Specific Receptor-mediated Process Research Articles

LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3.

Storage proteins are the major protein synthesized in the fat body, released into hemolymph and re-sequestered into the fat body before pupation in most insect species. Storage proteins are important amino acid and nutrition resources during the non-feeding pupal period and play essential roles for the metamorphosis and oogenesis of insects. The sequestration of storage protein is a selective, specific receptor-mediated process. However, to date, the potential receptor mediating the sequestration of storage protein has not been determined in Bombyx mori. In this study, we expressed and purified the first ligand binding domain of Bombyx mori vitellogenin receptor (BmVgR), LBD1, and found LBD1 could bind with an unknown protein from the hemolymph of the ultimate silkworm larval instar via pull-down assay. This unknown protein was subsequently identified to be the female-specific storage protein SP1 by mass spectrometry. Furthermore, far western blotting assay, immunoprecipitation and isothermal titration calorimetry analysis demonstrated LBD1 specifically bound with the female-specific SP1, rather than another unisex storage protein SP2. The specific binding of LBD1 with SP1 was dependent on the presence of Ca2+ as it was essential for the proper conformation of LBD1. Deletion mutagenesis and ITC analysis revealed the first and third ligand binding repeats LBR1 and LBR3 were indispensable for the binding of LBD1 with SP1, and LBR2 and LBR4 also had a certain contribution to the specific binding. Our results implied BmVgR may mediate the sequestration of SP1 from hemolymph into the fat body during the larval-pupal transformation of Bombyx mori.

Identifying an essential interaction between malaria parasites and erythrocytes unlocks the door to promising vaccine targets

Invited Commentary on key findings in the literature It has long been known that erythrocyte invasion by the merozoite stage of the malaria parasite is a specific receptor-mediated process. Over the last twenty years several laboratories have attempted to exploit novel molecular and biochemical approaches to identify the surface molecules on both the parasite and erythrocyte membranes that mediate erythrocyte invasion. Research has focused on two groups of proteins secreted from the rhoptries and micronemes, secretory organelles that, upon contact with the host cell, release proteins essential both for orientation of the parasite at the erythrocyte surface and for invasion and formation of the parasitophorous vacuole. A number of ligand:receptor interactions have been identified yet attempts to disrupt these interactions experimentally with specific antibodies or through competitive binding assays have shown an inhibitory effect only in a subset of parasite strains.1 Similarly, genetic knockouts of these parasite ligands have failed to completely block parasite invasion of erythrocytes because of the functional redundancy of different receptor-ligand pairs. Furthermore, many parasite ligands have a low affinity for their corresponding erythrocytes receptors yet achieve high overall avidity due to high density of both the receptors and ligands on the parasite and erythrocyte surfaces at the point of contact. Therefore, many biologically relevant parasite-erythrocyte interactions cannot be investigated using conventional biochemical assays such as co-immunoprecipitation because they are designed to reveal high affinity interactions. In a recent paper by Crosnier et al.2 a novel screen was employed to detect low-affinity protein interactions between PfRh5 (Reticulocyte-binding protein homologue 5), a gene previously shown to be essential for erythrocyte invasion,3 and a panel of all the surface-exposed protein domains putatively expressed on the erythrocyte. To detect biochemically a low affinity, high avidity interaction, the authors used a mammalian cell expression system to produce pentamers of candidate erythrocyte protein domains and checked for in vitro binding. A single protein was implicated by this screen, basigin, a member of the immunoglobulin superfamily that determines the Ok blood group antigen.4 The requirement for the interaction between PfRh5 and basigin in erythrocyte invasion was confirmed through several lines of experimental evidence: excess soluble basigin, anti-basigin antibodies and knockdown by RNAi all led to a drastic reduction in erythrocyte invasion by merozoites. Importantly, a similar effect was seen across all parasite strains tested, including several laboratory-adapted lines and six freshly isolated parasite isolates from the field in West Africa. If this interaction is so conserved and fundamental we might expect selection for mutations in basigin in malaria-endemic areas that diminish parasite binding. The authors assayed existing genetic basigin variants, some of which showed reduced binding affinity for PfRh5. None of these were isolated from malaria-endemic areas, however, and future studies will assess whether in malaria-endemic regions basigin gene variants are causally associated with malaria incidence. One glaring question that emerges from this research is whether the inhibitory effect on invasion mediated by antibodies against a (presumably) continually exposed erythrocyte antigen is mimicked by antibodies against the parasite antigen PfRh5, which might only be exposed fleetingly following release from the rhoptry on contact with the erythrocyte. In this light, very recent work showing that vaccine-induced anti-PfRH5 antibodies can potently inhibit parasite invasion in vitro5 is very encouraging and suggests that future malaria vaccine research will focus heavily on this basigin-PfRH5 interaction.

