Cell surface Trk receptors mediate NGF-induced survival while internalized receptors regulate NGF-induced differentiation - PubMed (original) (raw)

Cell surface Trk receptors mediate NGF-induced survival while internalized receptors regulate NGF-induced differentiation

Y Zhang et al. J Neurosci. 2000.

Abstract

Internalization and transport of a ligand-receptor complex are required to initiate cell body responses to target-derived neurotrophin. However, it is not known whether internalized receptors and cell surface receptors initiate the same signaling pathways and biological responses. Here we use a temperature-sensitive mutant of dynamin (G273D) to control the subcellular localization of activated NGF receptors (Trks). We show that dynamin function is required for ligand-dependent endocytosis of Trk receptors. In PC12 cells, nerve growth factor (NGF) stimulation promotes both survival and neuronal differentiation. These distinct biological responses to NGF are controlled by receptors signaling from different locations within the cell. Neuronal differentiation is promoted by catalytically active Trks within endosomes in the cell interior. In contrast, survival responses are initiated by activated receptors at the cell surface where they orchestrate prolonged activation of the kinase Akt. Thus, interactions between Trk receptor tyrosine kinases and intracellular signaling molecules are dictated both by phosphotyrosine motifs within the receptors and by the intracellular location of phosphorylated receptors.

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Figures

Fig. 1.

Fig. 1.

Dynamin is required for internalization of NGF and TrkA. a, TS-dynamin PC12 cells were stimulated with biotin-tagged NGF for 2 hr at the indicated temperatures, then fixed and permeabilized (Permeabilized) or not. Accessible biotin was visualized with streptavidin-Cy3. Scale bar, 50 μm. b, TS-dynamin PC12 cells were stimulated with125I-NGF alone or with excess unlabeled NGF for 2 hr at the indicated temperatures. Specific internalized counts (125I-NGF internalized minus 125I-NGF internalized in the presence of 200 ng/ml NGF) were similarly decreased at the nonpermissive temperature and when internalization was blocked at 4°C. c, TS-dynamin PC12 cells were stimulated with NGF (NGF+) or vehicle control (_NGF_−) for 15 min at the indicated temperature, then fixed and permeabilized (Permeabilized) or not. Cells were immunostained with anti-TrkA, followed by biotinylated secondary antibody and streptavidin-Cy3. Scale bar, 50 μm. d, Intensity of TrkA immunostaining was measured at 33 and 39°C for TS-dynamin or WT-dynamin PC12 cells 15 min after stimulation with NGF (+) or vehicle control (−). Values are mean surface TrkA per cell (±SEM) for each condition, normalized to total TrkA per cell. NGF induces a decrease in the percentage of TrkA at the surface at both temperatures in WT-dynamin cells. In TS-dynamin cells, NGF induces a decrease in surface TrkA at the permissive but not at the nonpermissive temperature (p < 0.001). Paired t test was used to compare surface TrkA at the permissive and nonpermissive temperatures.

Fig. 2.

Fig. 2.

Inhibition of internalization enhances NGF-dependent survival. a, TS-dynamin PC12 cells were incubated in serum-free media with NGF (+NGF) or vehicle control (−NGF) for 24 hr at the indicated temperatures. Dying cells were visualized by TUNEL stain. Scale bar, 50 μm. b, TS-dynamin PC12 lines (TS1_–_TS4) and wild-type dynamin PC12 cells (WT) were incubated in serum-free media with NGF (+NGF) or vehicle control (−NGF) for 24 hr at the indicated temperatures. Relative cell viability for each condition was assessed by LDH activity normalized to vehicle control. Each data point represents mean values (±SEM) from six separate experiments. Wilcoxon signed rank test was used to compare results at the two temperatures. *p < 0.05.

Fig. 3.

Fig. 3.

Trk internalization promotes neuronal differentiation. a, TS-dynamin and wild-type dynamin cells were incubated in DME with NGF (NGF+) for 3 d at the indicated temperatures, then visualized with a phase-contrast microscope. Representative examples of the TS-dynamin cells are shown. Scale bar, 50 μm. b, The percentage of cells with neurites was assessed for each condition. Each data point represents mean values of two to five separate experiments (±SEM). Wilcoxon signed rank test was used to compare results at the two temperatures. *p < 0.05, **p < 0.01, ***p < 0.001. c, TS-dynamin PC12 cells were cultured in DME alone or with 50 ng/ml NGF, or in DME with serum alone or with 50 ng/ml NGF for 24 hr in the presence of bromodeoxyuridine. At 24 hr, the cultures were fixed, and the percentage of cell nuclei labeled with BrdU was assessed for each condition. Wilcoxon signed rank test was used to compare results at the two temperatures.

Fig. 4.

Fig. 4.

Phosphorylation state of activation loop tyrosines is regulated by internalization. a, b, TS-dynamin PC12 cells were stimulated with NGF (+) or vehicle control (−) for the indicated times at the indicated temperature. Phosphorylation at Y490 (a) and Y674/675 (b) was assessed by immunoblot analysis with phosphospecific antibodies to Trk (Segal et al., 1996).c, d, Intensity of the bands visualized by ECF and the Storm System was quantified using Imagequant and expressed as NGF-induced fold stimulation relative to vehicle control. Each data point represents pooled data from five or six experiments. Wilcoxon signed rank test was used to compare results at the two temperatures. *p < 0.05.

Fig. 5.

Fig. 5.

Internalization enhances catalytic kinase activity of Trk. Ts-dynamin PC12 cells in serum-free medium were stimulated with NGF (+) or vehicle control (−) for the indicated times at the indicated temperature. Protein extracts were immunoprecipitated with anti-pan Trk and incubated with γ32P-ATP and Trk substrate peptide. Radioactivity incorporated into the Trk substrate peptides was quantified using the Storm System. Kinase activity was normalized to the maximal kinase activity in each experiment. Each data point represents the mean of five experiments ± SEM. Wilcoxon signed rank test was used to compare results at the two temperatures. *p < 0.05.

Fig. 6.

Fig. 6.

Phosphorylation of Akt, MEK-1, and Erk1 and Erk2 are modified by internalization. TS-dynamin PC12 cells in serum-free media were stimulated with NGF (+NGF) or vehicle control (−NGF) for the indicated times at the indicated temperatures. Phosphorylation of Akt (a), MEK-1 (b), or Erk1 and Erk2 (c) was assessed by immunoblot analysis with phosphospecific antibodies and visualized by enhanced chemifluorescence.

Fig. 7.

Fig. 7.

Internalization terminates Akt activation but has dual effects on the Ras-MAPK pathway. TS-dynamin PC12 cells in serum-free media were stimulated with NGF or vehicle control for 5 min, or 2 hr, at the indicated temperatures. Intensities of p-Akt, p-MEK, p-Erk1, p-Erk2, and p-CREB were quantified using Imagequant and expressed as fold induction relative to the relevant vehicle control, after correction for protein loading. Each data point represents mean values from five or six experiments ± SEM. Wilcoxon signed rank test was used to compare results at the two temperatures. *p < 0.05.

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