Abstract
Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level measured by 125I-EGF binding, tyrosine phosphorylation, and Western blotting. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of [35S]methionine-labeled EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140(trk) NGF receptor. However, in PC12nnr5 cells stably transfected with p140(trk), the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140(trk)-, Ras- and Src-dependent mechanism of NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells.
Highlights
Ʈ To whom correspondence should be addressed: Section on Growth Factors, NICHD, National Institutes of Health, Bldg. 49, Rm. 5A64, 9000 Rockville Pike, Bethesda, MD 20892
1 The abbreviations used are: Nerve growth factor (NGF), nerve growth factor; 6.24, a pheochromocytoma clone overexpressing human p140trk; PC12nnr5, a pheochromocytoma clone nonresponsive to nerve growth factor; GSrasDN6, dexamethasone-inducible, dominant-negative Ras PC12 variant cell line; M-M17-26, stable, dominant-negative Ras PC12 variant cell line; SrcDN2, stable, dominant-negative Src PC12 variant cell line; p140trk, high affinity nerve growth factor receptor; EGF, epidermal growth factor; PACAP, pituitary adenylate cyclase-activating peptide; In PC12 cells, NGF interacts with two distinct plasma membrane receptor proteins: p75NGFR, a cysteine-rich glycoprotein having a relatively low affinity for NGF [3], and p140trk, a receptor tyrosine kinase activated by NGF, which binds NGF with high affinity and mediates many of the biological activities of this neurotrophin [4]
NGF-induced Down-regulation of EGF Receptors—The level of 125I-EGF binding during NGF-induced differentiation of PC12 cells was examined (Fig. 1)
Summary
Vol 272, No 17, Issue of April 25, pp. 11026 –11034, 1997 Printed in U.S.A. Down-regulation of Epidermal Growth Factor Receptors by Nerve Growth Factor in PC12 Cells Is p140trk-, Ras-, and Src-dependent*. NGF treatment of PC12 cells in suspension induces downregulation of EGF receptors in the absence of neurite outgrowth These results strongly suggest a p140trk-, Ras- and Src-dependent mechanism of NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells. K-252a, 8R*,9S*,11S*-(Ϫ)-9-hydroxy-9-methoxycarbonyl-8-methyl2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7,11␣-triazadibenzo(a, g)cycloocta(c,d,e)trindene-1-one; Src, pp60c-src cytoplasmic tyrosine kinase; Ras, a guanine nucleotide-binding protein; Erk, extracellular signal regulated kinase; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco’s modified Eagle’s medium; FGF, fibroblast growth factor. Demonstrating an independence of this cellular process from NGF-induced neurite outgrowth
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