Abstract
All receptor tyrosine kinases share a common intracellular signaling machinery, including ras activation, whereas cellular responses vary from mitogenesis to cell differentiation. To investigate the structural basis for receptor tyrosine kinase action for nerve growth factor, the juxtamembrane region of TrkA was transferred to a corresponding region of the epidermal growth factor (EGF) receptor. The resulting chimeric receptor contains an additional Shc site, Tyr 490 , in the juxtamembrane region. In transfected PC12 cell lines, neuronal differentiation was observed with EGF treatment, as evidenced by increased neurite extension. The action of the chimeric receptor was correlated with prolonged activation of MAP kinases and a 3-4-fold increase in phosphatidylinositol 3-kinase activity. The effect of the juxtamembrane chimera was dependent upon the Shc site at Tyr 490 , because expression of a chimeric receptor containing a Y490F mutation resulted in a complete loss of neuritogenesis by EGF treatment. These findings indicate that the juxtamembrane region of the TrkA receptor serves as a key functional domain that can confer a dominant effect upon neuronal differentiation.
Cellular growth and differentiation are dependent upon multiple signaling pathways initiated by polypeptide growth factors. A predominant mechanism requires the activation of intracellular tyrosine phosphorylation through a specific ligandreceptor binding event, followed by recruitment of SH2 containing proteins that recognize specific tyrosine-phosphorylated sequences (1). A common outcome is the activation of ras, which regulates the enzymatic activities of serine-threonine kinases in the MAP kinase family. These regulatory events occur with every known receptor tyrosine kinase and lead to specific responses ranging from cell proliferation to cell survival and differentiation.

All receptor tyrosine kinases share a common intracellular signaling machinery, including ras activation, whereas cellular responses vary from mitogenesis to cell differentiation. To investigate the structural basis for receptor tyrosine kinase action for nerve growth factor, the juxtamembrane region of TrkA was transferred to a corresponding region of the epidermal growth factor (EGF) receptor. The resulting chimeric receptor contains an additional Shc site, Tyr 490 , in the juxtamembrane region. In transfected PC12 cell lines, neuronal differentiation was observed with EGF treatment, as evidenced by increased neurite extension. The action of the chimeric receptor was correlated with prolonged activation of MAP kinases and a 3-4-fold increase in phosphatidylinositol 3-kinase activity. The effect of the juxtamembrane chimera was dependent upon the Shc site at Tyr 490 , because expression of a chimeric receptor containing a Y490F mutation resulted in a complete loss of neuritogenesis by EGF treatment. These findings indicate that the juxtamembrane region of the TrkA receptor serves as a key functional domain that can confer a dominant effect upon neuronal differentiation.
Cellular growth and differentiation are dependent upon multiple signaling pathways initiated by polypeptide growth factors. A predominant mechanism requires the activation of intracellular tyrosine phosphorylation through a specific ligandreceptor binding event, followed by recruitment of SH2 containing proteins that recognize specific tyrosine-phosphorylated sequences (1) . A common outcome is the activation of ras, which regulates the enzymatic activities of serine-threonine kinases in the MAP kinase family. These regulatory events occur with every known receptor tyrosine kinase and lead to specific responses ranging from cell proliferation to cell survival and differentiation.
What determines the final biological outcome elicited from transmembrane signaling through tyrosine phosphorylation? The duration of MAP 1 kinase activity has provided an important insight into the difference between cellular proliferation and differentiation. In PC12 cells, NGF induces prolonged activation of MAP kinases lasting several hours, whereas EGF-mediated MAP kinase activation is transient in nature (2) (3) (4) . The time course of downstream signaling is therefore one of the major differences that accounts for the differentiation program elicited by NGF versus other mitogenic growth factors, such as EGF. The MAP kinase family plays a pivotal role, because the activation of these enzymes is both necessary and sufficient for NIH 3T3 cell transformation and PC12 cell differentiation (5) . Interestingly, overexpression of the EGF receptor in PC12 cells led to a long term activation of MAP kinases and neuronal differentiation by EGF (6) . In the retina, expression of EGF receptors using retroviral mediated gene transfer resulted in selective differentiation of glial cells (7) . These results suggest that overexpression may lead to an amplification of the signal, which leads to differential biological outcomes. An amplification of tyrosine phosphorylation signaling may be generated by several mechanisms. Receptors, by virtue of overexpression, may recruit multiple substrates to the receptor complex and may give rise to additional interactions that are not normally found. Alternatively, tyrosine kinase receptors, when expressed abundantly, may display less internalization, leading to more receptors that remain longer at the cell surface. This situation may also permit additional substrate recruitment and phosphorylation. It should be noted, however, that ligand induced autophosphorylation of the EGF receptor is actually higher than the TrkA NGF receptor in PC12 cells (8) , so that the level of receptor activation cannot solely account for the difference in growth factor action.
