Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding

The Mr 46,000 mannose 6-phosphate receptor is an integral membrane protein with its ligand-binding site in the ectoplasmic domain. By site-directed mutagenesis, a stop codon was introduced in the receptor cDNA at the border between the ectoplasmic and membrane-spanning domain. The truncated receptor was expressed in three different systems, Xenopus oocytes, COS cells and BHK-21 cells. In all three systems the truncated receptor behaved as a soluble protein. In oocytes only small amounts of the truncated receptor were secreted within 48 h after synthesis. Accumulation of endoglucosaminidase H-sensitive forms of the truncated receptor in oocytes suggested that exit from the endoplasmic reticulum was slowed down. In COS and BHK-21 cells, the truncated receptor was secreted and, as for wild-type receptor, most of the N-linked oligosaccharides were processed to complex forms. Both the intracellularly-retained (oocytes) and the secreted (COS and BHK-21 cells) truncated receptors bound to phosphomannan-Sepharose in a mannose-6-phosphate-dependent manner. Using chemical cross-linking, the truncated receptor was shown to be secreted as a homodimer.

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Binding of Delta1, Jagged1, and Jagged2 to Notch2 Rapidly Induces Cleavage, Nuclear Translocation, and Hyperphosphorylation of Notch2

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6913-6922.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6913-6922 ◽

Cited By ~ 122

Author(s):

Kiyoshi Shimizu ◽

Shigeru Chiba ◽

Noriko Hosoya ◽

Keiki Kumano ◽

Toshiki Saito ◽

...

Keyword(s):

Ligand Binding ◽

Notch Signaling ◽

Time Course ◽

Nuclear Translocation ◽

Reporter Genes ◽

Time Dependent ◽

Dependent Manner ◽

Notch Receptors ◽

Notch Protein ◽

Membrane Spanning

ABSTRACT Delta1, Jagged1, and Jagged2, commonly designated Delta/Serrate/LAG-2 (DSL) proteins, are known to be ligands for Notch1. However, it has been less understood whether they are ligands for Notch receptors other than Notch1. Meanwhile, ligand-induced cleavage and nuclear translocation of the Notch protein are considered to be fundamental for Notch signaling, yet direct observation of the behavior of the Notch molecule after ligand binding, including cleavage and nuclear translocation, has been lacking. In this report, we investigated these issues for Notch2. All of the three DSL proteins bound to endogenous Notch2 on the surface of BaF3 cells, although characteristics of Jagged2 for binding to Notch2 apparently differed from that of Delta1 and Jagged1. After binding, the three DSL proteins induced cleavage of the membrane-spanning subunit of Notch2 (Notch2TM), which occurred within 15 min. In a simultaneous time course, the cleaved fragment of Notch2TMwas translocated into the nucleus. Interestingly, the cleaved Notch2 fragment was hyperphosphorylated also in a time-dependent manner. Finally, binding of DSL proteins to Notch2 also activated the transcription of reporter genes driven by the RBP-Jκ-responsive promoter. Together, these data indicate that all of these DSL proteins function as ligands for Notch2. Moreover, the findings of rapid cleavage, nuclear translocation, and phosphorylation of Notch2 after ligand binding facilitate the understanding of the Notch signaling.

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Site-directed removal of N-glycosylation sites in the bovine cation-dependent mannose 6-phosphate receptor: effects on ligand binding, intracellular targetting and association with binding immunoglobulin protein

Biochemical Journal ◽

10.1042/bj2950841 ◽

1993 ◽

Vol 295 (3) ◽

pp. 841-848 ◽

Cited By ~ 22

Author(s):

Y Zhang ◽

N M Dahms

Keyword(s):

