Ligand-induced internalization of the p75 neurotrophin receptor: a slow route to the signaling endosome

Abstract

The nerve growth factor (NGF) family of neurotrophins binds two classes of cell-surface receptors, trk receptor tyrosine kinases and the shared p75 receptor. Rapid internalization and retrograde trafficking of neurotrophin-trk complexes have been demonstrated in a number of systems and are thought to transmit trophic signals from terminals to neuronal cell bodies. In contrast, the internalization and trafficking of neurotrophin-p75 complexes are not well understood. In this study, we used biotinylated NGF and a fluorescent-labeled anti-p75 antibody to follow the kinetics and route of ligand-induced internalization of the p75 receptor in cycling and differentiated PC12 cells. Binding of neurotrophins to p75 induced internalization at a rate approximately three times slower than that of transferrin and NGF-TrkA complexes in the same cells. The ligand-p75 complex was internalized via clathrin-coated pits into early endosomes and eventually accumulated in recycling endosomes in the cell body and vesicles colabeled by the cholera toxin B-subunit in the growth cones. Both internalized ligand and p75 were protected from proteolytic degradation and accumulated in vesicles that did not undergo acidification. Finally, NGF induced endosomal association of p75 and its MAGE interactors, necdin and NRAGE. These data suggest that signaling endosomes containing activated p75 are involved in neurotrophin signaling, and that such endosomes may be temporally and spatially distinct from those containing trk receptors.

Fig. 1.

Generation of a stably labeled bioactive derivative of NGF. A, NGF was labeled with biotin through the COOH groups using as reactants amino-biotin and EDC; the biotinylated NGF was separated by HPLC on a Vydac C4 reverse phase column. Inset, NGFb was run in an SDS-PAGE gel and silver stained or blotted and detected with avidin peroxidase. B, NGF and NGFb displacement of¹²⁵I-NGF bound to PC12 cells (EC₅₀ NGF, 2.8 ± 0.4 nm; EC₅₀ NGFb, 2.2 ± 0.6 nm). C, nnr5 cells were incubated at 4°C with 6 nm NGFb and different ratios of NGF, as shown. NGFb was precipitated from cell homogenates on streptavidin magnetic beads and visualized on Western blots with avidin peroxidase.D, Left panel, Western blot to show TrkA expression in transfected COS cells. Right panel, Control or TrkA-transfected COS cells were incubated with 4 nm NGFb at 4°C, with or without 100-fold excess NGF, and binding was visualized as described above. E, PC12 cells were incubated with different concentrations of NGF or NGFb for 15 min, and TrkA tyrosine phosphorylation was determined. F, Morphological differentiation of PC12 cells after 3 d of treatment with 2 nm NGF or NGFb concomitant with increased acetyl cholinesterase activity as a cholinergic differentiation marker.

Fig. 2.

p75 is internalized to a transferrin-positive endosome in a ligand-dependent manner. A, Visualization of the internalization of p75 (labeled with MC192-FITC; 3 μg/ml, green) in the presence of NGF (20 nm) and transferrin-Alexa-647 (red, 60 μg/ml) by confocal microscopy on PC12 cells. After incubation, cells were fixed and incubated with anti-mouse-RRX (1:250, blue) to label cell-surface p75.B, Binding of MC192 to PC12 cells at 4°C in the presence or absence of NGF (20 nm). C, Degree of colocalization of p75 and transferrin in the recycling endosome; 12–15 cells were analyzed for time point. D, Confocal three-dimensional reconstruction of serialz-planes of a PC12 cell incubated for 120 min at 37°C with MC192-FITC, NGF, and transferrin-Alexa-647. Different rotation angles are shown. Scale bar, 10 μm. E, Internalization kinetics of p75 labeled with MC192-FITC as shown in A(relative fluorescence, normalized to cell surface p75), and transferrin-Alexa-647 (arbitrary fluorescence units) in the presence of NGF (20 nm); 12–15 cells were analyzed for each time point. F, p75 internalization kinetics in differentiated PC12 treated with MC192-FITC (3 μg/ml) and 6 nm of the indicated neurotrophin. The plots show average ± SEM for 12–15 cells at each time point.

Fig. 3.

