Human Placental Receptor Research Articles

Effects of prenatal opioid and alcohol exposures on immune and serotonin factors in human placenta

PurposeOpioids and alcohol impact critical serotonin (5-HT) function in the developing placenta and fetus through the actions of immune proinflammatory factors. Yet, possible convergent effects of opioids and alcohol on human placental toll-like receptor 4 (TLR4) activation and subsequent 5-HT homeostasis remain entirely unknown. The purpose of this study was to examine the effect of prenatal exposure to opioids with or without prenatal alcohol exposure (PAE) on the expression of key placental immune and serotonin signaling factors in human placental tissue obtained from a well-characterized prospective cohort. MethodsData were collected from a subset of participants enrolled in the prospective pre-birth Ethanol, Neurodevelopment, Infant, and Child Health (ENRICH-1) cohort. Women were recruited and classified into four study groups: 1) PAE (n = 20); 2) those taking medications for opioid use disorder (MOUD; n = 28), 3) concurrent PAE and MOUD (n = 20); and 4) controls (HC; n = 20) based on prospective, repeated self-report, and biomarker analysis. Placenta samples underwent tissue processing to identify mRNA for TLR4, nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), serotonin transporter (SERT), tryptophan hydroxylase (TPH1), indoleamine 2,3-Dioxygenase 1 (IDO) as well as protein concentrations of TLR4, IL-1β, TNF-α, SERT. To consider the association between study group and mRNA/protein expression of our targets, multivariable regression models were developed with inclusion of a priori selected covariates. ResultsThere was a significant negative association between PAE and SERT mRNA (β = −0.01; p < 0.01) and a positive association with TPH1 mRNA expression (β = 0.78; p < 0.05). In addition, there was a negative association between MOUD and TNF-α protein expression (β = −0.12; p < 0.05). ConclusionsThis study provides the first evidence that PAE may inhibit SERT expression while simultaneously promoting increased TPH1 protein expression in human placenta. This may result in increased 5-HT in fetal circulation known to affect neurodevelopment. Our data suggest that opioids and alcohol may disturb the bidirectional, dynamic interaction between the placental immune and serotonin system. Given the implication for brain development and health across the life-span further investigation of these critical mechanisms in well-defined cohorts is required.

Human placental androgen receptor variants: Potential regulators of male fetal growth

IntroductionNumerous studies show that males have increased intrauterine growth compared to females, and that pregnancy complications may further these growth differences, but the regulatory mechanisms underlying these differences remain unknown. We propose that these growth outcomes may be due to sex-specific differences in androgen sensitivity – giving rise to altered growth signalling pathways – mediated by the differential expression of placental androgen receptor (AR) variants. MethodsPlacental protein and mRNA were used to identify AR protein variant levels and AR-downstream target gene expression, and were then analysed against neonatal measurements. Dihydrotestosterone (DHT)-induced AR protein variant expression and downstream growth factors were examined in vitro. ResultsFour known AR variants (AR-FL, AR-V1, AR-V7, and AR-45), and three unknown proteins (120, 90 and 55 kDa) immunoreactive to the anti-AR antibody were identified in human placentae. Male placentae from controlled asthmatic pregnancies had increased AR-45 and decreased AR-V1 and AR-V7 nuclear expression. Increased nuclear AR-45 expression was associated with increased insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), and IGF-binding protein 5 (IGFBP-5) mRNA expression and normal male growth. AR-45 mRNA and protein did not change in the presence of uncontrolled maternal asthma and associated with an increase in small for gestational (SGA) male fetuses. In vitro DHT stimulation increased AR-45 protein and IGF-1R and IGFBP-5 mRNA expression. ConclusionsCollectively, our data shows altered AR protein expression and downstream signalling targets may contribute to sex-specific fetal growth outcomes in response to an adverse environment, and that AR-45 appears central in mediating these changes.