Polycation/DNA complexes coated with oligonucleotides for gene delivery

Ternary nanoparticles with negatively charged surface were prepared by coating single-stranded oligonucleotides (5′–C 10A 20–3′) on histidine-conjugated polyallylamine (PAA-HIS)/DNA complexes for gene delivery. Characterization of PAA-HIS/DNA/oligonucleotide complexes demonstrated that nanoparticles possessed the negative surface charge −27 mV and size of around 100 nm when the molar ratio of oligonucleotide/PAA-HIS exceeded 1.5. The negatively charged oligonucleotide-coated PAA-HIS/DNA complexes could be entirely internalized by the living HeLa cells to exhibit high gene expression with low cytotoxicity and the resistance against erythrocyte agglutination and serum inhibition. Since the gene expression of PAA-HIS/DNA complexes was significantly inhibited by coating other polyanions and oligonucleotides, the ternary PAA-HIS/DNA/deoxyadenosine-rich oligonucleotide complexes were uptaken by specific receptor-mediated process. Additionally, the deposition of a layer of oligonucleotides onto the binary PAA-HIS/DNA complexes could effectively transfect various types of cells including HEK-293, HepG2 and Hs68 cells, indicating the technology of coating specific oligonucleotides on PAA-HIS/DNA complexes or other cationic binary DNA complexes might facilitate the use of nanoparticles for safe and efficient gene delivery and eventual therapy.

Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells.

We have previously demonstrated that human bronchial epithelial cells engulf apoptotic eosinophils. To compare and contrast the phagocytic capabilities of monocyte-derived macrophage and primary airway epithelial cells for apoptotic granulocytes. Here we compared phagocytosis of human apoptotic eosinophils and neutrophils by small and large airway epithelial cells (SAEC and LAEC) and monocyte-derived macrophages. Confocal microscopy of F-actin staining and scanning and transmission electron microscopy revealed phagocytic cup formation around apoptotic eosinophils by airway epithelial cells (AEC) membranes with evidence of their digestion. Resting and cytokine-stimulated AEC did not recognize and ingest apoptotic neutrophils. The latter were phagocytosed by macrophages that exhibited greater ingestion of and higher capacity for, apoptotic eosinophils over apoptotic neutrophils. Cytochalasin D completely abolished uptake of apoptotic eosinophils by SAEC, LAEC or macrophage monolayers. Ligation of epithelial cell CD44 receptors for 24 h increased phagocytosis of apoptotic eosinophils by SAEC and LAEC with a potency comparable with that of IL-1. Phagocytosis was a specific receptor-mediated process involving integrin- (alphavbeta3, alphavbeta5, CD36), phosphatidylserine receptor- and lectin-dependent mechanisms. No significant differences were observed in avarice for apoptotic eosinophils by SAEC or LAEC either resting, CD44 monoclonal antibodies- or cytokine- stimulated, or in their usage and expression of recognition receptors. These findings further suggest and define an important role for the bronchial epithelium in the selective removal of apoptotic eosinophils from the airways in asthma.