Slow internalization and limited recycling of p75-bound NGF. A, Confocal microscopy of nnr5 cells treated for 120 min at 37°C with MC192-FITC (green, 3 μg/ml) and transferrin-Alexa-594 (red, 60 μg/ml), in the presence of 6 nm NGF. Colocalized ligands appear in yellow. Scale bar, 10 μm. B, Top panels, PC12 and nnr5 cells incubated with 4 or 6 nm, respectively, of NGFb, at different internalization times before acid wash. NGFb was precipitated from cell homogenates on streptavidin magnetic beads and visualized on Western blot with avidin peroxidase. Bottom panel, Corresponding total p75 levels in cell lysates. C, Internalization kinetics of NGFb in PC12 versus nnr5 (average ± SEM; n = 3). D, Recycling of NGFb in PC12 and nnr5. The cells were incubated with 4–6 nm NGFb for 60 min at 4°C, followed by 2 hr at 37°C. Cells were then acid washed and incubated for 1 hr at 37°C with 100-fold excess of NGF. Approximately 15% of the NGFb was recovered in the incubation media in both cases. Approximately 90% of transferrin-HRP recycled under similar conditions in PC12 cells.

Fig. 4.

Distinct p75 and TrkA internalized vesicles in differentiated PC12 cells. A, Confocal microscopy of differentiated PC12 cells treated with MC192-FITC (3 μg/ml, green), the ligand-mimicking r-TrkA antibody (1:500), and BDNF (4 nm). Cells were incubated with primary antibodies for 60 min at 4°C, anti-rabbit-Cy5 (1:250, red) and BDNF (4 nm) for 30 min at 4°C, and for the indicated times at 37°C in the presence of BDNF (4 nm). B, Visualization of p75 and TrkA-positive vesicles in the growth cone of differentiated PC12 treated as in A (60 min internalization). Scale bar, 10 μm. Bottom panel, Confocal three-dimensional reconstruction of serial z-planes of the indicated region. The graph on the right shows fluorescence intensity levels along the line drawn through the three vesicles present in the boxed region of the growth cone. C, Quantification of the experiment shown inA. The presence of p75 or TrkA, or both, was quantified for individual vesicles by measuring the fluorescence intensity of each dye (FITC for p75 and Cy5 for TrkA), summing ∼15 vesicles per cell and 12–15 cells for each time point.

Fig. 5.

Internalized p75 compared with early endosome markers in the initial phase of internalization. A, Differentiated PC12 cells were incubated 30 min at 37°C with MC192-FITC (green, 3 μg/ml), NGF (6 nm), and 2 mg/ml HRP (a fluid phase marker) and then fixed and incubated with anti-HRP-RRX (1:100, red). Bottom panels, Magnification of the cell body and growth cone boxed in the top panel. Scale bar, 10 μm. Arrows indicate colocalization. B, Differentiated PC12 were incubated 60 min at 37°C with MC192-FITC (green, 3 μg/ml) and NGF (6 nm) and then fixed and immunostained for EEA1 (an early endosome marker). Arrows indicate vesicles positive for EEA1 and p75 in the periphery of the cell. Bottom panel, Fluorescence intensity levels of the two vesicles indicated in B corroborate p75 and EEA1 colocalization in these vesicles.

Fig. 6.

Internalized p75 compared with acidic organelle markers. A, Differentiated PC12 cells were incubated for 15 min at 37°C with 2 mg/ml HRP and then washed and incubated for 3 hr at 37°C with MC192-FITC (green, 3 μg/ml) and NGF (6 nm). Cells were fixed and incubated with anti-HRP-RRX antibody (1:100, red). B, C, Differentiated PC12 cells were incubated for 3 hr at 37°C with MC192-FITC (green, 3 μg/ml) and NGF (6 nm) and then fixed and incubated with anti-rab7 (B) or anti-cathepsin-D (C) and developed with anti-rabbit-RRX (1:500, red). D, Differentiated PC12 cells were incubated for 3 hr at 37°C with MC192-FITC (green, 3 μg/ml) and NGF (6 nm), followed by 5 min at 37°C with lysotracker Red DND-99, washed, and fixed. Scale bar, 10 μm.