Both the EGF and TrkA receptors share common substrates, including the recruitment of phospholipase C-␥, Grb2, and Shc (9 -11) . For the TrkA receptor, phospholipase C-␥1 binds to the carboxyl-terminal tyrosine residue, Tyr 785 (12) , and Shc binds to the juxtamembrane tyrosine residue, Tyr 490 (9, 10) . Shc can associate with a variety of signaling molecules, such as the Grb2 adaptor protein and PI3-kinase substrates, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-diphosphate (13) (14) (15) . The large number of potential interactions suggests that Shc may play a crucial role in substrate recruitment to activated tyrosine kinase receptors and that Shc phosphorylation may determine specific growth factor responses. In support of this conclusion is the finding that overexpression of Shc in PC12 cells is sufficient to induce neurite outgrowth (15) .
The juxtamembrane region of the TrkA NGF receptor contains a single Shc binding motif, IENPQpY, which is distinct from other Shc binding sites, such as those found in the EGF receptor (9, 10, 16, 17) . In addition, the neighboring amino acid sequences in this region are unique among tyrosine kinase receptors, suggesting that distinct functions are subserved by these sequences. We therefore undertook a domain swap approach to explore the functional significance of the juxtamembrane (JM) region of the TrkA NGF receptor. Here we demonstrate that the juxtamembrane domain of TrkA can function in the corresponding location of the EGF receptor to promote differentiation of PC12 cells. The effect of the juxtamembrane sequence is mainly mediated by the Shc site, IENPQpY, because a change of the tyrosine residue to phenylalanine (NPQY to NPQF) abrogated the differentiation signaling properties of the chimeric receptor. These results suggest that the juxtamembrane region, in particular the Shc site, is a major determinant of specificity for trk receptor tyrosine kinases.
EXPERIMENTAL PROCEDURES
Construction of Juxtamembrane Chimeras-The JM of the human EGF receptor was replaced with the corresponding domain of the human trkA receptor, using a hybrid PCR strategy (18) . Briefly, use of hybrid primers (see the primer sequences JMa and JMb shown below) from human trkA as a template generated a hybrid PCR fragment consisting of the trkA juxtamembrane region spanning amino acid residues 434 -509 (19) . This sequence was flanked at both the 5Ј and 3Ј end by sequences from the EGF receptor corresponding to amino acid residue 640 at the 5Ј end and 692 at the 3Ј end (20) . Three amino acids at the swap junctions of the resulting fragment were LFMnkc and kweLGS, where the lowercase letters designate trkA amino acid sequences. The second round of PCR reactions utilized the EGF receptor and the first PCR fragment as the templates. A PCR reaction using a primer complementary to amino acid residues 565-572 of the human EGF receptor cDNA and JMb generated a PCR fragment of 476 bp. Another PCR reaction using a primer that was complementary to amino acids 811-816 in the EGF receptor and the JMa primer resulted in a PCR fragment of 630 bp. A third round of PCR utilized the two fragments from the second round of PCR as templates to generate a final PCR fragment 839 bp in length. This fragment was digested with NaeI and ApaLI and cloned into the EGF receptor cDNA digested with the same enzymes. The entire fragment of 839 bp, which was subjected to PCR reaction, was sequenced for any potential mutations. The JM chimera was then subcloned into XbaI/SmaI sites of the pCMV5 expression vector. The hybrid primers used in the chimera are: JMa, GGGGATCGGCCTCTTCATGaacaaatgtggacggagaaac, and JMb, GCCGAACGCACCGGAGCCCAGctcccacttgagcacgatgtc. The uppercase letters represent the EGF receptor, and the lowercase ones represent the trkA receptor. The wild type human EGF receptor was also cloned into pCMV5 expression vector using XbaI/SmaI sites.
