Trk-signaling endosomes are generated by Rac-dependent macroendocytosis

Abstract

Neurotrophin (NT) control of neuronal survival and phenotype requires the persistent activation of signaling intermediates over hours and days, a result of which is long-term alterations in gene expression (1). The molecular basis for long-term NT signaling, as exemplified by the PC12 cell model, is by persistent signaling by NGF through TrkA-receptor tyrosine kinase effectors, including Ras, Rap, and the Erk/MAP kinases (2–4). Interestingly, and unlike the case for TrkA, the EGF receptor (EGFR) tyrosine kinase, which signals through the same Erk/MAP kinases in PC12 cells, mediates transient rather than sustained signaling. Indeed, transient signaling through Erk/MAP kinases has been invoked to explain the inability of EGFR to mediate alterations of neuronal phenotype and survival (see ref. 5). The mechanisms underlying sustained vs. transient Erk/MAP kinase signaling, however, have not been elucidated.

That sustained Erk signaling by Trk is dictated directly by the receptor is suggested by the preferential binding of signaling adaptors to TrkA, such as SNT/FRS2 and ARMS, and the stimulation of a Rap1-based signaling pathway (6). Consistent with this preference, long-term signaling by TrkA receptors has been hypothesized to be specifically mediated through endosomes associated with Rap1 (4, 7, 8). However, another characteristic of Trk endosomal signaling is long-lived endosomes, as exemplified by Trk endosomal signaling that persists from the nerve terminal to the soma (1, 9). These endosomes are apparently refractory to degradation during and after retrograde transport, and can signal through Erk kinases (10, 11). The Trk-signaling endosome responsible for retrograde signaling in neurons is formed by an unusual endocytic mechanism mediated by the membrane trafficking protein, Pincher (12, 13). Trk is specifically concentrated and endocytosed at complex plasma membrane ruffles by a process termed “macroendocytosis.” Pincher confers on Trk endosomes the ability to avoid lysosomal processing (13), although the underlying mechanism is not known.

In contrast to Trk, EGFR endosomes are short-lived and their signaling through Erk kinases is transient. These properties are consistent with EGFR's established clathrin-based endocytic pathway, wherein signaling is terminated by rapid lysosomal processing (14). Thus, the differences between EGFR and Trk raise the possibility that sustained signaling may be a function of a long endosome lifetime (15). The extent to which transient or sustained signaling is an intrinsic property of Trk receptors and/or a function of the endosomal process has yet to be determined.

An understanding of Trk endosomal processing would yield insights into the selective ability of Trk to mediate sustained retrograde signaling. However, with the exception of Dynamin (8) and Pincher, the molecular entities that mediate the formation, processing, and fate of specialized Trk signaling endosomes are relatively unexplored. Here, we define a Rac-based macroendocytic process for Trk, which results in Trk multivesicular bodies that tenaciously retain molecular characteristics of early endosomes, allowing for long-term Trk endosomal signaling.

Acknowledgments

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