Fig. 7.

p75 internalization involves clathrin-mediated endocytosis. A, Co-patching of p75 with clathrin. Control, PC12 cells were treated for 30 min with MC192-FITC (3 μg/ml, green) and NGF (4 nm) at 37°C, fixed, and immunostained for Clathrin HC (developed with anti-goat-Cy5, red). Patched, PC12 cells were treated for 15 min with MC192-FITC (3 μg/ml, green) and NGF (4 nm) at 37°C, followed by 30 min with rabbit-anti mouse (1:25) at 4°C and also incubated for 15 min at 37°C, fixed, and treated as in control. Arrows indicate co-patching of p75 and clathrin. Scale bar, 10 μm. B, Inhibition of clathrin-mediated endocytosis. PC12 cells were incubated with MC192-FITC (3 μg/ml, green) in the presence of NGF (6 nm) for 90 min at 37°C, and clathrin-dependent endocytosis was inhibited as described in Materials and Methods. After treatment, cells were fixed and incubated with an anti-mouse-RRX antibody to label p75 remaining on the cell surface (red). CCA, Control cytosol acidification; CA, cytosol acidification; KD, potassium depletion. The control for potassium depletion was similar to the CCA (data not shown). C, Quantification of the degree of internalization of p75, transferrin (Transf), or HRP under the different treatments.

Fig. 8.

Cointernalization of p75 and NGF in common endocytic vesicles. A, Confocal microscopy on cell bodies of differentiated PC12 cells treated with MC192-FITC (3 μg/ml, green) and NGFb (20 nm) for 90 min at 4°C, washed and incubated for 30–45 min at 4°C with streptavidin-Alexa-647 (1:500, red), and then incubated for the indicated times at 37°C. Scale bar, 10 μm. B, Merged confocal images of differentiated PC12 cells treated as described in A and incubated for 60 min at 37°C. Scale bar, 10 μm. C, Confocal three-dimensional reconstruction of serial z-planes of a growth cone of differentiated PC12 cell treated as described inA. Different rotation angles are shown. Scale bar, 5 μm. D, Top panel, Confocal microscopy on differentiated PC12 cells treated for 60 min at 37°C with MC192-FITC (green, 3 μg/ml) and CTX-B-Alexa-594 (red, 5 μg/ml) in the presence of 6 nm NGF. Scale bar, 10 μm. Bottom panels, Confocal three-dimensional reconstruction of serial z-planes of the region of the growth cone boxed in the top panel. Different rotation angles are shown. Scale bar, 5 μm.

Fig. 9.

NGF induces the association of p75 and its MAGE interactors in endosomes. PC12 cells were treated for 2 hr at 37°C with NGF (4 nm) and 15 min at 37°C with HRP (1 mg/ml) in the presence of NGF, or 1 hr at 37°C with transferrin-HRP (8 μg/ml). Cells were then homogenized in 320 mm sucrose, 20 mm HEPES, pH 7.4, buffer with a ball homogenizer. A postnuclear supernatant was prepared and centrifuged for 1 hr at 35,000 rpm in a TLS 55 rotor over a cushion of 1% Ficoll to obtain a P2 pellet. A, Plasma membrane contamination assessed by Western blot for the β1 subunit of Na⁺/K⁺-ATPase, 50 μg of protein per lane. B, P2 was resuspended in 1 ml of 320 mm sucrose, 20 mm HEPES, pH 7.4, and loaded in an 11 ml linear Ficoll gradient (1–16%). The gradient was centrifuged for 3 hr at 35,000 rpm in an SW41 rotor, and 1 ml fractions were manually collected from the top. HRP or transferrin-HRP was used to define the early/recycling compartment (gradient 1), and the three peak fractions indicated in black were pooled for a second fractionation. The second gradient was 9 ml of linear Ficoll (3–16%) and centrifuged for 3 hr at 35,000 rpm in an SW41 rotor. One milliliter fractions were manually collected from the top (gradient 2) and found to be free of detectable plasma membrane marker (data not shown). C, Western blots of gradient 2 fractions from cells treated with NGF for 2 hr. Aliquots (70 μl) were separated on 12% PAGE and probed for rab5, p75, NRAGE, or necdin. D, p75 levels in the peak endosomal pool of PC12 treated or not treated with NGF (equal protein loaded). E, Coimmunoprecipitation of MAGE interactors with p75 in endosomes. Fractions 5–10 of gradient 2 were pooled, diluted eightfold with 320 mm sucrose, 20 mm HEPES, pH 7.4, and centrifuged for 10–12 hr at 35,000 rpm in an SW41 rotor. The sedimented endosomes were additionally centrifuged for 1 hr at 55,000 rpm in a TLS 55 rotor and lysed with 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% Igepal, 10% glycerol, and protease inhibitors. p75 was immunoprecipitated with the MC192 antibody, and Western blots of precipitates were probed for NRAGE or necdin. Representative results from three (NRAGE) or two (necdin) experiments are shown.