Human Placental Receptor Research Articles

Solubilization and identification of human placental endothelin receptor

Endothelin-1 (ET-1) receptor was identified on the membranes from human placenta and 66% of original binding activity in the membranes was solubilized with 0.75% ( w v ) CHAPS. Binding studies of the solubilized membranes using 125I-ET-1 indicated the presence of a single class of high-affinity binding sites with an apparent Kd of 760 pM and a Bmax of 1.8 pmol/mg of protein. The binding was inhibited by addition of unlabeled ET-1 and ET-3 in dose dependent manner. The Ki values of solubilized membranes were 84 pM for ET-1 and 250 pM for ET-3, whereas particulate membranes had weaker affinities (Ki=410 pM for ET-1, 2500 pM for ET-3). Calcium channel blockers such as nicardipine, verapamil and diltiazem did not affect the binding of 125I-ET-1. Affinity labeling of the particulate and solubilized membranes with CHAPS revealed a specific binding protein with a Mr of 32,000.

Site-directed Anti-peptide Antibodies Define the Topography of the β-Adrenergic Receptor

Abstract Molecular cloning has revealed the primary structure of a number of G-protein-linked receptors. The organization and topography of these proteins predicted to have seven hydrophobic membrane-spanning domains, in contrast, have not been established. Antibodies were prepared against 11 peptides corresponding to each of the hydrophilic sequences of the hamster beta 2-adrenergic receptor. Each of the anti-peptide antibodies displayed immunoreactivity for its synthetic peptide antigen and beta 2-adrenergic receptor (Mr 65,000) on blots of cell membranes and of purified receptor. All but three anti-peptide antisera also displayed immunoreactivity toward human placental and rat fat cell beta 1-adrenergic receptors, reflecting the level of sequence identity that exists between the two subtypes, Chinese hamster ovary cells stably transfected with an expression vector harboring the cDNA encoding the hamster beta 2-adrenergic receptor provided a cell type with 2 million receptors/cell, suitable for in situ localization of the sequences used as antigens. Indirect immunofluorescence of intact and permeabilized cells performed with these site-directed anti-peptide antibodies permitted the assignment of the general topography of each of the hydrophilic sequences of this G-protein-linked receptor. The results support the predictive value of hydropathy analysis for one class of membrane proteins with multiple transmembrane-spanning domains.

Changes in placental atrial natriuretic peptide receptors associated with severe toxemia of pregnancy

We have previously demonstrated the presence of atrial natriuretic peptide (ANP) specific receptors in normal human placentas. Since toxemia of pregnancy may affect important metabolic, transport and hemodynamic functions of placentas, we have asked the question whether binding properties of human placental ANP receptors are changed in patients with severe toxemia of pregnancy when compared to normal patients. ANP receptors in plasma membranes from normal and severely toxemic patients were characterized by in vitro radioligand assays utilizing [125I]-alpha-hANP. In all cases, we identified specific, high affinity, low capacity ANP binding sites in a microsomal fraction of human placentas. Although the total concentration of receptors did not differ between the two groups, the dissociation constant, KD, was significantly higher in placentas from severely toxemic patients than from normal controls. From the above we conclude that placental ANP receptors are dynamically modulated and their characteristics may be altered in severe toxemia of pregnancy.

Biosynthesis and processing of the mannose receptor in human macrophages

The biosynthesis and processing of the human mannose receptor has been studied in monocyte-derived macrophages. Adherent cells were labeled for 60 min with Trans35S (a mixture of 35S-labeled methionine and cysteine), chased, and subjected to immunoprecipitation by antibody raised against the human placental receptor. The antibody immunoprecipitated a single protein of molecular mass 162 kDa; precipitation of the labeled receptor could be inhibited by placental receptor. The results presented demonstrate that the receptor is synthesized as a 154-kDa precursor which is processed to 162 kDa in 90 min. The precursor is a glycoprotein bearing endoglycosidase H-sensitive oligosaccharides; the 162-kDa form is endoglycosidase H-resistant but peptide:N-glycanase-sensitive. Desialylation of the mannose receptor with neuraminidase generates a protein which is recognized by peanut agglutinin, a lectin that specifically binds desialylated O-linked oligosaccharides. Thus, the human macrophage mannose receptor bears both N- and O-linked oligosaccharide chains. Newly synthesized mannose receptor exhibits a half-life of 33 h as determined by pulse-chase studies. This indicates that on the average, each molecule of receptor recycles between the cell surface and endosomes hundreds of times before degradation.