Biological actions of the free acid of hepoxilin A 3 on human neutrophils

In earlier reports and reviews, it was suggested that unlike its methyl ester, the free acid form of the 12-lipoxygenase-derived eicosanoid hepoxilin A 3 (HXA 3) does not enter neutrophils and other cells. Therefore, in the past, most studies on the biological activities of HXA 3 on human neutrophils were conducted with its methyl ester. Here, we present evidence that free HXA 3 is biologically active towards human neutrophils at submicromolar concentrations, which may occur under certain circumstances in vivo. Thus, HXA 3 caused chemotaxis at concentrations as low as 30–40 nM, an effect which was attenuated at higher concentrations of this eicosanoid. Its chemotactic potency proved to be comparable to that of leukotriene B 4, but higher than that of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP), and greatly exceeded that of the other 12-lipoxygenase metabolite, 12( S)-hydroxy-5,8,10,14-eicosatetraenoic acid, which was inactive at comparable concentrations. The chemotactic activity of HXA 3 was not abolished by serum albumin, but it was suppressed by pertussis toxin. Unlike fMLP, at this concentration range HXA 3 did not cause respiratory burst or aggregation of the neutrophils or activation of protein kinase C. These observations suggest a remarkably selective and specific receptor-mediated process. At concentrations higher than 1 μM, HXA 3 gives rise to an instantaneous release of calcium from intracellular stores which causes, however, only a slight, if any, liberation of arachidonic acid. On the other hand, pretreatment of the neutrophils with submicromolar concentrations of HXA 3 significantly blunts the liberation of arachidonic acid caused by fMLP.

17Beta-oestradiol enhances nitric oxide synthase activity in endothelium-denuded rat aorta.

1. It has been suggested that oestrogen-produced vasodilatation is due to induction of endothelial nitric oxide synthase (NOS), but there are many reports of direct effects on vascular smooth muscle. In the present study, these processes were investigated in rat aorta isolated from ovariectomized rats. 2. Short-term treatment (10 min) with 17beta-oestradiol (10 micromol/L) produced a small attenuation of the phenylephrine (PE)-induced constriction, which was unaffected by the nitric oxide synthase inhibitor L-N5(-1-iminoethyl)ornithine (NIO; 100 micromol/L). Long-term treatment (6 h) with 17beta-oestradiol (10 micromol/L) did not affect acetylcholine-mediated vasorelaxation in endothelium-intact aortic rings, but did attenuate PE-induced constriction. This attenuation was also observed in endothelium-denuded preparations after 17beta-oestradiol (10 micromol/L for 6 h) and was far greater than the acute effect of 17beta-oestradiol (10 micromol/L). 3. The attenuation produced by 17beta-oestradiol (10 micromol/L for 6 h) was significantly inhibited by concomitant treatment with cycloheximide (1 micromol/L), suggesting that protein synthesis was involved. NIO (100 micromol/L) also attenuated the effect, which suggests that the anti-constrictor effect of 17beta-oestradiol occurs through the increased production of nitric oxide (NO). 17Beta-oestradiol increased NO production, as assessed by the conversion of [3H]-arginine to [3H]-citrulline in rat aorta. These effects were prevented by cycloheximide and NIO. The anti-constrictor effect of oestrogen was blocked by the oestrogen receptor antagonist ICI 182 780 (100 nmol/L). 4. Western blotting using an antibody specific for inducible nitric oxide synthase (NOS) revealed that 17beta-oestradiol (10 micromol/L for 24 h) treatment induced the formation of inducible NOS protein in the aorta, an effect blocked by cycloheximide. The results indicate that 17beta-oestradiol can attenuate the vasoconstrictor effect of PE by a specific receptor-mediated process that involves induction of inducible NOS.

In vitro glial responses to nerve growth factor.

Nerve growth factor (NGF) stimulates expression of the low affinity neurotrophin receptor p75NGFR mRNA in primary cultures of neonatal rat cortical type I astrocytes. Nerve growth factor treatment altered glial morphology in glial fibrillary acidic protein positive (GFAP+) cell cultures derived from newborn (P0) and 3-day-old (P3) rat pups. When P0- or P3-derived primary glial cultures were serum-deprived, in the presence of 200 pM NGF for 5 days, the flat polygonal glia present in culture assumed a fibrous morphology, an effect not seen in the untreated serum-deprived controls. The NGF effect on astrocytic morphology was blocked by continuous serum treatment. Nerve growth factor did not stimulate astrocytic proliferation under these culture conditions, as assayed by cell cycle analysis using 3H thymidine autoradiography. P0-derived primary glial cultures expressed the signal transducing neurotrophin receptors p145trkB and p140trkA as determined by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products were identified by sequencing or restriction enzyme analysis. Astrocytes internalized 125I-NGF at 37 degrees C but not at 4 degrees C, consistent with energy requirements for internalization. Also, internalization of 125I-NGF was abolished by the addition of a 300-1,000-fold excess of unlabeled NGF. Thus, astroglial cells in culture internalize NGF through a specific receptor-mediated process, express trkA and full-length trkB mRNAs at low levels, and respond to exogenous NGF by expressing a fibrous morphology under serum-free culture conditions.