The JM Y490 F chimera was constructed using a hybrid PCR strategy with the JM chimera as a template. The primers used for the JM Y490F chimera are: JMc, ATCATCGAGAACCCACAATtCTTCAGTGATGCC-TGTGTT, and JMd, AACACAGGCATCACTGAAGaATTGTGGGTTCT-CGATGAT. The tyrosine to phenylalanine mutation is indicated by a TAC to TtC substitution. The PCR fragment containing the point mutation was introduced to the JM chimera using BstXI sites. The 712-bp PCR fragment was sequenced in its entirety to verify its integrity.
Cell Cultures and Generation of Stable Lines-PC12 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 5% horse serum. The 3T3-2.2 line lacking the endogenous EGF receptor was kept in Dulbecco's modified Eagle's medium with 10% calf serum. For generation of stable lines, cells were transfected with each construct plus MEX-neo plasmid at 10:1 ratio using lipofectamin reagents (Life Technologies, Inc.). After 2 days, cells were maintained in 0.4 mg/ml G418 for selection. Upon expanding the stable colonies, the positive clones were initially identified by 125 I-EGF binding and further confirmed by performing immunoprecipitation/Western analysis.
Neurite Extension-Stable PC12 lines were tested for neurite extension upon EGF/NGF treatment in Dulbecco's modified Eagle's medium without serum. After 5-6 days, cells were fixed in 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min and incubated for 1 h at room temperature in a blocking solution containing 10% horse serum and 0.1% Triton X-100 in 0.1 M phosphate buffer. Cells were stained with antibodies against MAP1B (generous gift from Dr. R. Vallee) at 1:6000 dilution overnight at 4°C. The secondary antibody (goat antimouse IgG, biotinylated, Vector) was used at 1:500 dilution in 2.5% horse serum and 0.1% Triton X-100 in 0.1 M phosphate buffer. Stained cells were then visualized by 3,3Ј-diaminobenzidine reaction (Vectastain, Vector).
In Vivo Metabolic Labeling-Metabolic labeling of PC12 stables with [ 35 S]methionine was performed according to Ref. 21 , except that mixture of R1 and 528 antibodies were used to pull down the humanspecific form of the EGF receptor or chimeras.
Immunoprecipitation and Western Analysis-To prepare cell lysates, cultured cells were typically starved for 3-4 h for PC12 stable cell lines and 18 -20 h for 3T3-2.2 stable cell lines. After induction, cells were washed twice in ice-cold phosphate-buffered saline and lysed for 15 min on ice in a buffer containing 1% Nonidet P-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 10 mM Na 2 P 2 O 7 , 10 mM NaF, 1 g/ml aprotinin, 10 g/ml leupeptin, 1 mM vanadate, and 1 mM phenylmethylsulfonyl fluoride. Upon lysis, the lysates were spun at 15,000 rpm for 15 min, and the supernatants were collected. For the immunoprecipitation of chimeric receptors from PC stables, the lysates were precleared twice by centrifugation at 100,000 ϫ g for 30 min and subsequent incubation for 1 h at 4°C with protein A-Sepharose (Pharmacia Biotech Inc.). For immunoprecipitations, lysates were incubated for 2 h with the primary antibodies and for an additional 1 h with protein A-Sepharose. The protein A beads were then washed three times in the lysis buffer and once with 50 mM Tris (pH 7.5). The samples were then boiled for 3-5 min in the Laemmli sample buffer before SDS-polyacrylamide gel electrophoresis. For Western analysis, the proteins were transferred onto nitrocellulose paper and blocked for 2-3 h at room temperature in a buffer containing 1% bovine serum albumin, 20 mM Tris (pH 7.5), 137 mM NaCl, and 0.1% Tween 20. Incubation with primary antibodies was carried out for 2 h at room temperature or overnight at 4°C in the same buffer as a blocking buffer. For the detection, an ECL chemiluminescence system (Amersham Corp.) was used with horseradish peroxidase-conjugated secondary antibodies (Boehringer Mannheim). Antibodies used in this work were as follows: R1, anti-EGF receptor (Santa-Cruz, CA); 528, anti-EGF receptor (a generous gift from Dr. H. Masui, Sloan-Kettering); 4G10, anti-phosphotyrosine (Upstate Biotechnology); PY20, anti-phosphotyrosine (Transduction Lab); MAP-1B (a generous gift from Dr. R. Vallee); anti-MAP kinase 1 and 2 and anti-Grb2 (Santa-Cruz); anti-Shc; and RK2, antihuman EGF receptor (provided by J. Schlessinger, New York University).