The Arg-Gly-Asp binding domain of the vitronectin receptor. Photoaffinity cross-linking implicates amino acid residues 61-203 of the beta subunit.

We have employed photoaffinity cross-linking to examine RGD recognition by the human placental vitronectin receptor. The peptide GRGDSPK was coupled to a thiol-cleavable radioiodinatable aryl azide (sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate. When 125I-sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate-GRGDSPK was cross-linked to the vitronectin receptor in solution, 80% of the label was associated with the beta subunit. Cross-linking to both subunits of the receptor was highly specific and dependent upon the presence of divalent cations. Ca2+ and Mg2+ promoted RGD recognition by the receptor; however, the effects of each divalent cation were kinetically distinct. We have also identified and determined the amino acid sequence of chymotryptic and V8 protease-generated peptides of the beta subunit that were radiolabeled as a result of cross-linking. The results of these studies demonstrate that amino acid residues 61-203 are proximal to the RGD binding domain of the vitronectin receptor.

Characterization of human placental opioid receptors by 3H-ethylketocyclazocine and 3H-naloxone binding

The human placenta contains membrane bound opioid receptors. The binding of the antagonist naloxone and the fairly kappa-selective agonist EKC were studied in the microvillous membrane fraction. In both cases high affinity binding sites were detected with K d values in the nanomolar range. A series of kappa-selective ligand (PD-117302, EKC, and U-50, 488H) were tested in displacement experiments, and found to be potent inhibitors of agonist and antagonist binding. It was confirmed that large percentage of the binding is associated with the kappa sub-type which is of current interest, in that it shows distictive pharmacology and distribution and is selective for the natural opioid polypeptide, dynorphin.

Nonbinding inhibitory antiinsulin receptor antibodies. A new type of autoantibodies in human diabetes.

Sera and their IgG from 10/104 diabetic patients (five with insulin-dependent and five with noninsulin-dependent diabetes, NIDDM), contained antibodies that bound 125I-labeled purified human insulin receptors. 9 of these 10 sera failed to inhibit insulin binding (to rat hepatocytes and human placental membranes), did not stimulate glucose oxidation (by isolated rat adipocytes), and did not bind human placental IGF-1 receptors. Only one serum (and its IgG) modestly inhibited insulin binding and stimulated glucose oxidation. We conclude (a) that sera from 9/104 diabetics (five insulin-dependent and four noninsulin-dependent) contained a newly identified species of IgG antiinsulin receptor autoantibodies (AIRA), which bound to the insulin receptor at a locus different from the insulin binding site and did not inhibit insulin binding; and (b) that only 1/104 diabetic sera contained low-titer "conventional" antiinsulin receptor autoantibodies that bound to the insulin receptor at or near the insulin binding site, inhibited insulin binding and caused a clinical condition, which was difficult to distinguish from typical NIDDM.

Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts.

Transforming growth factors of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung fibroblasts (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to 20-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.

Human placental LHRH receptor: agonist and antagonist labeling produces differences in the size of the non-denatured, solubilized receptor.

