Nerve Growth Factor Receptor TrkA, a New Receptor in Insulin Signaling Pathway in PC12 Cells*

Background: TrkA is a transmembrane receptor tyrosine kinase for nerve growth factor. Results: TrkA forms a molecular complex with insulin receptor and IRS-1 to induce Akt and Erk5 phosphorylation. Conclusion: The NGF-TrkA receptor influences insulin signaling. Significance: The TrkA receptor is involved in insulin signaling, and NGF may regulate neuronal glucose uptake as neurons are insulin-insensitive. TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.


TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF3 TrkA signaling might overlap with insulin3 INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent
upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.
Insulin plays a crucial role in brain functions (14) such as memory improvement (15,16) and energy metabolism (17,18). Insulin binds to the insulin receptor (INSR) on the cell surface, leading to its autophosphorylation (19). The activated INSR then binds and phosphorylates the intracellular substrates such as the insulin receptor substrate (IRS) family proteins. Tyrosine-phosphorylated IRS-1 interacts with SH 2 domain of various signaling proteins, including the 85-kDa regulatory subunit of the PI3K (p85⅐p110) that stimulates the production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3P). PIP3P recruits phosphoinositide-dependent kinase 1 and protein kinase B (Akt) to the membrane where phosphoinositide-dependent kinase 1 phosphorylates and activates Akt (20). Akt activation leads to increase in glucose uptake. In addition to PI3K, IRS-1 also interacts with sequestosome 1/p62, a scaffolding protein that is involved in the activation of Akt, Glut4 translocation, and glucose uptake (21). IRS-1 also interacts with growth factor receptor binding protein 2, leading to MAPK activation, which mediates cell survival and mitogenesis (22,23).
Neurons are classified as insulin-insensitive, as insulin is incapable of increasing glucose uptake in neurons (24) compared with muscle and fat cells (25). In this study, we report that NGF or insulin induces TrkA to form a molecular complex with INSR and IRS-1. The tyrosine phosphorylation of the INSR and IRS-1 requires functional TrkA kinase in PC12 cells. In addition, TrkA influences insulin signaling through the activation of Akt and Erk5, which reveals a novel overlapping signaling mechanism between NGF 3 TrkA and insulin 3 INSR.
Cell Culture-PC12 rat pheochromocytoma cells were maintained in DMEM with 10% heat-inactivated horse serum, 5% fetal bovine serum, and antibiotics (100 units/ml streptomycin and penicillin). Parental L6 cells were maintained in DMEM supplemented with 10% fetal bovine serum and penicillin/ streptomycin. Cells were incubated in a humidified atmosphere containing 5% CO 2 and 95% air at 37°C. Cells were transfected with using the cationic lipid method by using Lipofectamine TM 2000 transfection reagent (Invitrogen). The cells were deprived of serum in culture medium overnight at 37°C before cell lysis.
Immunoprecipitation and Western Blotting Analysis-Cells were stimulated with insulin (100 nM) or NGF (100 ng/ml) according to the experimental design. The cells were lysed with Triton lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 20 mM sodium pyrophosphate, 10 mM NaF, 20 mM ␤-glycerophosphate, 1% Triton X-100, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml leupeptin and aprotinin). Protein was estimated by the Bradford procedure (Bio-Rad) using bovine serum albumin (Sigma) as standard. Cell lysates (1 mg) were diluted in lysis buffer and incubated with 4 g of primary antibody. The immunoprecipitates were collected with protein A-Sepharose beads (Sigma) overnight at 4°C and then washed three times with PBS. Samples were boiled in SDS-PAGE sample buffer and resolved on 10% SDS-PAGE, transferred onto PVDF membranes (Millipore), and analyzed by Western blotting with the appropriate antibodies such that the immune complex was detected by enhanced chemiluminescence.
Neurite Outgrowth-PC12 cells were grown on a collagen and polylysine-coated 24-well plate and treated with NGF or insulin, and 200 cells were scored for the presence or absence of neuritis (11).