MAP Kinase Activity Assay-Assays of MAP kinase activity were performed using myelin basic protein as a substrate as described previously (22) .
PI3-Kinase Activity Assay-Assay of PI3-kinase activity was performed according to Soltoff and Cantley (23) using anti-phosphotyrosine antibodies, 4G10, for immunoprecipitation reactions.
Receptor Kinase Assay-EGF receptors and chimeric receptors were immunoprecipitated with 528 and RI anti-human EGF receptor, and the immune complexes were subjected to an in vitro auto-kinase assay in the presence of 50 mM Tris (pH 8.2), 50 mM NaCl, 25 mM KCl, 10 mM MgCl 2 , 10 mM MnCl 2 and [␥-
32 P]ATP. The reaction was subjected to gel electrophoresis and autoradiography.
RESULTS

Construction of Juxtamembrane Chimera-To examine the
TrkA JM domain in NGF signaling, we transferred the JM region of trkA (amino acids 434 -509) onto the corresponding region in the human EGF receptor, as shown in Fig. 1A . No mutations or new amino acids were introduced at the swap junction. The resulting JM chimera contains three Shc binding motifs compared with the wild type EGF receptor, which has two Shc binding sites at the carboxyl-terminal tail (24, 25) .
Stable PC12 lines expressing cDNAs encoding the chimeric receptor (JM-1 and JM-2), the wild type human EGF receptor (ER-1 and ER-2), and human trkA cDNA (Trk-1) were generated. PC12 cells were chosen as a recipient due to several considerations. First, PC12 cells display a well studied and characteristic neuronal phenotype after NGF treatment. Second, PC12 cells do not differentiate upon EGF treatment. Finally, we found that other potential recipient cell lines, such as mutant PC12 cells lacking the TrkA receptor, the PC12 nnr cell line (26) , displayed a higher number of EGF receptors, which would make an analysis based on EGF treatment more difficult to interpret.
The expression of the JM chimera and the human EGF receptor was examined after metabolic labeling with [ 35 S]methionine and immunoprecipitation with monoclonal antibodies, RI and 528, which specifically recognized epitopes in the extracellular domain of the human EGF receptor, and not the en-dogenous rat receptor. The transfected receptor could be specifically detected after immunoprecipitation with these antibodies (Fig. 1B) . The A431 line, which overexpresses the human EGF receptor, was used as a positive control. Native PC12 cells did not give a signal, indicating the antibody did not recognize the rat EGF receptor (Fig. 1B) . We found that PC12 cells expressed approximately 10,000 EGF receptors per cell, consistent with other measurements (6) and considerably less than A431 cells, which express nearly 10 6 receptors per cell (27) . The level of JM receptors was measured to be 5-15,000 receptors per cell, based upon binding measurements (data not shown). It should be emphasized that the level of receptors in the stable lines described here was well below the levels measured previously for insulin receptors (28) and EGF receptors (6) transfected in PC12 cells.
The JM Chimera Induces Neurite Extension in PC12 Cells upon EGF Treatment-The ability of the JM chimera to induce differentiation was evaluated by analyzing neurite outgrowth upon EGF treatment. To detect the morphology of neurites with an appropriate marker, transfected cells were stained for MAP1B immunoreactivity. MAP1B is a microtubule-associated protein that is expressed in PC12 cells (22) . Cell lines expressing the JM chimera (JM-1) were found to be responsive to EGF treatment (50 ng/ml) with increased neurite outgrowth ( Fig.  2A, panel J) . As a control, PC12 cells stably transfected with the wild type EGF receptor (ER-1) did not induce neurite extension ( Fig. 2A, F) . This response was quantitated by assessing the number of cells bearing neurites twice the length of the cell bodies in two independent lines that expressed similar level of receptors (Fig. 2B ). Both lines behaved similarly. The percentage of neurite-bearing cells in the two JM lines reached over 50% after 6 days of EGF treatment, whereas other cell lines failed to respond. Under the same condition, 60% of the native PC12 cells contained neurites upon NGF treatment (data not shown). Furthermore, the morphology of neurites generated by EGF treatment in the JM-1 line was the same produced by NGF treatment (compare panels J and K in Fig.  2A) , suggesting that the JM domain of TrkA was capable of conferring a differentiative signaling potential onto the EGF receptor.