Prostaglandin E2 and rat liver regeneration

Background: Prostaglandin (PG) is reported to be involved in hepatic regeneration. However, little is known about the detailed relation in PG-induced stimulation of the proliferation. The present study was attempted to elucidate the relation. Methods: The serial change of PGE2 level released from the regenerating rat liver and the effect of PGE2 on the proliferation of rat hepatocytes were studied, with special reference to PGE2 binding and cyclic AMP (cAMP). Results: The PGE2 level increased 3 hours and 10 hours after partial hepatectomy. Timings of these increases seemed to coincide with those of the first and second increase of cAMP in the liver before the initiation of DNA synthesis. DNA synthesis of hepatocytes in primary culture between 24 and 36 hours, 36 and 48 hours, and 48 and 60 hours of culture were significantly enhanced by addition of PGE2 between 4 and 24 hours of culture at concentrations of 2 nmol/L to 1 μmol/L, 2 nmol/L to 200 nmol/L, and 5 μmol/L to 10 μmol/L, and 2 nmol/L and 10 μmol/L, respectively. Enhancement of DNA synthesis with PGE2 at concentrations less than 1 μmol/L seemed to be associated with the high-affinity binding and that at high concentrations with the lowaffinity binding. Intracellular cAMP level in the hepatocytes increased during culture, and its increase was enhanced by PGE2 addition. Conclusions: It is suggested that PGE2 production in the liver increases biphasically during hepatic regeneration and that PGE2 enhances the proliferation of hepatocytes by a specific receptor-mediated process, which is largely associated with cAMP-dependent process.

Receptor-Mediated Endocytosis of an Extracellular Steroid-Binding Protein (TeBG) in MCF-7 Human Breast Cancer Cells*

Previous studies suggested that an extracellular steroid-binding protein, testosterone-estradiol-binding globulin (TeBG), can enter a variety of cells. Experiments were conducted to determine whether uptake of TeBG occurs by nonspecific fluid phase endocytosis or via a specific receptor-mediated process. In human breast carcinoma cells (MCF-7) maintained on serum-free medium, exposure to radiolabeled TeBG resulted in cellular uptake, which reached a plateau by 6 h and could be inhibited 80% by competition with unlabeled TeBG. Uptake was temperature dependent with cell-associated radioactivity at 37 C being 1.6-fold higher than at 4 C. Subsequent exposure of cells to pronase resulted in release of the cell-associated TeBG by 88% and 44% at 4 C and 37 C, respectively. After transfer to media devoid of TeBG, approximately 35% of cell-associated radioactivity was release into the medium at 37 C; it was not possible to distinguish whether this was released from the cell surface or from inside the cell. Investigation of the localization of TeBG-gold complexes by electron microscopy revealed that TeBG first binds to the plasmalemma. Within 15 min label appears in receptosomes, which fuse to form multivesicular endosomes. By 1 h all label is observed in multivesicular endosomes and lysosomes, most of which are in the Golgi zone. Localization of the internalized radioactivity using classical cell fractionation techniques showed it appears in a symmetrical band exhibiting the same buoyant density as the lysosomal marker acid phosphatase. The observations reported here show that: 1) TeBG binds to MCF-7 cells; 2) some of the bound TeBG is taken up via a mechanism with all the characteristics of receptor-mediated endocytosis; and 3) within these cells TeBG is localized in endosomes and lysosomes.