Ligand Binding ◽

Intracellular Transport ◽

Polypeptide Chain ◽

Site Directed Mutagenesis ◽

Pulse Labelling ◽

Wild Type ◽

Binding Ability ◽

Oligosaccharide Chain ◽

Glycosylation Sites ◽

Mannose 6 Phosphate

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) contains five potential N-linked glycosylation sites, four of which are utilized. To evaluate the function of these oligosaccharides, site-directed mutagenesis was used to generate glycosylation-deficient CD-MPR mutants lacking various potential glycosylation sites. The mutants were constructed in both a full-length and a soluble truncated (STOP155 construct) form of the receptor and their properties were examined in transfected COS-1 cells. The results showed that the presence of a single oligosaccharide chain, particularly at position 87, on the CD-MPR significantly enhanced its mannose 6-phosphate (Man-6-P)-binding ability when compared with non-glycosylated receptors. In addition, the presence of a single oligosaccharide chain at position 87, and to a lesser degree at position 31 or 81, promoted the secretion of the STOP155 CD-MPR. Pulse-labelling of transfected COS-1 cells followed by immunoprecipitation with binding immunoglobulin protein (BiP)-specific and CD-MPR-specific antibodies indicated that BiP associated with the non-glycosylated forms of the receptor but not with the wild-type CD-MPR. Furthermore, the association of the various glycosylation-deficient forms of the CD-MPR with BiP correlated inversely with their ability to bind Man-6-P. From these results we conclude that N-glycosylation of the bovine CD-MPR facilities the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and ligand binding.

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Exogenous Cripto-1 Suppresses Self-Renewal of Cancer Stem Cell Model

International Journal of Molecular Sciences ◽

10.3390/ijms19113345 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3345 ◽

Cited By ~ 1

Author(s):

Md Alam ◽

Ryota Takahashi ◽

Said Afify ◽

Aung Oo ◽

Kazuki Kumon ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cancer Stem Cells ◽

Cancer Stem Cell ◽

Stop Codon ◽

Site Directed Mutagenesis ◽

Soluble Form ◽

Dependent Manner ◽

Self Renewal ◽

Mesenchymal Transition

Cripto-1 is a glycophosphatidylinositol (GPI) anchored signaling protein of epidermal growth factor (EGF)-Cripto-1-FRL1-Cryptic (CFC) family and plays a significant role in the early developmental stages and in the different types of cancer cells, epithelial to mesenchymal transition and tumor angiogenesis. Previously, we have developed cancer stem cells (miPS-LLCcm) from mouse iPSCs by culturing them in the presence of conditioned medium of Lewis Lung Carcinoma (LLC) cells for four weeks. Nodal and Cripto-1 were confirmed to be expressed in miPS-LLCcm cells by quantitative reverse transcription PCR (rt-qPCR) implying that Cr-1 was required in maintaining stemness. To investigate the biological effect of adding exogenous soluble CR-1 to the cancer stem cells, we have prepared a C-terminally truncated soluble form of recombinant human CR-1 protein (rhsfCR-1), in which the GPI anchored moiety was removed by substitution of a stop codon through site-directed mutagenesis. rhsfCR-1 effectively suppressed the proliferation and sphere forming ability of miPS-LLCcm cells in a dose-dependent manner in the range of 0 to 5 µg/mL, due to the suppression of Nodal-Cripto-1/ALK4/Smad2 signaling pathway. Frequency of sphere-forming cells was dropped from 1/40 to 1/69 by rhsfCR-1 at 1 µg/mL. Moreover, rhsfCR-1 in the range of 0 to 1 µg/mL also limited the differentiation of miPS-LLCcm cells into vascular endothelial cells probably due to the suppression of self-renewal, which should reduce the number of cells with stemness property. As demonstrated by a soluble form of exogenous Cripto-1 in this study, the efficient blockade would be an attractive way to study Cripto-1 dependent cancer stem cell properties for therapeutic application.

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Parathyroid Ca2+-conducting currents are modulated by muscarinic receptor agonists and antagonists

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1997.273.5.e880 ◽

1997 ◽

Vol 273 (5) ◽

pp. E880-E890 ◽

Cited By ~ 1

Author(s):

Wenhan Chang ◽

Tsui-Hua Chen ◽

Stacy A. Pratt ◽

Benedict Yen ◽

Michael Fu ◽

...