Receptor Tyrosine Phosphorylation-The striking morphological response by EGF in the JM cell lines raises a question regarding the tyrosine kinase activity of the chimeric receptor. To assess this activity in PC12 cells, the receptor was immunoprecipitated with an antibody specific for the human EGF receptor, followed by blotting with anti-phosphotyrosine antibodies. In transfected PC12 cells, the JM chimeric receptors were found to be autophosphorylated after EGF treatment (Fig.  3A) . The extent of receptor autophosphorylation was comparable in ER-1 and JM-1 lines.
To determine whether the JM chimeric receptor contained intrinsic kinase activity, a 3T3 cell line that lacked endogenous EGF receptors, clone 2.2 (29), was used. Analysis of the JM receptor in these 3T3 cells indicated that ligand-dependent autophosphorylation of the chimeric receptor was not detectable compared with the wild type EGF receptor (Fig. 3B) . The expression of the wild type and JM receptors in stably transfected cells was verified by subsequent blotting with the RK2 antibody against the EGF receptor. The lack of kinase activity by the JM chimera was further supported by receptor immunoprecipitation and autokinase experiments with anti-human EGF receptor antibodies, RI and 528 (Fig. 3C) . These results indicated that the JM receptor lacked intrinsic tyrosine kinase activity.
The lack of detectable autophosphorylation by the JM receptor suggested that the domain swap rendered the chimeric receptor defective in its intrinsic kinase activity. Nevertheless, the results in PC12 cells indicated that the JM receptor could serve as a substrate for tyrosine phosphorylation (Fig. 3A) . The detection of JM autophosphorylation in PC12 cells suggests that the chimeric receptor underwent ligand-dependent phosphorylation by endogenous EGF receptors, which contain an active catalytic domain. A similar kind of receptor transactivation has been observed in a kinase-inactive ErbB3 receptor coexpressed with wild type EGF receptors (30) or other ErbB family members (31) .
Activation of the JM Chimera through Endogenous EGF Receptors-To investigate whether endogenous EGF receptors are responsible for the transphosphorylation of the JM chimeras, we utilized an inhibitor, AG1478, which is specfic for the EGF receptor (32) . When the JM-1 line was treated with EGF in the presence of 300 nM AG1478, cells failed to produce neurites ( Fig. 2A, L) . The effect of AG1478 on EGF treatment of the JM-1 cell line was specific, because NGF-induced differentiation of native PC12 cells was unaffected by the same dose of AG1478 ( Fig. 2A, D) .
The differential effect of AG1478 on the EGF receptor and the TrkA NGF receptor was further probed by examining the tyrosine phosphorylation status of the MAP kinases (Fig. 4) . Cells were treated with AG1478 for 2 h prior to a 5-min treatment with EGF. In both PC12 and JM-1 cells, tyrosine phosphorylation of MAP kinases was reduced as the dose of the drug increased with EGF treatment (Fig. 4) . In contrast, AG1478 treatment did not affect NGF-induced tyrosine phosphorylation of MAP kinases in PC12 cells overexpressing the TrkA receptor (Trk-1 line), even at the highest dose (Fig. 4) . Tyrosine phosphorylation of MAP kinases was also unaffected by NGF treatment in PC12 and JM-1 cells (data not shown). The specific inhibitory action of AG1478 on the EGF receptor and not the NGF TrkA receptor indicates that the phosphorylation of the JM chimera required the action of the endogenous EGF receptor and not the TrkA receptor.
A Long Term Activation of MAP Kinase in JM Chimeric Line-NGF-mediated differentiation in PC12 cells is characterized by a long term activation of MAP kinases (2-4).
To assess whether the JM domain from TrkA had an influence upon the time course of downstream signaling by the EGF receptor, we examined the temporal activation of MAP kinases in the JM-1 line by EGF. As shown in Fig. 5A , tyrosine phosphorylation of MAP kinases, pp44, was observed for at least 6 h in the JM-1 line but was significantly reduced after 1 h in the ER-1 line. MAP kinase activity was also measured by an immunoprecipitation kinase assay using myelin basic protein as a substrate. This assay indicated a similar temporal difference (Fig. 5B) . The observed correlation of neurite formation and sustained MAP kinase activation in the JM cells indicates that the TrkA juxtamembrane domain contains critical sequences for long term activation of MAP kinases.
