Down-regulation of Epidermal Growth Factor Receptors by Nerve Growth Factor in PC12 Cells Is p140 -, Ras-, and Src-dependent

Abstract

PC12 cells, derived from a rat pheochromocytoma, have been extensively used as a model of neuronal differentiation (1), because the cells acquire the phenotype of sympathetic neurons and stop dividing in response to nerve growth factor (NGF) 1 (2).In PC12 cells, NGF interacts with two distinct plasma membrane receptor proteins: p75 NGFR , a cysteine-rich glycoprotein having a relatively low affinity for NGF (3), and p140 trk , a receptor tyrosine kinase activated by NGF, which binds NGF with high affinity and mediates many of the biological activities of this neurotrophin (4). The binding of NGF to the p140 trk receptor stimulates rapid tyrosine autophosphorylation of the receptor and activation of several signal-transducing proteins (4). Activated p140 trk receptors bind to and tyrosine phosphorylate the signaling substrates phospholipase C␥1, phosphatidylinositol-3 kinase, and Src homologous collagen protein (4), resulting in their activation. The latter stimulates Ras activity which subsequently activates a series of serine/threonine kinases including B-Raf, myelin basic protein kinase kinase, the Erks, and p90 rsk (4). This Ras-Erk pathway plays a major role in the activation of transcriptional events by NGF and in NGFinduced neuronal differentiation (5), as illustrated by inhibition of NGF actions upon expression of dominant-negative HaRas (Asn-17) proteins (6, 7). In addition a role for pp60 c-src in NGF actions has been suggested (5) by experiments involving the expression of a dominant-interfering kinase-inactive Src (8).PC12 cells also express plasma membrane receptors for epidermal growth factor (EGF) (9, 10), a mild mitogen for these cells (9). PC12 cells treated with NGF exhibit an attenuated response to EGF (9) because EGF receptors are down-regulated (9, 11). The cellular mechanisms that mediate NGF-induced EGF receptor down-regulation are unknown and represent an interesting question in neuronal development. The existence of functional receptors for both NGF and EGF on PC12 cells and the recent advances in understanding the signaling pathways activated by these receptors make these cells a useful model for the study of cross-regulation between NGF and EGF receptors during differentiation.In this study, we have used a number of PC12 variant cell lines with different levels of p140 trk (12, 13) and EGF receptor expression and dominant-negative Ras (7,14,15) or Src (5,6,8) PC12 transfectants in an attempt to elucidate the molecular mechanisms of the receptor cross-talk required by NGF to down-regulate EGF receptors. Our findings implicate the involvement of p140 trk -, Ras-, and Src-dependent signaling pathways in NGF-induced down-regulation of EGF receptors while * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Permanent address:

Nerve Growth Factor Treatment Prevents the Increase in Superoxide Produced by Epidermal Growth Factor in PC12 Cells

“…One way in which the neurotrophins promote cellular differentiation may be to interfere with mitogenic signals. Indeed, previous data from this laboratory suggest that one action of NGF on PC12 cells is the long term down-regulation of mitogen receptors (9,10). The present experiments demonstrate that NGF also has profound and immediate effects on mitogen signaling.…”

Adenosine A2A Receptor mRNA Regulation by Nerve Growth Factor Is TrkA-, Src-, and Ras-dependent via Extracellular Regulated Kinase and Stress-activated Protein Kinase/c-Jun NH2-terminal Kinase

“…For comparison, the effects of NGF on A 2A AR expression were studied in clonal cell line 106. Clone 106, derived from PC12nnr5 cells stably transfected with TrkA, has levels of 125 I-NGF binding similar to those in native PC12 cells and differentiates in response to NGF (32). When cultures of clone 106 were treated with NGF for 1 or 3 d, both A 2A AR mRNA and protein levels were down-regulated to the same extent as native PC12 cells (Fig.…”

The Wilms' Tumor Gene Product WT1 Mediates the Down-regulation of the Rat Epidermal Growth Factor Receptor by Nerve Growth Factor in PC12 Cells

“…We have previously shown that down-regulation of EGFR in PC12 cells caused by NGF treatment is dependent on p140 trk , Ras, and Src pathways (5). It is quite reasonable to ask whether these pathways are involved in the down-regulation of WT1.…”

“…Previous studies have demonstrated that NGF-induced down-regulation of the EGFR was at the transcriptional level (4) and that the down-regulation is p140 trk -, Ras-, and Src-dependent (5). Recently, we isolated and characterized the rat EGFR promoter region and found that TCC repeat sequences in the promoter region of the rat EGFR are required for the down-regulation of rat EGFR by NGF during the differentiation of PC12 cells (6).…”