Membranes of human term placenta were labeled with several photosensitive and non-photosensitive analogues of LHRH. In both groups agonistic and antagonistic peptide structures were tested. The photolabeling amino acid azidophenylalanine was placed either in positions 2, 6 or 7. All compounds were specifically displacing iodinated Buserelin from human placental membranes and from rat pituitary membranes. The iodinated photolabels were displaced by cold Buserelin, some compounds however had a high non-specific binding. Photolabeling experiments on human placental membranes with radioactive photolabels produced all a band at 58,000 daltons in SDS-gel electrophoresis. Solubilization with a non-denaturing detergent and gel filtration produced a major radioactivity peak which was attributed to the LHRH receptor. All labeling experiments with agonist labels produced Kav's indifferent from each other but significantly different from the Kav's of antagonist labeled membranes. This result was confirmed with similar experiments carried out with radioactive Buserelin and a radioactive antagonist under non-photolytic conditions. It is therefore concluded that the placental LHRH receptor contains an LHRH binding component of 58,000 daltons but that the native receptor is composed of several proteins. It is also concluded that agonist occupation of the receptor induces the association of a further component which might be involved in the transmembrane signalling system. The agonist labeled, solubilized native LHRH receptor has a Stokes radius of 52 A and the same, antagonist labeled receptor a radius of 48 A.

Isolation and characterisation of a human calcitonin-gene-related-peptide receptor.

We describe the identification and purification of a receptor for calcitonin-gene-related peptide (CGRP) from human term placenta, using lectin and beta-CGRP-Affigel affinity chromatography. The membrane-bound receptor has an estimated Mr of 240,000, as determined by cross-linking 125I-labelled alpha-CGRP (125I-alpha-CGRP) using discuccinimidyl suberate and SDS/polyacrylamide gel electrophoresis, or of 263,000, as judged by sucrose gradient centrifugation of the soluble partially purified native receptor preparation. Cross-linking studies with disuccinimidyl suberate and N-hydroxysuccinimidyl-4-azidobenzoate using membrane-solubilised, partially purified and CGRP-affinity-purified preparations, show a number of 125I-alpha-CGRP binding subunit(s) of Mr 62,000-68,000. Silver staining of the purified CGRP receptor preparation showed two distinct doublets in this plus a number of minor doublets of lower Mr. The receptor binds human beta-CGRP with greater affinity than alpha-CGRP, and showed little affinity for human calcitonin. Adsorption isotherms and Scatchard analysis of 125I-alpha-CGRP binding to the membrane-bound or soluble purified receptor are consistent, under the conditions used, with a single binding site of high affinity. Molecular cloning at present in progress should define the amino acid sequence and subunit composition of the human placental CGRP receptor, since at present the observed heterogeneity of CGRP-binding proteins may be interpreted in a number of ways, for instance: receptor heterogeneity, variable glycosylation of one of two subunits, or limited proteolysis of a single subunit during purification.

Effect of perturbation of specific folate receptors during in vitro erythropoiesis.

Although antisera to specific placental folate receptors inhibits the uptake of 5-methyltetrahydrofolate into cultured malignant human cells, little is known of the functional significance of folate receptors in normal human cells. Human bone marrow cells were therefore assayed for erythropoietic burst-forming units in the presence of an antihuman placental folate receptor serum and preimmune serum to determine the role of such a receptor in erythroid differentiation. When marrow cells were assayed in the presence of anti-receptor antiserum, there was (i) a threefold increase in erythropoietic burst formation and a twofold increase in the number of cells per erythroid burst; (ii) morphological evidence for nuclear/cytoplasmic dissociation of orthochromatic normoblasts composing erythroid bursts (megaloblastic erythropoiesis); (iii) intracellular folate deficiency with a 70% reduction of intracellular folate in antiserum treated cells as compared with control cells; and (iv) complete reversal of antiserum-induced changes on preincubation of antiserum with purified human placental folate receptor. These data support the conclusion that folate receptors on marrow cells provide an important function in the cellular uptake of folates during in vitro erythropoiesis. This process of folate uptake also appears to play a pivotal role in the differentiation and proliferation of erythroid progenitor cells.

Amino acid sequence of the human fibronectin receptor.

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.

