Heparan sulfate is essential to amphiregulin-induced mitogenic signaling by the epidermal growth factor receptor.
Human amphiregulin (AR) is a heparin-binding growth factor which functions by binding to and activating the epidermal growth factor (EGF) receptor tyrosine kinase. AR contains an EGF-like domain (residues 44-84) and a Lys/Arg-rich NH2-terminal extension (residues 1-43). Synthetic peptides corresponding to residues 8-26, 26-44, and 68-84 of AR were tested for their ability to compete for the binding of AR to immobilized heparin. AR8-26 and AR68-84 had no significant effect on the binding of AR to heparin, whereas AR26-44 bound to heparin and blocked the binding of AR to heparin. Both soluble heparin and heparan sulfate inhibited AR-induced mitogenesis in MCF-10A human mammary epithelial cells with an IC50 of 5 and 2 micrograms/ml, respectively, whereas soluble chondroitin sulfate had only a slight inhibitory effect. When MCF-10A cells were grown in the presence of chlorate, an inhibitor of sulfation, or exposed to the glycosaminoglycan-degrading enzymes heparitinase or heparinase, the ability of AR to evoke mitogenesis in these cells was lost. Chlorate, heparitinase, or heparinase treatment inhibited AR-induced autophosphorylation of tyrosine residues in the EGF receptor. None of these treatments had any significant effect on EGF-triggered mitogenic signaling by the EGF receptor. These results indicate that extracellular heparan sulfate glycosaminoglycan is essential to AR-induced mitogenic signaling by the EGF receptor tyrosine kinase.
Johnson
G R
GR
Division of Cytokine Biology, Food and Drug Administration, Bethesda, Maryland 20892.
Wong
L
L
D066258:Amphiregulin; D001940:Breast / Q000166:cytology; D002455:Cell Division; D002478:Cells, Cultured; D002704:Chlorates / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D066255:EGF Family of Proteins; D004847:Epithelial Cells; D066246:ErbB Receptors / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000494:pharmacology; D006133:Growth Substances / Q000378:metabolism*; D019763:Heparin Lyase; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008934:Mitogens / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects
D066258:Amphiregulin; D002704:Chlorates; D066255:EGF Family of Proteins; D066246:ErbB Receptors; D006023:Glycoproteins; D006025:Glycosaminoglycans; D006133:Growth Substances; D019763:Heparin Lyase; D006497:Heparan Sulfate; D036341:Intercellular Signaling Peptides and Proteins; D008934:Mitogens; D010446:Peptide Fragments; D011133:Polysaccharide-Lyases; C587848:AREG protein, human; C013786:A73025; C021349:heparitinsulfate lyase