Human Placental Adenosine Receptor Expression is Elevated in Preeclampsia and Hypoxia Increases Expression of the A 2A Receptor

Placental hypoxia as a result of impaired trophoblast invasion is suggested to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine, and the actions of adenosine are mediated through four adenosine receptors, A 1, A 2A, A 2B and A 3. We investigated the presence, distribution and expression of adenosine receptor subtypes in the human placenta, the expression of the adenosine receptors in placentas from pregnancies complicated by preeclampsia, small for gestational age (SGA) infants and uncomplicated pregnancies, and the effect of hypoxia on placental adenosine receptor expression. Immunofluorescent microscopy localized A 1, A 2A, A 2B and A 3 adenosine receptors to the syncytiotrophoblast, endothelial cells and myofibroblasts within the human placenta. Adenosine receptor protein and message expression levels were significantly higher in placentas from preeclamptic pregnancies with or without SGA infants, but not different in pregnancies with SGA infants alone. In vitro exposure of placental villous explants to hypoxia (2% oxygen) increased the expression of A 2A adenosine receptor 50%. These data indicate that all four known adenosine receptors are expressed in the human placenta and adenosine receptor expression is significantly higher in pregnancies complicated by preeclampsia. These data are consistent with the hypothesis that differences in placental adenosine receptors may contribute to alterations in placental function in preeclampsia.

Placental Corticotrophin‐Releasing Hormone and its Receptors in Human Pregnancy and Labour: Still a Scientific Enigma

It is now accepted that, in humans, placental corticotrophin-releasing hormone (CRH) is involved in the mechanisms controlling the onset of labour; however, the precise biological role in foeto-maternal tissues remain enigmatic. Maternal plasma levels of CRH rise exponentially as pregnancy progresses towards term and peak during labour; however, evidence to link this with an active role in the onset and progression of labour, is still inconclusive. Certainly, one of the tissues targeted by CRH is the myometrial smooth muscle, which expresses a plethora of specific CRH receptors. This finding implicates CRH in the mechanisms preparing the myometrial microenvironment for the onset of labour and possibly in the regulation of active contractility during labour. Other gestational tissues also targeted by CRH include the placenta, foetal membranes and foetal adrenals, where CRH might regulate distinct physiological functions, ranging from control of vascular tone to adrenal steroidogenesis and prostaglandin synthesis and activity. Given the unique, among mammals, pattern of human placental CRH secretion and CRH receptor expression and signalling during pregnancy and labour, there are only limited biological tools available to delineate the actions of CRH in foeto-maternal tissues, primarily based on in vitro characterisation of the signalling and molecular events driven by CRH. This review will set in context the current concepts about the role of CRH and its receptors during pregnancy and labour, focusing on the unresolved questions and paradoxes that currently exist.

Lectin-induced alterations of the interaction of insulin and insulin-like growth factor 1 receptors with their ligands

In order to study whether the carbohydrate moieties of the human placental IGF-I receptor (IGF1R), IGF-II receptor (IGF2R) and insulin receptors (IRs) play a role in ligand binding, solubilised cell membrane preparations were incubated with 125I-labelled IGF-I, IGF-II and insulin in the presence of lectins with different sugar specificities. Three incubation procedures were tested: ligand-first, co-incubation and lectin-first incubation. Wheat germ agglutinin (WGA), concanavalin A (Con A) and phytohaemagglutinin (PHA) altered the binding of IGF-I and insulin to their high-affinity receptors in a lectin specific and dose-dependent manner, whereas these lectins did not affect the interaction of IGF-II with its receptor(s). Moreover, the same lectins either inhibited or enhanced IGF-I and insulin binding, depending on the incubation scheme. These results also suggest that IR-A and IR-B from human placenta might be differently glycosylated.