Binding and degradation of NH2-terminal parathyroid hormone by opossum kidney cells

The binding and cellular processing of NH2-terminal parathyroid (PTH) hormone by confluent monolayers of opossum kidney (OK) cells was characterized using radiolabeled PTH peptide analogues. Time- and temperature-dependent specific binding of 125I-labeled (Nle-8,18, Tyr-34)-NH2-bovine(b)PTH-(1-34) was accompanied by the appearance of degraded radiolabel in the cell medium. Degrading activity was observed to be a specific consequence of binding by PTH receptors. Degrading activity was inhibited by monensin, chloroquine, and NH4+ but not by chymotrypsin inhibitors. Acid washing demonstrated that greater than 80% of total cell-associated specific binding at equilibrium was located in a rapidly internalized (acid-resistant) pool. Monensin pretreatment led to increased acid-resistant binding, presumably through inhibition of turnover of internalized receptor ligand and indicated that the degradation of radiolabel was probably associated with processing of the receptor-ligand complex. Release of intact radiolabel from the acid-resistant pool indicated that some of the internalized peptide was recycled out of the cell in an undegraded form (retroendocytosis). Acid-resistant binding and degradation of 125I-(Nle-8,18, Tyr-34)-NH2-bPTH-(3-34) was minimal, indicating that this ligand was not internalized. It is concluded that the binding and internalization of PTH-(1-34) fragment by confluent OK cells is a specific receptor-mediated process. Cellular processing of PTH-(1-34) conforms to established models of internalization by receptor-mediated endocytosis.

Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro

Following suggestions that transferrin present in fetal-bovine serum, a common supplement used in tissue-culture media, may not bind well to human cells, we have isolated the protein and investigated its interaction with both human and bovine cells. Bovine transferrin bound to a human cell line, K562, at 4 degrees C with a kd of 590 nM, whereas human transferrin bound with a kd of 3.57 nM, a 165-fold difference. With a bovine cell line, NBL4, bovine transferrin bound with the higher affinity, kd 9.09 nM, whereas human transferrin bound with a kd of 41.7 nM, only a 5-fold difference. These values were reflected in an 8.6-fold difference in the rate of iron delivery by the two proteins to human cells, whereas delivery to bovine cells was the same. Nevertheless, the bovine transferrin was taken up by the human cells by a specific receptor-mediated process. Human cells cultured in bovine diferric transferrin at 40 micrograms/ml, the concentration expected in the presence of 10% fetal-bovine serum, failed to thrive, whereas cells cultured in the presence of human transferrin proliferated normally. These results suggest that growth of human cells in bovine serum could give rise to a cellular iron deficiency, which may in turn lead to the selection of clones of cells adapted for survival with less iron. This has important consequences for the use of such cells as models, since they may have aberrant iron-dependent pathways and perhaps other unknown alterations in cell function.

Haem transport to the liver by haemopexin. Receptor-mediated uptake with recycling of the protein.

Rat [(59)Fe]haem-(125)I-labelled haemopexin complexes (700pmol/rat) associate rapidly and exclusively with the liver after intravenous injection into anaesthetized rats. The two isotopes exhibit different patterns of accumulation. Liver (125)I-labelled haemopexin is maximum 10min after injection (20+/-4.9pmol/g of liver) and then declines by 2h to the low values (about 3pmol/g of liver) seen after injection of the apoprotein. In contrast, [(59)Fe]haem accumulates in the liver for at least 2h. Haemopexin undergoes no extensive proteolysis during 2h of haem transport as shown by precipitation with acid (98%) and specific antiserum (92%) and by electrophoresis. Moreover, only 1-2% of the dose is located in extrahepatic tissues, and there is no significant urinary excretion of either (125)I or (59)Fe. Hepatic uptake at 10min is saturable, reaching 200pmol of haemopexin/g of liver and 350pmol of haem/g of liver at a dose of 9nmol/rat, whereas uptake of the apoprotein is 3-5% of the dose. This suggests that the interaction of haem-haemopexin with the liver is a specific receptor-mediated process. The complex probably interacts via the protein moiety, since the haem analogues mesohaem and deuterohaem do not affect association of the protein with the liver but the species of haemopexin does. Increasing amounts of protein are associated with the liver 5min after injection in the order: human>rabbit>rat, and haem uptake is consistently increased. For both rat and rabbit haemopexin saturation is reached at the same concentration of protein, i.e. 180-200pmol/g of liver, indicating that the different protein species bind to a common receptor. We propose that haemopexin transports haem to the liver by a specific receptor-mediated process and then returns to the circulation.