The Activation of PI3-Kinase-Previous studies implicated sequences in the juxtamembrane region of TrkA in the generation of ras-independent signaling (33) . In addition, analysis of the Shc binding site at position Tyr 490 revealed that this site was linked to PI3-kinase activity (34) . We therefore measured PI3-kinase activity in the transfected cell lines by an immunoprecipitation kinase assay using anti-phosphotyrosine antibod- ies and phosphoinositide as a substrate with [␥ 32 P]ATP. The enzymatic activity of PI3-kinase was found to be higher in JM-1 cells than the control ER-1 cell line, suggesting the increased activity was linked to the JM receptor. After 1 and 5 min of EGF treatment, there was a 3-4-fold difference between the two cell lines in absolute values of PI3-kinase activity (Fig. 6) .
The peak values of PI3-kinase activity in the JM cell lines was comparable with the one obtained in the Trk-1 line with NGF treatment, indicating that the TrkA receptor is also capable of recruiting PI3-kinase to the same extent as the JM chimera (Fig. 6) . Hence, some PI3-kinase activity appears to be contributed by the juxtamembrane region of the chimeric receptor.
PI3-kinase activity has been shown to be associated with the TrkA receptor (35) and displays a prolonged time course in PC12 cells overexpressing TrkA receptors (36) . It is clear that activation of PI3-kinase can exert multiple effects during the NGF signaling. For instance, continuous treatment of PC12 cells continuously with wortmannin, an inhibitor of PI3-kinase, led to an inhibition of neurite outgrowth (37) . This result is consistent with our findings, where differentiation correlated with an increase in PI3-kinase activity. Activation of PI3-kinase may also be involved in survival of serum-deprived PC12 cells (38) . In this regard, it is important to note that our neurite outgrowth assays were performed in serum-free conditions. Under these conditions, an elevation of PI3-kinase activity by the JM chimera may have exerted an effect upon cell survival. Preliminary results indicate that the survival of JM cells was reduced by 50% with EGF treatment, when LY294002, a PI3-kinase inihibitor, was added at a concentration of 10 M (data not shown). This indicates that activation of PI3-kinase may exert effects at the level of both cell survival and differentiation.
Essential Role of NPQY 490 Site-The JM domain of TrkA confers a differentiative function upon the EGF receptor in PC12 cells, which correlated with a prolonged MAP kinase activation and an increase in PI3-kinase activity. To investigate which residues are responsible for neurite outgrowth in the chimeric receptor, the tyrosine at position 490 was mutated to a phenylalanine residue in the JM chimeric receptor. The Tyr 490 residue represents the only tyrosine phosphorylation site within the juxtamembrane region of TrkA. Previous mapping studies localized Shc binding to this phosphorylation site (9, 10) and biophysical measurements have confirmed that Shc binds to the phosphotyrosine residue at NPQY 490 through its phosphotyrosine binding (PTB) domain (13, 17) . This interaction is responsible for activation of ras (15) .
PC12 stable lines expressing the JM Y490F mutation were assessed for neurite outgrowth. The level of transfected receptors and the extent of tyrosine phosphorylation was similar to the JM stable transfectants (data not shown), and both sets of cell lines responded to NGF normally (Fig. 7) . However, in contrast to the JM cell lines, neuritogenesis with EGF was not observed in lines expressing JM Y490F , even with concentrations of EGF at 100 ng/ml (Fig. 7) . In JM Y490F cell lines, EGF was capable of inducing MAP kinase activity, but the activation of MAP kinase was not observed beyond 60 min ( Fig. 8B ; compare with Fig. 5A ). Similarly, tyrosine phosphorylation of the p66 isoform of Shc was also reduced significantly after 60 min in JM Y490F cells, whereas it lasted up to 2 h in JM-1 cells. In terms of which isoform of Shc is bound to the receptors, no preference has been observed (data not shown). These results point to a The results reported here suggest that the opposite actions of the EGF and NGF receptors in PC12 cells may be due to their inherent structural differences, which can lead to amplified activation of MAP kinases and PI3-kinase by NGF. It has been shown that a prolonged MAP kinase activity and neuronal differentiation can occur when EGF receptors are expressed at greater than 50-fold excess in PC12 cells (6) . The effects of EGF receptor overexpression may be potentially explained by a longer receptor half-time at the cell surface, which would allow for adaptor proteins to assemble on the receptor. Consistent with this model are experiments indicating that an EGF receptor mutant defective in internalization induced prolonged MAP kinase activation in 293 cells. 2 The same internalization mutant of the EGF receptor induced transformation in NIH 3T3-derived NR6 cells (39) . Differential amplification may be achieved by recruiting a different array of signaling proteins to the activated receptor. Our results favor the last possibility. That is, Shc plays a critical role in TrkA signaling by serving as a pivotal protein that has the potential of bringing both the ras-SOS and perhaps PI3-kinase components to the receptor after ligand binding.