Autoantibodies Against Folate Receptors in Women With a Pregnancy Complicated by a Neural Tube Defect

Even if in the absence of clinical folate deficiency, providing supplemental folic acid in the periconceptional period lowers the risk that the infant will have a neural tube defect (NTD). Findings that antifolate receptor antiserum leads to resorption of embryos as well as malformations in rats suggested that autoantibodies against these receptors could dispose to defects. The study group included 12 women who were, or had been, pregnant with an affected fetus and 24 control women (20 with a normal current or prior pregnancy and 4 nulligravidas). Serum specimens were analyzed for antifolate receptor autoantibodies by incubating samples with purified human placental folate receptors radiolabeled with 3 H-folic acid. Serum and autoantibodies isolated from serum were incubated with placental membranes, ED27 human placental cells, and KB cells, a human epidermoid carcinoma cell line, which express folate receptors. No significant demographic or clinical differences were found between the index subjects and control subjects. Serum antibodies against folate receptors were identified in 9 of the 12 index women (75%). Two of them were currently pregnant with a fetus exhibiting a NTD, whereas 7 had a history of such a pregnancy. Two of 20 control women (10%) had a current or previous infant with a NTD. The mean value for folate receptors bound by antiantibody was significantly greater in the index subjects. Only sera testing positive for autoantibody-blocked folic acid binding to placental membrane receptors, ED27 cells, and KB cells incubated at 4°C. Autoantibodies also blocked the uptake of 3 H-folic acid by KB cells incubated at 37°C. No anticardiolipin, antisingle-stranded DNA, or antithyroid autoantibodies were identified in sera from index subjects. Autoantibodies against folate receptors are present in sera from women whose pregnancies are complicated by a NTD. It remains to be determined whether these autoantibodies are pathogenic.

Hormonal regulation of human trophoblast differentiation: a possible role for 17β-estradiol and GnRH

We have examined the role of 17β-estradiol and gonadotropin releasing hormone (GnRH) in the regulation of functional differentiation in human trophoblasts. In contrast to its recognized functions as a proliferation-promoting hormone in a variety of cell types, we found that 17β-estradiol induced terminal differentiation in human trophoblastic cells, and that this event was estrogen-receptor-mediated. This process involved a loss in expression of Cyclins A2 and E, and a coincident increase in p27 Kip1. The anti-proliferative effects of 17β-estradiol were annulled by specific transforming growth factor-beta 1 (TGFβ1)-neutralizing antibody, suggesting that 17β-estradiol may mediate its growth-inhibitory actions, through TGFβ1 activity. Following exposure to Buserelin, cultured human trophoblastic cells stopped proliferating and formed functionally mature syncytiotrophoblasts. This differentiation event, that involved a drastic loss in expression of proliferating-cell-nuclear-antigen, could be blocked by Cetrorelix, suggesting the involvement of functional GnRH receptors. Preliminary studies on the characterization of the human placental GnRH receptor, indicate the presence of multiple receptor isoforms across human gestation.

Autoantibodies against folate receptors in women with a pregnancy complicated by a neural-tube defect.

In the absence of clinical folate deficiency, periconceptional supplementation with folic acid reduces a woman's risk of having an infant with a neural-tube defect. Since antiserum to folate receptors induces embryo resorption and malformations in rats, we hypothesized that autoantibodies against folate receptors in women may be associated with pregnancy complicated by a neural-tube defect. Serum from 12 women who were or had been pregnant with a fetus with a neural-tube defect and from 24 control women (20 with current or prior normal pregnancies and 4 who were nulligravid) was analyzed for autoantibodies by incubation with human placental folate receptors radiolabeled with [3H]folic acid. The properties of these autoantibodies were characterized by incubating serum and the autoantibodies isolated from serum with placental membranes, ED27 cells, and KB cells, which express the folate receptors. Serum from 9 of 12 women with a current or previous affected pregnancy (index subjects) and 2 of 20 control subjects contained autoantibodies against folate receptors (P<0.001). The autoantibodies blocked the binding of [3H]folic acid to folate receptors on placental membranes and on ED27 and KB cells incubated at 4 degrees C and blocked the uptake of [3H]folic acid by KB cells when incubated at 37 degrees C. Serum from women with a pregnancy complicated by a neural-tube defect contains autoantibodies that bind to folate receptors and can block the cellular uptake of folate. Further study is warranted to assess whether the observed association between maternal autoantibodies against folate receptors and neural-tube defects reflects a causal relation.

