Nerve growth factor receptor TrkA, a new receptor in insulin signaling pathway in PC12 cells

Abstract

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.

Keywords: Akt; IRS-1; Insulin; Insulin Receptor; NGF; Neurotrophins; Protein-Protein Interactions; Signal Transduction; TrkA.

FIGURE 1.

Amino acid sequence similarities between human insulin receptor (INSR) and NGF receptor TrkA. The solid box represents the NPXY motif, and the dashed line box represents the triple Tyr (Y) amino acids in INSR and the TrkA receptor.

FIGURE 2.

TrkA receptor interacts with IRS-1. PC12 cells were either stimulated with insulin (100 nm) for 10 min or with NGF (100 ng/ml) for 1, 10, or 15 min. A, the cells were lysed and immunoprecipitated (IP) with anti-TrkA or anti-IRS-1 and Western blotted with anti-IRS-1 or anti-TrkA. B, the lysates were immunoprecipitated with TrkA antibody and Western blotted for TrkA (E-6) antibody that recognizes the phosphotyrosine 496 of TrkA. C, the lysates were Western blotted with anti-IRS-1 or anti-TrkA. Experiments were replicated three times with similar results.

FIGURE 3.

INSR interacts with TrkA receptor. A, PC12 cells were either stimulated with NGF (100 ng/ml), insulin (100 nm), or both for 10 min and 15 min. The cell lysates were immunoprecipitated (IP) with anti-INSR or anti-TrkA and Western blotted with anti-TrkA or anti-INSR. B, PC12 cells were transfected with control or TrkA siRNA and stimulated with either NGF, insulin, or both. The cells were lysed and immunoprecipitated with anti-INSR and Western blotted with anti-TrkA or anti-INSR. C, cells were transfected with control or INSR siRNA and stimulated as above. TrkA was immunoprecipitated and immunoblotted with TrkA or INSR antibody. D, PC12 cells treated with NGF, insulin, or both were immunoprecipitated with INSR and blotted for phospho-INSR antibody that recognizes the tyrosine 1146 or 1150/1151 of INSR. E, the lysates were blotted for phospho-INSR (Tyr-1146) or (Tyr-1150 or -1151), total INSR, and TrkA. F, PC12 cells were transfected with control or INSR siRNA and stimulated with or without insulin for 15 min. TrkA was immunoprecipitated and Western blotted for anti-TrkA (E-6) that recognizes the phosphotyrosine 496 of TrkA. G, cells were transfected with control or TrkA siRNA and treated with NGF for 15 min. The cell lysates were immunoprecipitated with anti-INSR and blotted for phospho-INSR (Tyr-1146) antibody. H, the PC12 cells were treated either with NGF (50 ng/ml) or insulin (100 nm) followed by assessment of neurite outgrowth 3 days post addition of NGF or insulin. The cells were counted, and the percentage of cells with neurites was determined (values expressed as mean ± S.D.). Experiments were replicated three times with similar results.

FIGURE 4.

Activation of INSR and IRS-1 requires functional TrkA kinase. A, PC12 cells were transfected with HA-wild-type TrkA or kinase-inactive TrkA (KD) followed by stimulation of insulin (100 nm) or NGF (100 ng/ml) or both for 10 min. The lysates were Western blotted with phospho-INSR antibody that recognizes the tyrosine 1146 or 1150/1151 of INSR and total INSR. The expression of wild-type TrkA or kinase inactive (KD) was verified by blotting with HA tag. B, the phosphorylation of IRS-1 was determined by blotting the PC12 cell lysates with phospho-IRS-1 antibody that recognizes the tyrosine 632 and total non-phospho-IRS-1. C, L6 cells were cotransfected with HA-wild-type or kinase-inactive TrkA (KD) and treated with insulin or NGF or both as above. The lysates were Western blotted with phospho-INSR (Tyr-1146 and Tyr-1150/1151) antibody and total INSR. D, L6 cell lysates were blotted with phospho- and non-phospho-IRS-1 antibody. Experiments were replicated three times with similar results.

FIGURE 5.

Kinase activity of TrkA is required for the interaction with INSR and IRS-1. A, PC12 cells were transfected with HA-wild-type TrkA or kinase-inactive TrkA (KD) followed by insulin (100 nm) or NGF (100 ng/ml) stimulation for 10 min. The lysates were immunoprecipitated with anti-HA followed by Western blotting with INSR-β, IRS-1, pTrkA, or HA-tagged antibody. The lysates were Western blotted with INSR-β, IRS-1, and HA antibody to verify the protein expression levels. B, PC12 cells were pretreated with K252a (100 nm) for 1 h prior to stimulation with insulin or NGF for 10 min. The cell lysates were immunoprecipitated with anti-TrkA and Western blotted with INSR-β, IRS-1, pTrkA, and TrkA antibody. Cell lysates was analyzed by blotting with INSR-β, IRS-1, or TrkA antibody. Experiments were replicated three times with similar results.

FIGURE 6.

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.

FIGURE 7.

Erk5 activation and glucose uptake in PC12 cells. A, PC12 cells were stimulated with NGF (100 ng/ml), insulin (100 nm), or both for 10 min and 15 min. The activation of Erk5 was determined by immunoprecipitating the lysates with Erk5 and Western blotting with PY20 antibody that recognizes the tyrosine phosphorylation and Erk5. B, cells were pretreated with K252a (100 nm) for 1 h prior to stimulation with insulin or NGF for 10 min. Erk5 was immunoprecipitated in the lysates and Western blotting with PY20 or Erk5 antibody. C, PC12 cells were stimulated with NGF (100 ng/ml), insulin (100 nm), or both for 20 min, and the rates of 2-deoxy-d-[3H]glucose uptake were determined. Each bar in the graph indicates the percentage change relative to the control cells. Differences from the control value treated with NGF, both NGF and insulin are statistically significant (*, p < 0.001). Error bars indicate S.D. Experiments were replicated three times with similar results.