Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: dependence on interactions with cell surface heparan sulfate

Heparin-binding EGF-like growth factor (HB-EGF) is a smooth muscle cell mitogen composed of both EGF receptor and heparin-binding domains. To better understand the function of its domains, intact HB-EGF or its heparin-binding (HB) domain (amino acids 1–45) were fused to a mutant Pseudomonas exotoxin with an inactivated cell-binding domain. The resulting chimeric toxins, HB-EGF-PE* and HB-PE*, were tested on tumor cells, proliferating smooth muscle cells and a mutant Chinese hamster ovary cell line deficient in heparan sulfate proteoglycans (HSPGs). Two targets were found for HB-EGF-PE*. Cells were killed mainly through EGF receptors, but the HB domain was responsible for killing via HSPGs. HB-PE* did not bind to the EGF receptor and thus was cytotoxic by interacting exclusively with HSPGs. We conclude that the HB domain of HB-EGF is able to mediate internalization through HSPGs, without requiring the EGF receptor.

Download Full-text

The heparin-binding domain of amphiregulin necessitates the precursor pro-region for growth factor secretion

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1635-1646.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1635-1646

Author(s):

B A Thorne ◽

G D Plowman

Keyword(s):

Growth Factor ◽

Egf Receptor ◽

Transforming Growth Factor ◽

Binding Domain ◽

Biologically Active ◽

Structural Motif ◽

Heparin Binding ◽

Active Growth ◽

Heparin Binding Domain ◽

Pro Region

The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.

Download Full-text

Receptor-Based Antidote for Diphtheria

Infection and Immunity ◽

10.1128/iai.70.5.2344-2350.2002 ◽

2002 ◽

Vol 70 (5) ◽

pp. 2344-2350 ◽

Cited By ~ 13

Author(s):

Jeong-Heon Cha ◽

Joanna S. Brooke ◽

Mee Young Chang ◽

Leon Eidels

Keyword(s):

Growth Factor ◽

Egf Receptor ◽

Binding Domain ◽

Amino Acid Residues ◽

Heparin Binding ◽

Soluble Receptor ◽

Binding Studies ◽

Diphtheria Antitoxin ◽

Heparin Binding Domain ◽

Epidermal Growth

ABSTRACT Although equine diphtheria antitoxin may be an effective therapy for human diphtheria, its use often induces serum sickness. We describe here a strategy for developing an alternative treatment based on the human diphtheria toxin (DT) receptor/heparin-binding epidermal growth factor-like growth factor (HB-EGF) precursor. Recombinant mature human HB-EGF acts as a soluble receptor analog, binding radioiodinated DT and preventing its binding to the cellular DT receptor/HB-EGF precursor. However, the possibility existed that radioiodinated DT-HB-EGF complexes associate with cells due to the binding of the heparin-binding domain of recombinant HB-EGF to cell surface heparan sulfate proteoglycans. This possibility was confirmed by performing DT binding studies in the presence of heparin. A recombinant truncated HB-EGF (residues 106 to 149), which lacks most of the heparin-binding domain, showed an essentially heparin-independent binding of radioiodinated DT to cells. Furthermore, it was a more effective inhibitor of DT binding than was recombinant mature HB-EGF. Since mature HB-EGF is a known ligand for the EGF receptor and is thus highly mitogenic (tumorigenic), we then changed amino acid residues in the EGF-like domain of the recombinant truncated HB-EGF and demonstrated that this DT receptor analog (I117A/L148A) displayed a low mitogenic effect. The truncated (I117A/L148A) HB-EGF protein retained high DT binding affinity, as confirmed by using surface plasmon resonance. Our results suggest that the truncated (I117A/L148A) HB-EGF protein could be an effective, safe antidote for human diphtheria.

Download Full-text

The heparin-binding domain of amphiregulin necessitates the precursor pro-region for growth factor secretion.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1635 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1635-1646 ◽

Cited By ~ 44

Author(s):

B A Thorne ◽

G D Plowman

Keyword(s):

Growth Factor ◽

Egf Receptor ◽

Transforming Growth Factor ◽

Binding Domain ◽

Biologically Active ◽

Structural Motif ◽

Heparin Binding ◽

Active Growth ◽

Heparin Binding Domain ◽

Pro Region

The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.

Download Full-text