scholarly journals Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.

1995 ◽  
Vol 6 (8) ◽  
pp. 967-980 ◽  
Author(s):  
K Goishi ◽  
S Higashiyama ◽  
M Klagsbrun ◽  
N Nakano ◽  
T Umata ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Vero Cell ◽  
Half Life ◽  
Immunofluorescent Staining ◽  
Simultaneous Increase ◽  
Cell Contact ◽  
Heparin Binding ◽  
Paracrine Growth Factor ◽  
Rapid Processing

Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.

Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system

1992 ◽  
Vol 102 (3) ◽  
pp. 543-555 ◽  
Author(s):  
L.P. Baker ◽  
Q. Chen ◽  
H.B. Peng
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Acetylcholine Receptor ◽  
Hot Spots ◽  
Hot Spot ◽  
Muscle Cells ◽  
Membrane Receptors ◽  
Heparin Binding ◽  
Polystyrene Beads ◽  
Myotomal Muscle

Aneural muscle cells in culture often form acetylcholine receptor (AChR) clusters, termed hot spots, which are similar to those found at the postsynaptic membrane both in structure and in molecular composition. Although hot spots form on both dorsal and ventral surfaces of the cell, the ventral ones are better characterized because of their association with sites of cell-substratum contact. To understand the stimuli and mechanisms involved in ventral hot spot formation, native, uncoated polystyrene beads were applied to cultured Xenopus myotomal muscle cells to create local membrane-substratum contacts. These beads were able to induce a postsynaptic-type development as evidenced by the clustering of AChRs and the development of a set of ultrastructural specializations, including membrane infoldings and a basement membrane. Whereas these native beads were effective in inducing clustering, beads coated with bovine serum albumin or treated with serum-containing medium were ineffective. Native beads were also capable of inducing clusters in serum-free medium, indicating that their effect was mediated by endogenous molecules that were locally presented by the beads, rather than by bead adsorption of components in the medium. Heparan sulfate proteoglycan (HSPG) is a major component of the muscle extracellular matrix and our previous study has shown that basic fibroblast growth factor (bFGF), a member of the heparin-binding growth factor (HBGF) family, and its receptor are present in Xenopus myotomal muscle during the period of synaptogenesis. Therefore, we tested the involvement of HBGF in bead induction. The results of this study show the following: (1) preincubation of cultures in heparin, which solubilizes matrix-bound HBGFs, suppressed the bead-induced AChR clustering. (2) Suramin, which interferes with the interaction between several growth factors and their receptors, also inhibited bead-induced clustering. (3) Tyrphostin, which blocks tyrosine kinase activity associated with a number of growth factor receptors, was also inhibitory to the bead effect. (4) The percentage of bead-induced AChR clusters was significantly enhanced by pretreating the cultures with bFGF prior to bead application. This exogenously applied bFGF could be largely removed by treatment of cultures with heparin, suggesting its association with HSPG at the cell surface. (5) An anti-bFGF neutralizing antiserum significantly reduced the efficacy of the bead stimulation. These data suggest that uncoated beads, which adhere to the cell surface and can mimic the cell-substratum interaction, effect a local presentation of HBGFs, such as bFGF, residing with the HSPG to their membrane receptors, thereby locally activating receptor-associated tyrosine kinases.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The Membrane-anchoring Domain of Epidermal Growth Factor Receptor Ligands Dictates Their Ability to Operate in Juxtacrine Mode

2005 ◽  
Vol 16 (6) ◽  
pp. 2984-2998 ◽  
Author(s):  
Jianying Dong ◽  
Lee K. Opresko ◽  
William Chrisler ◽  
Galya Orr ◽  
Ryan D. Quesenberry ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Resonance Energy ◽  
Structural Basis ◽  
Cell Contact ◽  
Heparin Binding ◽  
Epidermal Growth ◽  
Membrane Anchoring

All ligands of the epidermal growth factor (EGF) receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparin-binding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF, still required proteolytic release for activity, whereas ligands with the membrane-anchoring domain of HB-EGF could elicit full biological activity while still membrane anchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus. However, cell-mixing experiments and fluorescence resonance energy transfer studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.

scholarly journals Notch increases the shedding of HB-EGF by ADAM12 to potentiate invadopodia formation in hypoxia