The lack of differentiation with receptors containing a mutated Shc site at Tyr 490 differs from other observations, in which a mutation of the same Shc binding site in trkA did not affect neurite outgrowth, except when a corresponding mutation was also made at the phospholipase-␥ site at Tyr 785 (10) . Potential explanations for the different requirements for Shc in neurite outgrowth by TrkA signaling may be explained by the different mutations employed; the levels of receptor expression (40) ; or differences in the cell lines with regard to the expression of other signaling components. It is important to emphasize that overexpression of any components of the ras signaling pathway may lead to increased recruitment of substrates. Preliminary data indicate that a mutation in the Shc Tyr 490 binding site in trkA also resulted in loss of NGF-dependent differentiation in hippocampal neurons. 3 Hence, the effects of specific mutations in the trkA receptor may be dependent upon the responsiveness of the cells being analyzed.
An alternative explanation for the difference in the findings is that alternative pathways may exist. For example, a small deletion of three amino acids in the TrkA juxtamembrane region has been shown to affect signaling through a ras-independent pathway (33) . Indeed, the activity of PI3-kinase was 2 S. Green, personal communication. reduced as a result of the mutant NPQY 490 site (data not shown).
Shc adaptor proteins bind to the TrkA NGF receptor and the EGF receptor, but the mechanism of binding is different. When binding to TrkA at tyrosine 490, Shc utilizes its PTB domain rather than its SH2 domain (16) . Shc binding to TrkA is one of the highest affinity interactions measured between an adaptor protein and receptor tyrosine kinase (17) Shc binding to Tyr 490 was previously linked to activation of PI3-kinase activity. The activity of PI3-kinase was also reduced in JM Y490 cell lines compared with JM lines (data not shown). However, PI3-kinase does not bind directly to the EGF receptor or the TrkA NGF receptor (23, 30, 42) . Therefore, PI3-kinase may require an adaptor molecule to be recruited to the activated receptor. One candidate for the adaptor molecule is Grb2, because PI3-kinase activity has been detected in immunoprecipitation reactions using anti-GST/Grb2 antibodies (11, 34) .
We have attempted to examine the potential association of PI3-kinase with the chimeric receptor using co-immunoprecipitation reactions with antibodies against the adaptor proteins and PI3-kinase subunits. We have found that the Grb2 protein can be found to be associated either with Shc or PI3-kinase in JM-1 cells in a ligand-dependent manner (data not shown). However, due to the multiple Shc and Grb2 sites on the EGF receptor, it is difficult to assign the immunoprecipitated products exclusively to a single site in the intracellular domain of the chimeric receptor.
Our data indicate that the juxtamembrane domain of TrkA contains instructive cues required for neuronal differentiation and that the Tyr 490 Shc binding site provides a crucial determinant in signal amplification. In addition, the juxtamembrane domain may recruit other signaling molecules. One candidate is SNT, or the Suc-associated neurotrophic factorinduced tyrosine phosphorylated target. SNT is tyrosine phosphorylated in PC12 cells by NGF and not by EGF (43) . A direct association between SNT and TrkA has not been established, but a Grb2 docking protein, FRS2, has been recently purified and cloned. FRS2 is phosphorylated upon NGF treatment and is closely related to SNT (44) . Another potential interacting component is Src, which can induce differentiation of PC12 cells (45, 46) . Src appears to act upstream of ras in NGF signaling (47) and may be involved in a ras-independent mechanism.
In summary, we have demonstrated that the juxtamembrane domain of the TrkA receptor serves as a crucial structural determinant in mediating trophic factor-induced neuronal differentiation. The structural feature is attributed, in part, to the Shc binding site, which is present in the juxtamembrane region of the TrkA receptor and not in the corresponding region of the EGF receptor. The EGF/NGF chimera led to differentiation of PC12 cells, which was not observed with the EGF receptor itself and gave a greater activation of PI3-kinase and prolonged MAP kinase activity. Because EGF and NGF receptors share several common substrates, a major question will be to define how PI3-kinase and Shc adaptor proteins are able to dictate such distinctive outcomes.

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