mechanism of secretion is not specific to the LHCGR. The data presented here support previous work describing the abundant extracellular appearance of LH receptor and its natural variants lacking either transmembrane or intracellular domains, or both, following transfection with cDNA [11, 12]. It is noteworthy that these authors speculated that the secreted receptor proteins could be packaged in membrane vesicles . Additionally, while biochemical  and cell biological  studies demonstrated the release of LH receptor and hormone-receptor complex, our current data in conjunction with recent reports on the constitutive secretion of microvesicles from placenta throughout the human pregnancy , provide the possible mechanistic basis of the secretion of LHCGR from cells and tissues.
Questions have been raised regarding the conflicting results on LH receptor secretion described in the literature. In the light of the data presented in this manuscript, two explanations can be proposed that together might account for this discrepancy. Firstly, the methods used for in vitro expression and detection of the secreted receptor may be factors that affect experimental outcome. Secondly, the duration of experiments could be crucial. The reports of Tsai-Morris et al., and VuHai-LuuThi et al., [11, 12] both describe clear-cut secretion of the soluble LH receptor proteins where experiments were carried out using transient transfection of cells (via calcium phosphate) and by assaying for the soluble LH receptor via ligand-binding assays and by Western blot. The method of transient expression [11, 12] instead of stable cell lines [15, 23], detection of the soluble receptor by immunoprecipitation  instead of ligand-binding assays might therefore have influenced the different experimental outcomes described by these groups. The duration of experiments could be pertinent to the failure to detect soluble LH receptor. Our data show that receptor expression, measured by Western blot on supernatants concentrated by ultrafiltration, following 72h of transfection, was barely detectable (Figure 3). Indeed, immunoprecipitation of 48h and 72h transfected supernatants with anti-FLAG monoclonal antibody failed to detect sLHCGR when the blots were probed with anti-LHCGR monoclonal antibody (data not shown). Therefore, a combination of the methods and biochemical conditions of in vitro expression of LH receptor, sensitivity of the assay detecting soluble receptor and the duration of the experiment together could explain the conflicting data in the literature.
Direct translocation of the cytoplasmic receptor isoforms across the plasma membrane or protease cleavage of membrane-bound extracellular domains are the mechanisms that have been proposed for circulating soluble receptors . We demonstrate that LHCGR is released from transfected cells and placental explants packaged in microvesicles. Such secreted proteins released from tissues into the extracellular space fail to exhibit typical N-terminal secretory signal sequences as well as post-translational modifications necessary for traffic through Golgi/ER . Human tissues under oxidative stress , following cellular activation such as p53  and apoptosis release microvesicles ranging from relatively smaller (50-150nm) exosomes and plasma membrane-coated microparticles (100nm-1000nm), which represent fragments of cell membranes [28, 29, 30, 31, 32]. It should be noted however, that while the structural and immune-electron microscopic data indicate that released microvesicles could represent exosomes, future gradient density and biochemical analyses would be needed to confirm this.
The pleiotropic effects of microvesicles in inter-cellular communications have been intensely investigated in recent years. Given that microvesicles contain membrane proteins, mRNAs, miRNAs, growth factors, cytokine and chemokine receptors, it has been proposed that these circulating vesicles form a storage pool of bio-effectors or macromolecules for long-distance trans-cellular signaling/adhesion in endothelial modifications, angiogenesis, antigen presentation, immuno-suppression, differentiation and apoptosis in a variety of patho-physiological conditions including cancer [20, 21, 33, 34, 35, 36, 37]. Human placental syncytotrophoblasts constitutively secrete microvesicles throughout the pregnancy. Immune activation, immune-suppression by Fas-FasL-mediated apoptosis at the fetal-maternal interface and systemic vasculo-endothelial damage by microparticles and exosomes released from the placenta are emerging as modes of immune and vascular regulation in human pregnancies [22, 38, 39]. Our data showing that microvesicles released from the human placental explants contain full-length as well as truncated LHCGR raises the possibility that these vesicle-associated receptors, capable of binding LH and hCG hormones, may freely circulate in the blood. As such, the circulating membrane-bound LHCGR could cause reduced hormone bioactivity and may mediate abnormal activation of tissues through mobile receptor fusion. We propose that LHCGR-bearing microvesicles neutralize the natural ligands (LH or hCG) by forming soluble complexes which prevent the interactions of these hormones with tissue-specific target receptors. By altering hormone-mediated cellular activation of target tissues, the secreted, circulating sLHCGR may act as a physiological modulator of hormone function and/or as an inhibitor of hormone function that leads to pathology. This might explain the altered ratios of bioactive to immunoreactive LH or hCG that are frequently described in patients with hypogonadism , poor fertility [41, 42, 43], ovarian dysfunction [42, 43], miscarriage [44, 45, 46] and other early pregnancy complications . The possibility that circulating microvesicles bearing LHCGR might fuse with vasculo-endothelial or other cell types thereby making fused cells subject to LH/hCG activation, may be another level of complication that disrupts cell signaling. There is precedence for microvesicle-mediated transfer of the transferrin receptor , but whether the horizontal transfer of LHCGR occurs will require further investigation.
This work was supported in part by Research and Development grants from Advantage Proof of Concept (APOC) and Advantage West Midlands (AWM), West Midlands, UK. We appreciate and thank Theresa Morris for help with electron microscopy and Walter Mills for critical reading of the manuscript. We would like to thank anonymous reviewers for suggestions that improved the presentation of the manuscrpt.
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