Trk-signaling endosomes are generated by Rac-dependent macroendocytosis

Abstract

Why neurotrophins and their Trk receptors promote neuronal differentiation and survival whereas receptor tyrosine kinases for other growth factors, such as EGF, do not, has been a long-standing question in neurobiology. We provide evidence that one difference lies in the selective ability of Trk to generate long-lived signaling endosomes. We show that Trk endocytosis is distinguished from the classical clathrin-based endocytosis of EGF receptor (EGFR). Although Trk and EGFR each stimulate membrane ruffling, only Trk undergoes both selective and specific macroendocytosis at ruffles, which uniquely requires the Rho-GTPase, Rac, and the trafficking protein, Pincher. This process leads to Trk-signaling endosomes, which are immature multivesicular bodies that retain Rab5. In contrast, EGFR endosomes rapidly exchange Rab5 for Rab7, thereby transiting into late-endosomes/lysosomes for degradation. Sustained endosomal signaling by Trk does not reflect intrinsic differences between Trk and EGFR, because each elicits long-term Erk-kinase activation from the cell surface. Thus, a population of stable Trk endosomes, formed by specialized macroendocytosis in neurons, provides a privileged endosome-based system for propagation of signals to the nucleus.

Fig. 1.

Rac mediates the formation of plasma membrane ruffles from which activated TrkA is internalized. TrkA-PC12 cells were cotransfected with Rac-T7 and Pincher-HA (A). PC12 cells were transfected with TrkA (B Left) or cotransfected with TrkA and Rac-T7 (B Right) or with TrkA and RacV12-GFP (C). Cells were or were not treated with NGF for the indicated times and fixed and stained as described in Materials and Methods. P-Trk (red, A Upper, B, and C), phalloidin (Actin, green, A), anti-T7 (Rac-T7, cyan in A, green in B), RacV12-GFP (green in C), anti-Pincher (red in A Lower). (Scale bar: 2 μm.)

Fig. 2.

TrkA, but not EGFR, internalization is Rac-dependent. PC12 cells were triple-transfected with TrkA-YFP, Pincher-HA, and RacN17-T7 (A Upper) or with TrkA-YFP, PincherG68E-HA, and Rac-T7 (A Lower); cotransfected with EGFR-GFP and RacN17-T7 (C); or transfected with TrkA-YFP (A Left) or EGFR-GFP (C Left). TrkA-PC12 cells were cotransfected with Pincher-HA and DynaminK44A-GFP (B) or transfected with Pincher-HA alone (B, Control). Cells were treated with NGF for 10 min (A and B) or treated with EGF for 30 min (C) and then fixed and stained as in Materials and Methods. TrkA-YFP (green, A), P-TrkA (green, B), EGFR-GFP (green, C), RacN17-T7 (red, A and C) and Rac-T7 (red, A), Pincher-HA (red, B), Pincher-HA (cyan, A) and PincherG68E-HA (cyan, A), DynaminK44A-GFP (cyan, B). (Scale bar: 2 μm.)

Fig. 3.

Internalization of EGFR, but not Trk, is mediated by CCPs and CCVs in a Pincher-independent manner. (A) Cultured hippocampal neurons were infected with adenoviruses expressing TrkB-GFP and treated with BDNF for 15 min (Upper) or EGFR-GFP and treated with EGF for 15 min (Lower), and immunogold-electron microscopy by using antibodies against GFP carried out as described in Materials and Methods. Immunogold-labeled TrkB-GFP (Upper) was associated with plasma membrane ruffles (Left) and macroendosomes (Right) but not CCPs (arrowhead, Right), whereas EGFR-GFP (Lower) was gold-labeled at CCPs (arrowhead, Left) and CCVs (arrowhead, Right). (B) PC12 cells were transfected with TrkA-YFP (Left Upper) or EGFR-GFP (Left Lower) or cotransfected with PincherG68E-HA and either TrkA-YFP (Right Upper) or EGFR-GFP (Right Lower). Cells were treated with NGF for 20 min (Upper) or EGF for 30 min (Lower) and fixed and stained as in Fig. 1. TrkA-YFP (green, Upper), EGFR-GFP (green, Lower), PincherG68E-HA (red). (Scale bars: 0.2 μm in A, 2 μm in B.)

Fig. 4.

Pincher-mediated P-TrkA endosomes and multivesicular bodies associate with the early-endosome effector Rab5 but avoid the late-endosome effector, Rab7. TrkA-PC12 cells were cotransfected with Pincher-HA and Rab5-GFP and treated with NGF for 30 min (A). PC12 cells were triple-transfected with TrkA, Pincher-HA, and Rab7-GFP and treated with NGF for 60 min (C Upper), or cotransfected with EGFR and Rab7-GFP and treated with EGF for 60 min (C Lower). Cells were fixed and stained as in A. P-TrkA (red, A; green, C), Rab5-GFP (green, A), EGFR (green, C), Rab7-GFP (red, C). (Scale bars in A and C: 2 μm.) PC12 cells were cotransfected with Pincher-HA and Rab5-GFP (B), treated with NGF, and immunogold-EM-analyzed by using antibodies against GFP or Pincher as described in Materials and Methods. Ultrathin serial sections were alternately labeled with anti-GFP antibody for Rab5-GFP (B) or with anti-Pincher antibody for Pincher-HA as indicated. Rab5-GFP was associated with Pincher within the same macroendosomes. (Scale bar: 200 nm.)

Fig. 5.

Sustained signaling through Erk1/2 kinases is differentially mediated by compartmentalized TrkA and EGFR receptors. (A) PC12 cells were infected with an adenovirus expressing dominant-negative Dynamin (DynK44A, Lower) or expressing GFP (Control, Upper). Forty-eight hours after infection, cells were starved overnight and then treated with EGF as indicated. (B) PC12 cells stably expressing a temperature-sensitive mutant Dynamin (DynG273D), were starved overnight and preincubated either at the permissive (33°C, top panel) or nonpermissive temperature (39°C, bottom panel) for 20 min. Cells were treated with EGF as indicated. (C) Endosomal signaling was assessed in TrkA-PC12 cells (Upper) or EGFR-PC12 cells (Lower) pretreated with the MEK inhibitor U0126 for 15 min as indicated and then also treated with NGF or EGF as indicated for 5 min. Ligands and inhibitor were removed and the cells washed and reincubated for the indicated times, all as described in Materials and Methods. In all experiments, whole cell lysates were blotted and probed with an anti-P-Erk1/2 antibody.