Embryo implantation and GnRH antagonists: the search for the human placental GnRH receptor.

Hypothalamic gonadotrophin-releasing hormone (GnRH) plays a major role in the endocrine control of reproduction. Acting through its high affinity receptors on pituitary gonadotrophs, it regulates the secretion of gonadotrophins. In addition, GnRH also functions as a local regulator in a number of other cell lines and tissues, including the placenta. In a manner analogous to hypothalamic GnRH stimulation of LH and FSH from the anterior pituitary, GnRH was found to cause a dose-dependent release of human chorionic gonadotrophin (HCG) from the placenta. So began the search for a putative GnRH receptor in the human placenta. Although early radio-receptor studies reported specific binding, the properties of these 'putative' GnRH binding sites were found to differ significantly from those of their pituitary counterparts in several important respects. This was followed by a series of contradictory reports that led to more questions and opened up avenues for further investigations. Even after nearly two decades of research, the human placental GnRH receptor has not been characterized beyond all reasonable doubt. This review recalls the discovery, the controversies and unanswered questions concerning the human placental GnRH receptor.

Effects of alcohol on the human placental GnRH receptor system.

Isolation of human term placental membranes in the presence or absence of protease inhibitors indicated that protease inhibitors significantly reduced the amounts of [(125)I]-labelled gonadotrophin-releasing hormone (GnRH) binding to membrane GnRH-receptors in vitro by approximately 20%. This decrease was largely due to the ethanol used to dissolve the serine protease inhibitor, phenylmethylsulphonylfluoride (PMSF). Ethanol alone decreased the specific binding of [(125)I]-labelled GnRH isoform (IC(50), 7.9 +/- 0.8 mg/ml; n = 6) or agonist tracers (IC(50), 10.0 +/- 1.4 mg/ml; n = 6) to human placental membranes in a dose-dependent manner. Other alcohols also interfered with [(125)I]-GnRH isoform or agonist binding: inhibition increased with increasing carbon chain length and was dependent on the isomeric position of the hydroxyl group. Fractionation of term placental cytosol by gel chromatography demonstrated the presence of a high molecular weight fraction ( approximately 60-70 kDa) which inhibited [(125)I]-GnRH binding to human placental membranes. However, placental cytosol fractions did not cross-react significantly with a specific anti-GnRH antibody. Surprisingly, re-assay of cytosol fractions in the presence of a cocktail of protease inhibitors generated a factor (molecular weight approximately 40-50 kDa) which did cross-react strongly with the GnRH antibody. The generation of this factor was due to the ethanol solvent rather than to the protease inhibitors per se, as treatment of pooled 'latent' cytosol fractions with ethanol alone generated GnRH-like immunoactivity (irGnRH) which competed in parallel with GnRH standard. The amount of irGnRH generated depended on the concentration of ethanol added to the 'latent' cytosol fractions. However, ethanol had no effect on the assay in the absence of cytosol fraction, or with inactive cytosol fractions. Thus, ethanol can perturb the human placental GnRH/GnRH-receptor system in vitro in two distinct ways: by inhibition of GnRH binding to receptor, and by dissociation of complexed endogenous GnRH-like factor(s) from a GnRH-binding protein. It is postulated that high alcohol consumption in vivo may interfere with placental GnRH secretion/action and affect placental secretion of factors important to the establishment and maintenance of pregnancy.

Human placental Fc receptors and the trapping of immune complexes

Monomeric maternal antibodies are transmitted to the fetus, but most immune complexes are trapped in the placental stroma. The receptors responsible for trapping immune complexes appear to be FcγRIa, FcγRIIa, and FcγRIIIa on stromal macrophages, and FcγRII on fetal capillary endothelial cells.