Plasma concentrations and receptor binding of RU 486 and its metabolites in humans

Using Chromosorb® chromatography and HPLC, we measured the plasma concentrations of RU 486, and its monodemethylated (RU 42633), didemethylated (RU 42848) and alcoholic nondemethylated (RU 42698) metabolites up to 72 h following oral ingestion of 100 mg of RU 486 by five female volunteers. The peak plasma level of RU 486 (4.5 μmol/l) occurred within 1 h after ingestion of the compound; at this point significant amounts of the metabolites were also present in the plasma. After the initial redistribution within 6 h the plasma concentrations of RU 486 and three of its metabolites measured remained stable for 24 h. Concentrations of the monodemethylated metabolite exceeded those of the parent steroid during the time period measured, whereas the concentrations of the didemethylated and alcoholic metabolites were lower than those of RU 486, but still notable. At 72 h the concentrations of all the four steroids were still in the micromolar range. The relative bincling affinities of these metabolites to human endometrial and myometrial progesterone receptors as well as to human placental glueocorticoid receptors were determined in vitro. The affinity of RU 486 for the human uterine progesterone receptor ( K d = 1.3 × 10 −9 M for RU 486) was higher than that of progesterone but lower than that of ORG-2058, a potent synthetic progestin. The relative binding affinities of the monodemethylated, alcoholic and didemethylated metabolites to the progesterone receptor were 21, 15 and 9%, respectively, compared with the parent compound RU 486; each was lower than that of progesterone (43%). RU 486 had an approx. 4-fold higher relative bincling affinity to the glucocorticoid receptor than dexamethasone. Interestingly, the relative bincling affinities of the metabolites studied to the human glucocorticoid receptor exceeded those of dexamethasone or cortisol. Compared with the parent compound RU 486, they were 61, 48 and 45% for the monodemethylated, alcoholic and didemethylated metabolites, respectively; each was higher than that of dexamethasone (23%). The affinity of dexamethasone to the human glucocorticoid receptor was 1.6 × 10 −9 M. These data indicate that the pool of certain metabolites of RU 486 may contribute to a significant extent to the antiprogestagenic (23–33%) and even greater extent to the antiglucocorticoid (47–61%) effects of RU 486.

Improved purification of the human placental transferrin receptor and a novel immunoradiometric assay for receptor protein

A simplified method for the purification of human placental transferrin receptor is described. The procedure involves chromatography of a detergent extract of placenta on immobilized iron-loaded transferrin. Knowledge of the physiology of the interaction between transferrin and its receptor is applied to enable bound receptor to be eluted under mild conditions and essentially free of contaminating transferrin. Purified transferrin receptor and a monoclonal antibody to the receptor were used to developed a novel immunoradiometric assay for the receptor in which the monoclonal antibody is the radiolabelled species. A competition between two populations of receptor, one immobilized on a particulate support and the other in solution, provides the basis for the assay. Using this assay we have measured transferrin receptor levels in placental and hepatic tissue and in three cell lines during both the logarithmic and stationary phases of cell growth.

Nondetectable concentrations of human placental ah receptors are associated with potent induction of microsomal benzo[ a]pyrene hydroxylase in individuals exposed to polychlorinated biphenyls, quaterphenyls, and dibenzofurans

Human exposure to polychlorinated biphenyls, quaterphenyls, and dibenzofurans occurred in an accidental food poisoning episode in Taiwan in 1979. Our objective was to evaluate the role of Ah receptors in the production of biochemical effects detected in placentas from exposed subjects. Placentas were obtained from pregnant women who were identified from a registry of exposed individuals. Benzo[ a]pyrene (BaP) hydroxylase was quantified in placental microsomes and Ah receptor was investigated in placental cytosolic and nuclear fractions by a variety of receptor-binding techniques using 2,3,7,8-[1,6- 3H]tetrachlorodibenzo- p-dioxin ([ 3H]TCDD) as the ligand. Microsomal BaP hydroxylase was dramatically elevated in placentas of exposed subjects compared to those of nonexposed, nonsmoking control subjects. However, concentrations of displaceable [ 3H]TCDD binding in placental preparations from exposed or control subjects were uniformally low; approximately 1.0 fmol/mg cytosolic protein. Further evaluation of displaceable TCDD binding by sucrose-gradient sedimentation and hydroxylapatite column chromatography revealed that binding properties were different than those for the Ah receptor. These data suggest that extremely low concentrations of Ah receptors in human placenta may be sufficient to markedly elevate microsomal BaP hydroxylation. Alternatively, induction in human placenta may be mediated by a mechanism not requiring Ah receptor.

