A novel endocytic recycling signal distinguishes biological responses of Trk neurotrophin receptors

Abstract

Endocytic trafficking of signaling receptors to alternate intracellular pathways has been shown to lead to diverse biological consequences. In this study, we report that two neurotrophin receptors (tropomyosin-related kinase TrkA and TrkB) traverse divergent endocytic pathways after binding to their respective ligands (nerve growth factor and brain-derived neurotrophic factor). We provide evidence that TrkA receptors in neurosecretory cells and neurons predominantly recycle back to the cell surface in a ligand-dependent manner. We have identified a specific sequence in the TrkA juxtamembrane region, which is distinct from that in TrkB receptors, and is both necessary and sufficient for rapid recycling of internalized receptors. Conversely, TrkB receptors are predominantly sorted to the degradative pathway. Transplantation of the TrkA recycling sequence into TrkB receptors reroutes the TrkB receptor to the recycling pathway. Finally, we link these divergent trafficking pathways to alternate biological responses. On prolonged neurotrophin treatment, TrkA receptors produce prolonged activation of phosphatidylinositol 3-kinase/Akt signaling as well as survival responses, compared with TrkB receptors. These results indicate that TrkA receptors, which predominantly recycle in signal-dependent manner, have unique biological properties dictated by its specific endocytic trafficking itinerary.

Figure 1.

Trk receptors exhibit differential ligand-induced trafficking to degradative pathways. PC12 cells stably expressing FLAG-tagged TrkA (A) or TrkB (B) were surfaced biotinylated and incubated at 37°C for the indicated times in the absence or presence of 50 ng/ml ligand (NGF for A and BDNF for B). Surface-labeled receptors were detected by streptavidin pull-down followed by anti-Trk immunoblotting. Representative anti-Trk immunoblots are shown for TrkA- and TrkB-expressing cells. (C) Colocalization of TrkA and TrkB receptors relative to LAMP1, visualized by confocal microscopy in PC12 cells fixed after 75-min incubation at 37°C in the presence of ligand.

Figure 2.

Trk receptors exhibit similar ligand-induced internalization rates. (A) Schematic of internalization assay. PC12 cells stably expressing FLAG-tagged TrkA or TrkB were incubated at 37°C for 0, 5, 10, 20, 30, or 60 min in the presence or absence of 50 ng/ml ligand (NGF for TrkA and BDNF for TrkB) and then incubated at 4°C for 1 h with fluorescently labeled anti-FLAG antibodies. The cells were then assayed for ligand-dependent (B) and ligand-independent (C) internalization of receptors by flow cytometric determination of surface immunofluorescence. Squares, TrkA; circles, TrkB.

Figure 3.

Determination of ligand-dependent Trk receptor recycling rates. (A) PC12 cells stably expressing FLAG-tagged TrkA or TrkB were incubated at 37°C for 15 min with fluorescenated (Alexa-488) anti-FLAG antibodies (2 μg/ml) and then incubated at 37°C with 50 ng/ml ligand (NGF for TrkA and BDNF for TrkB) for 30 min. Afterward, the noninternalized fluorescenated anti-FLAG antibodies were stripped with EDTA, and cells incubated in the presence of 10 μg/ml anti-Alexa-488 antibodies for the indicated times before fixation. To obtain the zero-minute time point, cells were fixed after the EDTA strip step. Unquenched fluorescence was detected by wide-field microscopy. Relative fluorescence intensities per cell are plotted as a function of time of incubation with anti-Alexa-488. Dotted lines represent steady-state levels of immunofluorescence intensity/cell. Squares, TrkA; circles, TrkB. (B) Representative images from such an experiment, showing Alexa-488 fluorescence after 0 min or 45 min with anti-Alexa-488, or 45-min chase without anti-Alexa-488. (C) Colocalization of internalized TrkA and TrkB receptors relative to transferrin receptor visualized by confocal microscopy. PC12 cells stably expressing FLAG-tagged TrkA or TrkB were incubated at 37°C for 15 min with fluorescenated (Alexa-488) anti-FLAG antibodies and then incubated at 37°C with 50 ng/ml ligand (NGF for TrkA, BDNF for TrkB) and fluorescenated transferrin (rhodamine-transferrin) for 15 min. The noninternalized fluorescenated anti-FLAG antibodies were stripped with PBS/EDTA. Cells were reincubated at 37°C in the presence of rhodamine-transferrin, fixed for 30 min, and imaged by confocal microscopy. Representative micrographs are shown. The proportion of colocalization between internalized Trk and transferrin receptors is presented as a mean ± SEM determined from analysis of four independent experiments (^* represents p < 0.01, Student's t test).