Nucleotide sequence analyses predict that human pituitary and human placental gonadotropin-releasing hormone receptors have identical primary structures.

Gonadotropin-releasing hormone (GnRH) interacts with a putative receptor in human placenta to cause the dose-dependent release of human chorionic gonadotropin (hCG) in a manner analogous to hypothalamic GnRH stimulation of luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by the pituitary gland. However, GnRH agonists bind a placental binding site at a lower affinity than they bind the pituitary GnRH receptor, suggesting the two sites differ. To address this issue, human placental GnRH receptor mRNA from an 8-wk placental sample was amplified using primers based on the sequence of the human pituitary receptor, cloned, and sequenced. The sequence of the placental transcript was found to be identical to its pituitary counterpart. Thus, as with the pituitary receptor, this placental receptor transcript appears to encode a single polypeptide chain with seven intramembranous domains and two glycosylation sites. However, it is possible these receptors differ in their posttranslational processing or there is more than one placental GnRH receptor. In addition, subsequent gene amplification studies, modified to increase the sensitivity of the method, revealed a band of the predicted size (approximately 1051 bp) in a 6-wk and two 8-wk placental samples, but not in 5-wk and 7-wk samples. The failure to amplify GnRH receptor mRNA at these gestational ages did not appear to be an issue of cell health or sample degradation, since GnRH, alpha hCG, and beta hCG mRNA were amplified from each of these tissues. Instead these data suggest GnRH receptor mRNA may have a short half-life or the mRNA may be translated as rapidly as it is transcribed, with the result that it has not accumulated in the cell.

Expression of Angiotensin II Type-I Receptor and Phospholipase C-Linked Gαq/11 Protein in the Human Placenta

A recent study on the distribution of angiotensin II (ANG II), ANG II (AT) receptors, and human placental lactogen in human placental tissues from term pregnancies showed positive correlations among these indices, suggesting an important role for ANG II in placental endocrinology. However, nothing is known about the ontogenesis of this functional role for ANG II during pregnancy. Therefore, the aim of this study was to investigate the placental AT receptor expression at various trimesters of pregnancy. We also studied the ontogenesis of phospholipase C-linked G alpha q/11 protein, which is known to transduce ANG II type-1 (AT1) receptor signal. Western blot analysis of placental membrane proteins was performed using a chemiluminescence kit and specific antibodies against AT1 receptor and G alpha q/11 protein. Northern blot analyses of AT1 receptor and G alpha q/11 mRNA expression were accomplished using random primed [32P]dCTP-labeled specific probes. The autoradiographs of AT1 receptor mRNAs (2.4 kb) and proteins (83 kDa) showed a progressive 4.8-fold and 2.6-fold increase, respectively, during pregnancy, with maximal levels at term. We also observed progressive 1.8-fold and 4.5-fold increases of G alpha q/11 protein (43 kDa) and mRNAs (4.5, 6.0, and 7.1 kb), respectively, during pregnancy, with maximal levels observed at term. Our results demonstrate that both the human placental AT1 receptor content and the G alpha q/11 protein level increase during pregnancy, and suggest that the AT1 receptor pathway may play a role in human placental physiology.

Direct evidence for functional coupling between human placental folate transporter and folate receptor in a heterologous expression system

Direct evidence for functional coupling between human placental folate transporter and folate receptor in a heterologous expression system

Measurement of luteal and placental gonadotrophin-releasing hormone (GnRH) binding sites: role of inactivation of GnRH tracer.