Keyword(s):

Muscarinic Receptors ◽

Inositol Phosphate ◽

Receptor Protein ◽

Dependent Manner ◽

Rt Pcr ◽

Pipette Solution ◽

Pcr Products ◽

Inositol Phosphate Accumulation ◽

Receptor Cdna ◽

Dose Dependent

Parathyroid cells express Ca2+-conducting cation currents, which are activated by raising the extracellular Ca2+ concentration ([Ca2+]o) and blocked by dihydropyridines. We found that acetylcholine (ACh) inhibited these currents in a reversible, dose-dependent manner (50% inhibitory concentration ≈10−8 M). The inhibitory effects could be mimicked by the agonist (+)-muscarine. The effects of ACh were blunted by the antagonist atropine and reversed by removing ATP from the pipette solution. (+)-Muscarine enhanced the adenosine 3′,5′-cyclic monophosphate (cAMP) production by 30% but had no effect on inositol phosphate accumulation in parathyroid cells. Oligonucleotide primers, based on sequences of known muscarinic receptors (M1-M5), were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify receptor cDNA from parathyroid poly (A)+ RNA. RT-PCR products displayed >90% nucleotide sequence identity to human M2- and M4-receptor cDNAs. Expression of M2-receptor protein was further confirmed by immunoblotting and immunocytochemistry. Thus parathyroid cells express muscarinic receptors of M2 and possibly M4 subtypes. These receptors may couple to dihydropyridine-sensitive, cation-selective currents through the activation of adenylate cyclase and ATP-dependent pathways in these cells.

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Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

BMC Biology ◽

10.1186/s12915-020-00946-6 ◽

2021 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Jothi K. Yuvaraj ◽

Rebecca E. Roberts ◽

Yonathan Sonntag ◽

Xiao-Qing Hou ◽

Ewald Grosse-Wilde ◽

...

Keyword(s):

Ligand Binding ◽

Bark Beetle ◽

Pest Control ◽

Binding Sites ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

Odorant Receptors ◽

Functional Evolution ◽

General Importance ◽

Insight Into

Abstract Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

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Role of Cysteine Residues in the N-Terminal Extracellular Domain of the Human VIP 1 Receptor for Ligand Binding A Site-directed Mutagenesis Studya

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1996.tb17524.x ◽

2006 ◽

Vol 805 (1) ◽

pp. 585-589 ◽

Cited By ~ 4

Author(s):

PASCALE GAUDIN ◽

ALAIN COUVINEAU ◽

JEAN-JOSÉ MAORET ◽

CHRISTIANE ROUYER-FESSARD ◽

MARC LABURTHE

Keyword(s):

Ligand Binding ◽

Extracellular Domain ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

A Site

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Isolation and characterization of the two glycosylation isoforms of low molecular weight mannose 6-phosphate receptor from bovine testis. Effect of carbohydrate components on ligand binding.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)47417-6 ◽

1991 ◽

Vol 266 (26) ◽

pp. 17621-17630

Author(s):

M.M. Li ◽

G.W. Jourdian

Keyword(s):

Molecular Weight ◽

Ligand Binding ◽

Low Molecular Weight ◽

Isolation And Characterization ◽

Mannose 6 Phosphate ◽

Carbohydrate Components

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Tissue plasminogen activator is a ligand of cation-independent mannose 6-phosphate receptor and consists of glycoforms that contain mannose 6-phosphate

Scientific Reports ◽

10.1038/s41598-021-87579-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

James J. Miller ◽

Richard N. Bohnsack ◽

Linda J. Olson ◽

Mayumi Ishihara ◽

Kazuhiro Aoki ◽

...

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Dependent Manner ◽

Plasminogen Activation ◽

Urokinase Plasminogen Activator Receptor ◽

Plasminogen Activation System ◽

Extracellular Region ◽

Glycosylation Sites ◽

Tissue Plasminogen ◽

Mannose 6 Phosphate

AbstractPlasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

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