Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes.

Opresko, Lee K.; Wiley, H. Steven

doi:10.1083/jcb.111.4.1661

Cited by 33 publications

(26 citation statements)

References 62 publications

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“…A possible explanation for the activity of low concentrations of GM-CSF in the oocyte system could relate to the density of receptors expressed in the oocyte. Binding studies allowed us to estimate 5 x 108 to 1 x 1010 sites per oocyte, which corresponds to about 1000 to 20,000 sites per human cell, based on an average cell-surface area calculation (24,39). The dose-response curve for GM-CSF was, however, not related to the number of expressed receptors.…”

Section: Discussionmentioning

confidence: 99%

The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway.

Ding

Rivas²,

Heaney³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of an a and B3 subunit, which together form the high-affinity receptor. The a subunit by itself binds ligand at low affinity, whereas the isolated (8 subunit does not bind GM-CSF. It is generally believed that the high-affinity receptor is responsible for the multiple functions of GM-CSF and that the isolated a subunit (GMRa) does not transduce a signal. Xenopus laevis oocytes injected with RNA encoding human GMRa expressed up to 1010 low-affinity sites for GM-CSF (Kd = 6 nM). GM-CSF binding to the a subunit expressed in Xenopus oocytes caused activation of 2-deoxyglucose transport through endogenous glucose transporters. 2-Deoxyglucose transport was stimulated by similar low concentrations of GM-CSF in HL-60 leukemia cells as well as normal human neutrophils and Xenopus oocytes expressing GMRa. Engagement of the isolated a subunit in oocytes did not lead to protein phosphorylation or tyrosine phosphorylation of mitogen-activated protein kinase (MAP kinase). Staurosporin and genistein inhibited GM-CSFinduced tyrosine phosphorylation of MAP kinase in human neutrophils and HL-60 cells without affecting GM-CSFstimulated uptake of 2-deoxyglucose. These results provide direct evidence that the isolated a subunit signals for hexose transport and can do so without engagement of the kinase cascade. Our data also indicate that signaling for hexose uptake may occur in a phosphorylation-independent manner in cells expressing the high-affinity GM-CSF receptor.The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of an a and ( subunit, and the a subunit (GMRa) by itselfbinds ligand at low affinity (Kd = 3-7 nM) (1-6). The isolated 8 subunit (GMR(3) does not bind GM-CSF; however, it associates with GMRa to form a high-affinity receptor (Kd = 20-40 pM) (3-6). GM-CSF receptors are present on myeloid progenitors and mature neutrophils, eosinophils, and mononuclear phagocytes (7)(8)(9)(10)(11)(12)(13). GMRa is also present in some nonhematopoietic cells, such as placenta and melanoma cells (1,14,15), while the GMR(8 is mainly restricted to hematopoietic cells (3-5, 12).It is generally believed that the biological functions exerted by GM-CSF are mediated by the high-affinity receptor and that the isolated GMRa does not transduce a signal (3-6, 16, 17). GMRa, however, binds GM-CSF and in association with GMR,8 transduces signals involving protein phosphorylation (2,(18)(19)(20). To examine the functional capability of the isolated a subunit, we expressed it in Xenopus laevis oocytes and discovered that it could signal for glucose transport without involving the phosphorylation of mitogen-activated protein kinase (MAP kinase). MATERIALS AND METHODSComplementary DNAs for the human GMRa (21) and the human glucose transporter 4 (GLUT4) (22) cloned in pBluescript (Stratagene) were linearized by digestion with restriction enzymes, and the sense or antisense RNA was transcribed in vitro (23). Stage V and VI oocy...

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Section: Discussionmentioning

confidence: 99%

The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway.

Ding

Rivas²,

Heaney³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…SER and SER TKϪ -pcDNA3.1 plasmids were linearized by the enzyme PmeI, and pOBER plasmid (19) containing the human EGF-R (HER) was digested by NotI. cRNAs transcribed from 1 g of each linearized plasmid were precipitated by 2.5 M LiCl, washed in 70% ethanol, resuspended in 20 l of diethyl pyrocarbonate-treated water, and then quantified by spectrophotometry.…”

Section: Methodsmentioning

confidence: 99%

Conservation of Epidermal Growth Factor Receptor Function in the Human Parasitic Helminth Schistosoma mansoni