Purification and characterization of the human brain insulin receptor.

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.

Insulin-like growth factors I and II in fetal and adult bovine serum. Purification, primary structures, and immunological cross-reactivities.

Insulin-like growth factors I and II (IGF-I and IGF-II) were prepared from fetal calf serum and adult bovine serum by gel filtration, immunoaffinity chromatography, chromatofocusing, and reverse-phase high pressure liquid chromatography, and their complete amino acid sequences determined. IGF-I and -II are found in both adult and fetal serum. The sequence of bovine IGF-I is found to be identical to that of human IGF-I, whereas 3 out of 67 amino acid residues are found to be different between bovine and human IGF-II. The differences are located in the C-peptide region of the molecule. Bovine IGF-II shows less than 10% immunological cross-reactivity with antisera against human and rat IGF-II, but is equipotent to human IGF-II in displacing human 125I-labeled IGF-II from human placental receptor. Bovine IGF-I was equipotent to human IGF-I in both radioimmunoassays and radioreceptor assays within the limits of the assay.

Correlation between the number of placental opioid receptors, mode of delivery, and maternal narcotic use.

Human placental opioid receptors were assayed using the radioactive opioid agonist, etorphine, to determine the number of binding sites in villous tissue membrane preparations. Significant differences in receptor concentration per milligram of protein of tissue were found between placentas obtained following vaginal or abdominal delivery (P less than 0.002). Labor itself did not alter apparent receptor numbers. In patients with maternal narcotic abuse during pregnancy, no opioid binding could be detected regardless of the mode of delivery, suggesting possible receptor down-regulation.

Affinity of tocolytic agents on human placental and myometrial beta-adrenergic receptors.

The beta-adrenoreceptor antagonist [3H]-dihydroalprenolol (DHA) has been used to label adrenoreceptors in membranes from human pregnant myometrium and placenta. Six tocolytic drugs were tested for their ability to bind to the beta-adrenergic receptor of placental and myometrial membranes. Tocolytic agents competed with [3H]-DHA binding in the following order of potency: clenbuterol greater than fenoterol greater than ritodrine greater than isoxsuprine greater than orciprenaline greater than terbutaline. All drugs competed for [3H]-DHA binding sites with HILL coefficients greater than unity indicating heterogeneity of binding. In general, binding of beta-adrenoreceptor agonists was weaker in myometrium than in placenta homogenates. According to our results it is very likely that the placenta may play an important role in the pharmacological mechanism of tocolytic agents in inhibitory of premature labor.

The influence of extracellular folate concentration on methotrexate uptake by human KB cells. Partial characterization of a membrane-associated methotrexate binding protein.

Methotrexate accumulation, subcellular distribution, metabolism, and cytotoxicity were studied in human epidermoid carcinoma (KB) cells that were exposed to a low extracellular concentration of methotrexate (25 nM) following culture in widely differing concentrations of folic acid. KB cells cultured in standard medium with a high folic acid concentration (2.3 microM) had high levels of cellular folate (21.4 pmol/10(6) cells). Five passages through low folate (2.7 nM) medium reduced the level of cellular folate to near physiologic levels (0.4-1.0 pmol/10(6) cells). In contrast to KB cells cultured in standard medium, in KB cells cultured in low folate medium, 1) methotrexate inhibited growth; 2) methotrexate uptake was markedly increased; 3) methotrexate polyglutamation was almost complete; 4) methotrexate binding to dihydrofolate reductase was markedly enhanced; and 5) significant methotrexate binding to a previously undescribed membrane-associated protein occurred. The amount of methotrexate bound to the membrane-associated protein from KB cells cultured in low folate medium equaled the quantities bound by dihydrofolate reductase. Further characterization of this membrane-associated protein indicated that it was soluble in solutions containing Triton X-100, was capable of binding folic acid as well as methotrexate, had an apparent Mr of 160,000 by gel filtration in the presence of Triton X-100, and was precipitated by antiserum to human placental folate receptor. This membrane-associated protein may play an important role in the uptake and metabolism of methotrexate under physiologic conditions.