The ability of radiolabelled gonadotrophin-releasing hormone (GnRH) and GnRH analogues to bind to homogenates and membranes of rat, rabbit and sheep pituitary and to luteal homogenates and membranes from a number of species was measured. In addition, inactivation of tracer during binding incubation was estimated by measurement of the ability of the unbound tracer fraction to bind to fresh GnRH-receptor or anti-GnRH antibody. Following incubation of rat, sheep and rabbit pituitary gland with a radiolabelled GnRH agonist tracer, appreciable amounts of specific binding of GnRH agonist to fresh rat pituitary or human placental GnRH receptors could still be demonstrated. In contrast, no specific binding of [125I]-labelled GnRH analogues were measured following incubation of the tracer with homogenates or membranes of bovine, sheep and porcine luteal tissue, nor with rabbit and rat placental homogenates. However, during incubation of GnRH tracers with these tissues, almost complete inactivation of GnRH tracers occurred. There was an inverse relationship between binding and inactivation of [125I]-labelled GnRH for a number of human corpora lutea. We conclude that degradation of GnRH tracers may prevent the measurement of specific GnRH binding sites in some tissues.

Functional reconstitution of the human placental transferrin receptor into phospholipid bilayers leads to long tubular structures proceeding from the vesicle surface.

We have reconstituted the human placental transferrin receptor (hTfR) into phospholipid vesicles using either dialysis, dilution, or gel filtration. Several different detergents and phospholipids and a variety of lipid-to-protein weight ratios were tested. Preformed vesicles as well as detergent-solubilized phospholipids were used. Reconstituted mixtures were fractionated by sucrose density gradient centrifugation and screened for ferritransferrin binding activity, and peak fractions were analyzed by electron microscopy. The efficiency of reconstitution was strongly influenced by the choice of the phospholipids used and the reconstitution method. Best results were obtained when hTfR was dissolved with octylpolyoxyethylene, mixed with detergent-solubilized soy bean lecithin, and reconstituted by slow dialysis. The data show that hTfR capable of binding ferritransferrin was reconstituted into vesicles with an irregular surface and many protrusions. In addition the reconstitution of hTfR resulted in the formation of tubular structures proceeding from the vesicle surface, which may serve as an in vitro model for future studies on the relevance of self-assembly processes for cellular endocytosis.

Endothelin-1 conversion and receptor characterization in human placental arteries

Endothelin-1-(1-21), a potent pressor peptide, is transcribed as big endothelin-(1-38) and converted to active peptide by endothelin-converting enzyme. The current investigation tested the hypothesis that human fetoplacental blood vessels convert big endothelin-1 to active peptide and that fetoplacental blood vessels respond to endothelin-1 by binding of the peptide to specific receptor sites. In the isolated perfused placental cotyledon the addition of big endothelin-1 to the perfusate caused a time-dependent increase in perfusion pressure that corresponded to the appearance of endothelin-1 in the perfusate. The properties of human placental endothelin-1 receptors were defined in binding studies performed on a plasma membrane fraction of small arteries (<1.0 mm) dissected from the placenta. Binding was saturable, reached steady state by 3 h at 25 degrees C, and was linear with protein concentration. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a dissociation constant of 27.6 +/- 2.3 pM and a density of 856 +/- 119 fmol/mg protein (n = 5). The potency order for competitive inhibition of the binding of 125I-labeled endothelin-1 [endothelin-1 = endothelin-2 > endothelin-3 = sarafotoxin S6b >> big endothelin-1 (human) = big endothelin-1 (porcine)] is most consistent with a type A endothelin receptor subtype. Phenylephrine, bradykinin, norepinephrine, atrial natriuretic factor, diltiazem, U-46619, and angiotensin II did not displace 125I-endothelin-1 binding. Endothelin receptors were shown to have an approximate molecular weight of 36,600 by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

The binding of human serum transferrin to its specific receptor reconstituted into liposomes