2013 ◽  
Vol 201 (2) ◽  
pp. 279-292 ◽  
Author(s):  
Begoña Díaz ◽  
Angela Yuen ◽  
Shinji Iizuka ◽  
Shigeki Higashiyama ◽  
Sara A. Courtneidge
Keyword(s):  
Growth Factor ◽  
Signaling Pathway ◽  
Cancer Cell ◽  
Cell Invasion ◽  
Egf Receptor ◽  
Cell Contact ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Cancer Cell Invasion ◽  
Cellular Invasion

Notch regulates cell–cell contact-dependent signaling and is activated by hypoxia, a microenvironmental condition that promotes cellular invasion during both normal physiology and disease. The mechanisms by which hypoxia and Notch regulate cellular invasion are not fully elucidated. In this paper, we show that, in cancer cells, hypoxia increased the levels and activity of the ADAM12 metalloprotease in a Notch signaling–dependent manner, leading to increased ectodomain shedding of the epidermal growth factor (EGF) receptor (EGFR) ligand heparin-binding EGF-like growth factor. Released HB-EGF induced the formation of invadopodia, cellular structures that aid cancer cell invasion. Thus, we describe a signaling pathway that couples cell contact–dependent signaling with the paracrine activation of the EGFR, indicating cross talk between the Notch and EGFR pathways in promoting cancer cell invasion. This signaling pathway might regulate the coordinated acquisition of invasiveness by neighboring cells and mediate the communication between normoxic and hypoxic areas of tumors to facilitate cancer cell invasion.

scholarly journals The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF)

2002 ◽  
Vol 367 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Véronique HOSPITAL ◽  
Eiichiro NISHI ◽  
Michael KLAGSBRUN ◽  
Paul COHEN ◽  
Nabil G. SEIDAH ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Surface ◽  
Active Site ◽  
Regulatory Element ◽  
Enzymic Activity ◽  
Heparin Binding ◽  
Epidermal Growth

Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met49. Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc—HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca2+, via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase.

Mouse preimplantation blastocysts adhere to cells expressing the transmembrane form of heparin-binding EGF-like growth factor

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 637-645 ◽  
Author(s):  
G. Raab ◽  
K. Kover ◽  
B.C. Paria ◽  
S.K. Dey ◽  
R.M. Ezzell ◽  
...  
Keyword(s):  
Growth Factor ◽  
Egf Receptor ◽  
Transmembrane Protein ◽  
Mouse Cell ◽  
Cell Contact ◽  
Luminal Epithelium ◽  
Heparin Binding ◽  
Heparin Binding Domain ◽  
Epidermal Growth ◽  
Adhesion Factor

Previous studies have shown that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) mRNA is synthesized in the mouse uterine luminal epithelium temporally, just prior to implantation, and spatially, only at the site of blastocyst apposition (Das, S. K., Wang, X. N., Paria, B. C., Damm, D., Abraham, J. A., Klagsbrun, M., Andrews, G. K. and Dey, S. K. (1994) Development 120, 1071–1083). HB-EGF is synthesized as a transmembrane protein (HB-EGF TM) that can be processed to release the soluble growth factor. An antibody that cross-reacts only with the transmembrane form detected HB-EGF TM in uterine luminal epithelium in a spatial manner similar to that of HB-EGF mRNA. HB-EGF TM is a juxtacrine growth factor that mediates cell-cell contact. To ascertain if HB-EGF TM could be an adhesion factor for blastocysts, a mouse cell line synthesizing human HB-EGF TM was co-cultured with mouse blastocysts. Cells synthesizing HB-EGF TM adhered to day-4 mouse blastocysts more extensively than parental cells or cells synthesizing a constitutively secreted form of HB-EGF. Adhesion of cells synthesizing HB-EGF TM to blastocysts was inhibited by excess recombinant HB-EGF but less so by TGF-alpha. Adhesion was also inhibited by the synthetic peptide P21 corresponding to the HB-EGF heparin binding domain, and by incubating the blastocysts with heparinase. In addition, adhesion to delayed implanting dormant blastocysts, which lack EGF receptor (EGFR), was diminished relative to normal blastocysts. These results suggested that adhesion between blastocysts and cells synthesizing HB-EGF TM was mediated via interaction with both blastocyst EGFR and heparan sulfate proteoglycan (HSPG). It was concluded that HB-EGF TM, which is synthesized exclusively in the luminal epithelium at the site of blastocyst apposition, and which is a juxtacrine adhesion factor for blastocysts, could be one of the mediators of blastocyst adhesion to the uterus in the process of implantation.

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