Glucose Uptake Assay-The glucose uptake in PC12 cells was measured by the method described previously (26,27).  Fig. 1). NPEY motif in the juxtamembrane domain of INSR is an IRS-1 binding site (28 -30). This motif is also involved in binding several adaptor proteins such as Shc and Frs2 through their phosphotyrosine binding domains (30,31). In addition, TrkA receptor has YSTDYY similar to YETDYY in INSR (dashed line box in Fig. 1). The triple tyrosine residues in INSR are essential to interact with IRS-2 (32). Autophosphorylation of the triple tyrosine amino acids enhances the catalytic activity of the receptor tyrosine kinases (33).  min or NGF (100 ng/ml) for 1, 10, and 15 min. TrkA or IRS-1 was immunoprecipitated and Western blotted with anti-IRS-1 and anti-TrkA. Insulin and NGF stimulation (10 and 15 min) induced the interaction between TrkA and IRS-1 ( Fig. 2A). The same lysates were also used to check whether TrkA is tyrosinephosphorylated by insulin and NGF stimulation. The receptor was immunoprecipitated and blotted with TrkA (E-6) antibody that recognizes the phosphorylation of tyrosine residue at 496 of TrkA. Stimulation of PC12 cells with insulin or NGF induced tyrosine phosphorylation of the TrkA receptor (Fig. 2B). The lysates were also Western blotted with TrkA and IRS-1 antibody to check their expression level (Fig. 2C). These results suggest that stimulation of PC12 cells with insulin or NGF leads to the association of TrkA receptor with IRS-1 and tyrosine phosphorylation of TrkA. These results are in parallel to previous findings suggesting that IRS-1 and IRS-2 could be substrates of TRK-T1 and TrkA (34). TrkA Receptor Interacts and Phosphorylates INSR-Because insulin stimulation induced the phosphorylation of TrkA receptor (Fig. 2B), the TrkA receptor may interact with INSR. To investigate this possibility, we performed coimmunoprecipitation experiments in cells treated with either NGF, insulin, or both for 10 and 15 min. INSR or TrkA was immunoprecipitated and Western blotted with TrkA or INSR antibody. Stimulation with NGF, insulin, or both induced the association of TrkA with INSR (Fig. 3A). However, the interaction might be indirect through other unknown proteins. To avoid artifacts due to the antibody, the expression of TrkA or INSR was reduced by using corresponding siRNA, and the interaction of TrkA with INSR was determined. When TrkA or INSR was knocked down, the interaction was lost as shown in Fig. 3, B and C. Because NGF induced the interaction, we aimed to determine whether NGF would lead to tyrosine phosphorylation of INSR. PC12 cell lysates treated with either NGF, insulin, or both were immunoprecipitated with INSR and immunoblotted for phospho-INSR antibody. Interestingly, NGF phosphorylated the INSR at tyrosine 1146 and tyrosine 1150/1151 in the kinase activation loop similar to insulin (Fig. 3D). The lysates were also checked for phospho-INSR, total INSR, and TrkA expression (Fig. 3E). These results indicate that NGF induced the interaction of TrkA with INSR and tyrosine phosphorylation of INSR. As both NGF and insulin can induce the phosphorylation of TrkA (Fig. 2B) and INSR (Fig. 3E), we want to confirm whether NGF and insulin action is only through their respective receptors. We knocked down TrkA or INSR expression by siRNA and stimulated it either with insulin or NGF as shown in Fig. 3, F and G. When INSR expression was depleted, insulin failed to activate TrkA, and similarly, NGF did not activate INSR in absence of TrkA.

TrkA Receptor and IRS-1 Form a Molecular Complex-As
NGF is known to induce differentiation of PC12; here, we wished to determine whether insulin can mediate the differentiation as well. PC12 cells were treated with either NGF, insulin or both for 3 days. The majority of cells treated only with NGF and along with insulin developed a network of neurites. Insulintreated cells failed to develop neurites compared with NGF. The cells were scored for neurite outgrowth, and the percentage of cells with neurites was determined (Fig. 3H).
Functional TrkA Kinase Is Required for the Activation of INSR and IRS-1-Because NGF induced the tyrosine phosphorylation of INSR, we sought to determine whether TrkA kinase domain is essential for the activation of INSR. Therefore, PC12 cells were transfected with HA tag wild-type TrkA or kinaseinactive form of TrkA and stimulated with insulin, NGF, or both, and phosphorylation of INSR was determined by Western blotting. INSR was phosphorylated at tyrosine 1146 and 1150/ 1151 on insulin, NGF, or both in presence of wild-type TrkA, but the phosphorylation was much reduced in the presence of kinase-inactive TrkA (Fig. 4A). The expression of total INSR and the expression of TrkA constructs were also verified (Fig.  4A). TrkA interacts with IRS-1 as well, so we also explored whether the IRS-1 activation is dependent upon the TrkA kinase domain. IRS-1 was tyrosine-phosphorylated in the presence of insulin and NGF in presence of wild-type TrkA, whereas overexpression of kinase-inactive TrkA decreased the activation of IRS-1 (Fig. 4B). Thus, the tyrosine phosphorylation of INSR and IRS-1 is dependent upon the kinase domain of the TrkA receptor. Similar results were also obtained in L6 rat muscle cells (Fig. 4, C and D).