Human placental transferrin receptor (HPTR), purified following a procedure based on affinity chromatography step, was reconstituted by the detergent dialysis method into various kinds of phosphatidylcholine vesicles and the receptor ability to bind 125I-labeled human serum transferrin (HST) was then evaluated. In our experimental conditions, the binding of the labelled protein to its specific receptor showed several features, in particular: (1) in cholesterol/1-α-dipalmitoyphosphatidyl choline (CHO/DPPC) liposomes, a positive cooperativity of the transferrin binding resulted at the lowest cholesterol/phospholipids (C/P) ratio; 1-α-dioleylphosphatidyl choline (DOPC) and phosphatidic acid (PA) containing liposomes showed an opposite binding curve trend; (2) the apparent dissociation constant ( K′ d) did not change significantly as a function of the lipid composition, being always around 1.00 × 10 −6 M; (3) the encapsulation capacity of liposomes decreased from 27% to about 13% with increasing amounts of cholesterol and was around 20% in the presence of DOPC or PA; about 8–13% of this receptor was found to be functional; (4) receptor-loaded liposomes treated with polyclonal anti-HPTR rabbit antibodies showed a remarkable binding decrease for transferrin. All these results seem to point out the crucial role played by the environment in the binding behaviour of the transferrin receptor.

Characterization of human placental insulin-like growth factor-I/insulin hybrid receptors by protein microsequencing and purification

Protein microsequencing of human placental IGF-I receptors purified by immunoaffinity chromatography using IGF-I receptor specific monoclonal antibody revealed amino acid sequences of both IGF-I and insulin receptors. Since this finding indicated the presence of IGF-I/insulin receptor hybrids, hybrid receptors were further purified by immunoaffinity chromatography using insulin receptor specific monoclonal antibody. The molecular size of the nonreduced hybrid receptor was approximately 350K, indicating that the IGF-I and insulin receptor alpha beta halves were disulfide-linked. The ratio of IGF/insulin binding activity of purified hybrid receptors was approximately 25 when measured using tracer amounts of radioactive ligands. 125I-IGF binding to these receptors was inhibited by IGF-I and insulin with IC50s of approximately 2 and approximately 1000 nM, respectively. 125I-Insulin binding to these receptors was similarly inhibited by IGF-I and insulin with IC50 of approximately 3 nM. Autophosphorylation and kinase activities of the hybrid receptor were stimulated by IGF-I more effectively than insulin in a dose-dependent manner. Thus, the present studies indicate that hybrid receptors purified from human placenta have the functional properties of an IGF-I receptor.

Expression of the Human Placental Folate Receptor Transcript is Regulated in Human Tissues: Organization and Full Nucleotide Sequence of the Gene

The primary structures of the human KB cell (FR-KB1) folate receptor (FR) and of a human placental (FR-P2) FR, proteins important in cellular accumulation of folates, have been deduced from cDNA sequences. Herein, we report a novel human FR cDNA (FR-P3) isolated from a placental library and the chromosomal organization of the human FR-P3 gene. Compared to the FR-P2 cDNA, the composite 1084 base-pair (bp) FR-P3 cDNA is homologous, but contains a unique 5′ terminus and sequence differences within the open reading frame (ORF) and at the exon I-II junction. Polymerase chain reaction and RNase protection assays demonstrate that the FR-P3 cDNA represents the major transcript, and suggest that the FR-P2 cDNA is encoded by an independent FR gene. The nucleotide sequences of two non-overlapping human genomic clones contain the FR-P3 gene, which spans 5148 bp, is composed of five exons, and is polymorphic relative to 5′ restriction sites. The transcript size (1084 bp) predicted from structural analysis of the FR-P3 gene correlates with the size (1100 bp) determined by Northern blots. Based on RNase protection assays, both FR-P3 and FR-KBl transcripts are expressed in human fetal and adult tissues, and the abundance of each transcript varies among the tissues studied. These results indicate that the FR transcripts are products of independent, conserved genes; that neither FR gene is preferentially expressed during fetal development; and that specific FR transcripts are differentially expressed in human tissues, suggesting that transcription of each FR gene is regulated independently. The isolation of the FR-P3 gene will permit functional analysis of the cis and trans regulatory elements of the FR-P3 gene and the mechanisms involved in tissue-specific FR gene expression.