Vicogne

Cailliau

Tulasne

et al. 2004

Journal of Biological Chemistry

122

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The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni The epidermal growth factor receptor (EGF-R) 1 is a major key mediator of cell communication during animal development and homeostasis. EGF-R was the first receptor tyrosine kinase to be cloned (1), and its structure and activation pathways have been studied extensively. EGF-R represents the archetype of receptor tyrosine kinase with an extracellular ligand-binding part with two cysteine-rich repeats and an intracellular domain containing tyrosine kinase activity (2). In mammals, four isoforms of EGF-R have been characterized (EGF-R/ErbB-1, HER2/ErbB-2, HER3/ErbB-3, and HER4/ ErbB-4), and a number of different ligands, including epidermal growth factor (EGF)-like molecules, can selectively bind each isoform (3). Ligand binding activates the receptor by inducing the formation of homo-heterodimers. Dimerization triggers trans-phosphorylation and subsequent autophosphorylation of receptor molecules on tyrosine residues that provide docking sites for diverse effector and adaptor proteins. These partners (Grb2/Sos, p85-PI3K, PLC␥, and JAK) are active in different signal transduction cascades, such as the mitogenactivated protein kinase (MAPK), phosphoinositol 3-kinase, antiapoptotic kinase Akt, and several transcriptional regulatory pathways (reviewed in Ref. 4). Different homodimer-heterodimer combinations formed by EGF-R family members drive a complex signaling network within the MAPK pathway. The ERK pathway is the most recurrent and is mainly responsible for the mitogenic action of EGF receptors. Dysregulation of EGF-R signaling is therefore strongly oncogenic, and the direct implication of EGF-R isoforms in various cancers has been widely demonstrated. For this reason, EGF-R currently represents one of the major drug targets in human cancer therapy (5).In invertebrates, EGF-R isoforms appeared to be expressed in more limited numbers. A single isoform has been characterized in Caenorhabditis elegans (LET-23) (6) as well as in Drosophila melanogaster (DER) (7,8). A single cognate ligand (LIN-3) would be present in the worm (9), and four distinct cognate ligands (Vein, Gurken, Spitz, and Argos) would be present in the fly (10). These observations indicated that the EGF-R signaling module has grown in complexity from invertebrates to mammals. However, except for C. elegans and D. melanogaster models, few data are available at present about the role of the EGF-R family in invertebrate development.SER, the Schistosoma mansoni EGF-R homologue, is one of the three receptor tyrosine kinases that have been characterized in this trematode parasite (11,12). SER is present predominantly in schistosome muscles, suggesting that it could

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“…We therefore decided to gain further insight into the in vivo pathway of synthesis of human ET-1 by using the Xenopus oocyte system. Xenopus oocytes have proved to faithfully reproduce several steps of processing and transport on mammalian proteins synthesized upon mRNA microinjection (9)(10)(11). We report that the expression of human preproET-1 in oocytes results in Golgi-mediated secretion of molecules that have an elution profile identical to that of authentic ET-1, when fractionated by HPLC coupled to an RIA, and are active when tested in a contractile bioassay.…”

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confidence: 99%

Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

Fabbrini

Vitale

Patrono

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes njected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured.These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.Endothelin (ET), a 21-residue peptide, is one of the most potent vasoconstrictors known (1). In addition to its cardiovascular effects, recent studies indicate that ET could also act as a neurotransmitter or neurosecretory hormone (2). The regulation of ET biosynthesis in mammalian tissues is largely unknown. Interestingly, the peptide sequence shows a high degree of homology to the N terminus of a-scorpion neurotoxins (2) and particularly to the snake sarafotoxins that affect the heart (3), suggesting that similar mechanisms of synthesis and processing of the precursors could have survived in organisms as distant as mammals and reptiles. Sequencing of a human preproET-1 cDNA clone revealed that ET originates from a 212-amino acid precursor (4). The first 17 amino acids showed the sequence characteristics of a signal peptide for the translocation into the endoplasmic reticulum. Human ET spans the region from Cys53 to Trp73 and a 38-amino acid intermediate, termed big ET-1, has a 17-amino acid extension after Trp73. A possible pathway of ET biosynthesis has been proposed (1). In this pathway, the precursor is cleaved at a dibasic pair of residues and further processed by a specific ET converting enzyme that cleaves between Trp2l and Val22 of the last intermediate big

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Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes.

Cited by 33 publications

References 62 publications

The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway.

The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway.

Conservation of Epidermal Growth Factor Receptor Function in the Human Parasitic Helminth Schistosoma mansoni

Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

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