Analysis of Mutant Platelet-Derived Growth Factor Receptors Expressed in PC12 Cells Identifies Signals Governing Sodium Channel Induction during Neuronal Differentiation

Abstract

The mechanisms governing neuronal differentiation, including the signals underlying the induction of voltage-dependent sodium (Na ؉ ) channel expression by neurotrophic factors, which occurs independent of Ras activity, are not well understood. Therefore, Na ؉ channel induction was analyzed in sublines of PC12 cells stably expressing platelet-derived growth factor (PDGF) ␤ receptors with mutations that eliminate activation of specific signaling molecules. Mutations eliminating activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C ␥ (PLC ␥ ), the GTPase-activating protein (GAP), and Syp phosphatase failed to diminish the induction of type II Na ؉ channel ␣-subunit mRNA and functional Na ؉ channel expression by PDGF, as determined by RNase protection assays and whole-cell patch clamp recording. However, mutation of juxtamembrane tyrosines that bind members of the Src family of kinases upon receptor activation inhibited the induction of functional Na ؉ channels while leaving the induction of type II ␣-subunit mRNA intact. Mutation of juxtamembrane tyrosines in combination with mutations eliminating activation of PI3K, PLC ␥ , GAP, and Syp abolished the induction of type II ␣-subunit mRNA, suggesting that at least partially redundant signaling mechanisms mediate this induction. The differential effects of the receptor mutations on Na ؉ channel expression did not reflect global changes in receptor signaling capabilities, as in all of the mutant receptors analyzed, the induction of c-fos and transin mRNAs still occurred. The results reveal an important role for the Src family in the induction of Na ؉ channel expression and highlight the multiplicity and combinatorial nature of the signaling mechanisms governing neuronal differentiation.Efforts to understand the mechanisms controlling the development and maintenance of the nervous system have identified a number of growth factors with profound effects on neurons. These factors include the neurotrophin family of growth factors, which promote the survival and differentiation of distinct yet overlapping populations of neurons (for reviews, see references 42, 52, and 70). Of the neurotrophins, nerve growth factor (NGF) is the most extensively characterized and has served as the prototype for understanding their actions (for a review, see reference 45). However, other factors, including epidermal growth factor and fibroblast growth factor, also appear to play important roles in the nervous system (22, 82, 84; for reviews, see references 29 and 74). In addition, plateletderived growth factor (PDGF) is expressed in the nervous system and can enhance neuronal survival, neurite outgrowth, and differentiation (14,55,64,68).The effects of growth factors are often mediated through membrane-spanning receptors that exhibit intrinsic tyrosine kinase activity and undergo ligand-dependent autophosphorylation (for a review, see reference 86). The tyrosine-phosphorylated regions in the cytoplasmic domain of the receptor then serve as binding sites for the Src homology 2 (SH...

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth

“…This is in contrast to the other major receptor addback, generated from the PDGFb receptor (Valius and Kazlauskas, 1993), which still encodes tyrosines capable of interacting with signaling proteins, or that can compensate for the loss of a crucial tyrosine. A second weakness of the PDGFb receptor system is the presence of the Src family association sites, which cannot be eliminated without a loss of receptor kinase activity (Fanger et al, 1997). Trk also has the advantage that it will tolerate the addition of any four amino acid SH2 binding motif within a ten amino acid cassette into the kinase insert region.…”

Differential Neurotrophic Regulation of Sodium and Calcium Channels in an Adult Sympathetic Neuron

“…2, I and J) adds further support to the involvement of this pathway. A ras-independent increase in Na ϩ density in PC12 cells was previously reported (Fanger et al 1993(Fanger et al , 1997Hilborn et al 1998). However, in the latter report (Fanger et al 1997) it was suggested that the ras-independent pathway may involve members of the src nonreceptor tyrosine kinase family rather than PI3K.…”

“…However, in the latter report (Fanger et al 1997) it was suggested that the ras-independent pathway may involve members of the src nonreceptor tyrosine kinase family rather than PI3K. One possible reason for this difference was that our experiments were done with native TrkA on intact adult neurons, whereas the experiments of Fanger et al (1997) were done on PC12 cells expressing surrogate platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signaling molecules. Alternatively, it could be argued that our data, which depend on the supposed selectivity of inhibitors, are less reliable than those obtained using molecular and cell biological techniques.…”

“…A ras-independent increase in Na ϩ density in PC12 cells was previously reported (Fanger et al 1993(Fanger et al , 1997Hilborn et al 1998). However, in the latter report (Fanger et al 1997) it was suggested that the ras-independent pathway may involve members of the src nonreceptor tyrosine kinase family rather than PI3K. One possible reason for this difference was that our experiments were done with native TrkA on intact adult neurons, whereas the experiments of Fanger et al (1997) were done on PC12 cells expressing surrogate platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signaling molecules.…”

“…Expression of ion channels on a given neuronal type is not static and may change during development or aging or as a result of injury or disease (Craner et al 2002;Cummins and Waxman 1997;Jassar et al 1993Jassar et al , 1994Lhuillier and Dryer 2002;Martin-Caraballo and Dryer 2002). Various neurotrophic factors have therefore been studied as important determinants of ion channel expression in adult neurons and in cell lines (Chalazonitis et al 1987;Fanger et al 1997;Khorkova and Golowasch 2007;Levine et al 1995;Pollock and Rane 1996;Pollock et al 1990). Target-derived nerve growth factor (NGF) is responsible for the maintenance of tetrodotoxin (TTX)-sensitive and TTX-insensitive sodium currents (I Na ) and of N-and L-type calcium currents (I Ca,N and I Ca,L ) in B-neurons of adult bullfrog sympathetic ganglia (BFSG) (Lei et al 1997(Lei et al , 1998(Lei et al , 2001Petrov et al 2001).…”