Figure 4.

Determination of ligand-dependent Trk receptor recycling in PC12 cells. (A) Cells expressing FLAG-tagged TrkA or TrkB were incubated at 37°C for 15 min with fluorescenated (Alexa-488) anti-FLAG antibodies and then incubated at 37°C with 50 ng/ml ligand (NGF for TrkA, BDNF for TrkB, and EGF for EGFR) for 30 min. Afterward, the noninternalized fluorescenated anti-FLAG antibodies were stripped with EDTA, and cells were incubated in the presence of Cy3 anti-mouse at 37°C for 45 min before fixation. (B) Representative images from such an experiment with PC12 cells. The control condition was a parallel coverslip incubated for 30 min in the absence of ligand and without an EDTA stripping step. The strip condition was a parallel coverslip in which cells were fixed immediately after the EDTA-mediated stripping step. (C) The recycling behavior was quantitated as described in Materials And Methods, and the error bars represent the SEM of five independent experiments.

Figure 5.

Determination of ligand-dependent Trk receptor recycling in cortical neurons. (A) Representative images from recycling experiment described in Figure 4 with primary cortical neurons expressing FLAG-tagged TrkA receptors. (B) Representative images from such an experiment with primary cortical neurons expressing FLAG-tagged TrkB receptors. (C) Quantitation of recycling in A and B as described in Materials and Methods, and the error bars represent the SEM of five independent experiments.

Figure 6.

Identification of a sequence in TrkA responsible for ligand-dependent receptor recycling. (A) PC12 cells were transfected with TrkB chimeras containing corresponding regions of TrkA. (TrkB^ecA, TrkA extracellular; TrkB^tmA, TrkA transmembrane; TrkB^jmA, TrkA juxtamembrane; TrkB^tkA, TrkA tyrosine kinase; and TrkB^ctA, TrkA carboxy terminal). TrkA and TrkB was also transfected as controls. Steady-state ligand-dependent recycling was measured in these cells as described in Figure 4. (B) PC12 cells were transfected with TrkA constructs lacking the following intracellular regions: carboxy terminus, TrkAΔCT; tyrosine kinase, TrkAΔTK; juxtamembrane, TrkAΔJM, as well as smaller subregions of the juxtamembrane region (TrkAΔBox1-3), and steady-state ligand-dependent recycling was measured in these cells as described in Figure 4. (C) PC12 cells were transfected with TrkA constructs containing point mutations in the tyrosine kinase region (K546A), and the juxtamembrane region (N496A and Y499A), and steady-state ligand-dependent recycling was measured in these cells as described in Figure 4. In all experiments, error bars represent the SEM of five independent experiments. (^* represents p < 0.01, Student's t test).

Figure 7.

Trk receptors mutants exhibit differential ligand-induced trafficking to degradative pathways. PC12 cells stably expressing TrkA (A) and TrkB (B) mutants were surfaced biotinylated and incubated at 37°C for the indicated times in the absence or presence of 50 ng/ml ligand (NGF for A and BDNF for B). Surface-labeled receptors were detected by streptavidin pull-down followed by anti-Trk immunoblotting. Representative anti-Trk immunoblots are shown for TrkA- and TrkB-expressing cells.

Figure 8.

Effect of prolonged neurotrophin treatment on Trk-dependent survival. PC12 cells stably expressing TrkA or TrkB mutants were serum starved for 8 h and then surfaced biotinylated (A and B) and incubated at 37°C with 50 ng/ml ligand (NGF for TrkA and BDNF for TrkB) for 24 h. Surface-labeled receptors were detected by streptavidin pull-down followed by anti-Trk immunoblotting. Total receptors were detected by Trk immunoprecipitation followed by anti-Trk immunoblotting. Representative anti-Trk immunoblots are shown for TrkA- and TrkB-expressing cells. (C) Native PC12 cells (WT) or PC12 cells stably expressing TrkA or TrkB mutants were serum starved for 8 h and then incubated at 37°C with or without 50 ng/ml ligand (NGF for TrkA and BDNF for TrkB) for 5 min or 24 h. Levels of phospho-Trk, Trk, phospho-Akt, and Akt were determined by immunoblotting. (D) Native PC12 cells (WT) or PC12 cells stably expressing TrkA or TrkB mutants were serum starved for 8 h and then incubated at 37°C with 50 ng/ml ligand (NGF for TrkA and BDNF for TrkB) for 24 h, fixed, and processed for TUNEL analysis. The proportion of TUNEL-positive cells was scored for each culture condition, compared with WT PC12 cells (-NGF). Error bars indicate SEM from three independently conducted experiments.