INSR and IRS-1 Interacts with TrkA Receptor in a Phosphotyrosine-dependent Manner-Because the TrkA kinase domain is essential for the tyrosine phosphorylation of INSR and IRS-1, we sought to determine whether the association of the TrkA receptor with INSR and IRS-1 is dependent upon the activation of TrkA. PC12 cells were co-transfected with HA-tagged wild-type TrkA or kinase-inactive TrkA. The cells were treated with insulin (100 nM) or NGF (100 ng/ml) for 10 min, followed by immunoprecipitation of HA, and Western blotted with INSR, IRS-1, pTrkA (Tyr-496), and HA antibodies. We observed that wild-type TrkA was only tyrosine-phosphorylated and interacted with INSR and IRS-1 upon insulin or NGF stimulation, whereas the kinase-inactive form of TrkA was not phosphorylated and did not interact with INSR and IRS-1 (Fig. 5A). In parallel, PC12 cells were pretreated with K252a (100 nM), the kinase inhibitor that impairs the tyrosine phosphorylation of TrkA receptor (35,36) for 1 h prior to the stimulation with insulin or NGF for 10 min. We performed immunoprecipitation with anti-TrkA followed by Western blotting with INSR, TrkA Influences the Insulin Signaling-Insulin signaling plays a role in the activation of two distinct signaling cascades, such as the PI3K/Akt (protein kinase B) and MAPK pathway. To examine the effects of TrkA on insulin signaling, we determined the activity of Akt and Erk5 as downstream targets. PC12 cells were stimulated with insulin for 10 min or NGF for 1, 10, or 15 min. Equivalent amount of cell lysates were Western blotted with phospho-Akt (Ser-473 or Thr-308), stripped, and rep-robed with total Akt antibody. Both insulin and NGF induced the activation of Akt (Fig. 6A). Because the functional TrkA kinase domain is essential to interact with INSR and IRS-1, we sought to explore whether it influences the Akt activation as well. HA-wild-type TrkA or kinase-inactive TrkA were transfected, treated with insulin or NGF and the phosphorylation of Akt was determined by Western blot. Overexpression of kinase-inactive TrkA caused a marked reduction in the phosphorylation of Akt on insulin or NGF stimulation compared with the wild-type TrkA (Fig. 6B). We next set out to examine the effect of TrkA on the activation of Erk5. PC12 cells were stimulated with NGF, insulin or both and the activation of Erk5 was determined. Erk5 was immunoprecipitated and Western blotted with PY20 antibody that recognizes the tyrosine phosphorylation. Erk5 was tyrosine phosphorylated upon insulin and/or NGF treatment (Fig. 7A). Tyrosine phosphorylation of TrkA was abrogated by treating the cells with inhibitor K252a prior to the addition of insulin or NGF and Erk5 activation was analyzed. K252a severely blocked the NGF and insulin-induced activation of Erk5 (Fig. 7B). These results indicate that TrkA  . Functional TrkA kinase is required for Akt activation. A, PC12 cells were either stimulated with insulin (100 nM) for 10 min or with NGF (100 ng/ml) for 1, 10, or 15 min. Equivalent cell lysates were Western blotted (WB) with phospho-Akt antibody (Ser-473 or Thr-308) and non-phospho-Akt antibody. B, cells were cotransfected with HA wild-type TrkA or kinase-inactive TrkA and treated with insulin (100 nM) or NGF (100 ng/ml) for 10 min. The lysates were Western blotted with phospho-Akt antibody and non-phospho-Akt antibody. Experiments were replicated three times with similar results. kinase activity is required for Akt and Erk5 activation. We also examined the effect of insulin, NGF or both on glucose uptake. The cells were incubated with 2-deoxy-D-[3H]glucose, and the uptake of glucose was measured. Insulin alone did not show significant change in glucose uptake compared with control. In contrast, NGF alone or along with insulin significantly elevated the glucose uptake (Fig. 7C). Altogether, these findings reveal that TrkA participates in insulin receptor signaling pathway.
Our studies revealed a novel function for the TrkA tyrosine kinase in regulating insulin signaling. We provide evidence that TrkA, INSR and IRS1 (and presumably other proteins) form a complex that permits cross-phosphorylation. The activated NPEY motif in the juxtamembrane domain of INSR interacts with phosphotyrosine binding domain of IRS-1 (28,29). The juxtamembrane domain of TrkA receptor also has NPQY motif similar to INSR. NGF stimulation is capable of phosphorylating the tyrosine residues at kinase regulatory loop, YETDYY in INSR and tyrosine 632 of IRS-1, which is essential for binding and activating the p85 subunit of PI3K similar to insulin. Insulin binding to the INSR is known to activate PI3K, Akt (20), and MAPK (23) downstream signaling cascades. The Akt pathway mediates a "metabolic" effect (20), and the MAPK pathway mediates "mitogenic" responses of insulin (22). Our results suggest that activation of Akt and Erk5 by insulin or NGF is dependent upon the phosphorylation of TrkA. We propose that TrkA participates in insulin signaling pathway to regulate cell survival and mitogenesis.