Differential endocytic sorting of p75NTR and TrkA in response to NGF: a role for late endosomes in TrkA trafficking

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Abstract

NGF binds to two receptors, p75NTR and TrkA. The endosomal trafficking of receptors is of emerging importance for the understanding of their signaling. We compared the endocytic trafficking of the two NGF receptors in PC12 cells. Both p75NTR and TrkA were internalized in response to NGF and colocalized with early endosomes. However, surprisingly, the subsequent endosomal trafficking paths of both NGF receptors diverged: whereas p75NTR recycled back to the surface, TrkA moved to late endosomes and underwent lysosomal degradation. By performing subcellular fractionations of NGF stimulated PC12 cells, tyrosine-phosphorylated TrkA was recovered in fractions corresponding to late endosomes. This implicates these organelles as novel endosomal NGF signaling platforms. Furthermore, the trafficking of NGF receptors could be manipulated by pharmacological means. Disrupting p75NTR recycling diminished TrkA activation in response to low concentrations of NGF, demonstrating a functional role for the recycling of p75NTR.

Introduction

Much progress has been made in understanding the endosomal trafficking of signaling receptors such as receptor tyrosine kinases (RTKs) (Sorkin and Von Zastrow, 2002). After endocytosis by clathrin-coated pits (CCPs) into clathrin-coated vesicles (CCVs), signaling receptors move into early endosomes (according to kinetical considerations), which are also referred to as sorting endosomes (according to their function) (Gruenberg, 2001). At this trafficking checkpoint, important sorting decisions are made: from early endosomes, RTKs may either return to the plasma membrane via recycling endosomes for re-utilization, or–alternatively–they proceed further along the degradative pathway in a microtubule- and motor-dependent fashion, reaching multivesicular bodies (MVBs)/late endosomes. Often, MVBs are regarded as a subpopulation of late endosomes (Sorkin and Von Zastrow, 2002), as both MVBs and late endosomes share a similar morphology consisting of an outer, limiting membrane and multiple internal vesicles (Stahl and Barbieri, 2002). Trafficking of RTKs from the outer membrane into the inner membranes in MVBs/late endosomes is thought to terminate RTK function by disrupting access of the RTK's catalytic domain to cytoplasmic substrates (Sorkin and Von Zastrow, 2002). Subsequently, the physical destruction of RTKs by proteolysis takes place in lysosomes.

Nerve growth factor (NGF) is the prototypic neurotrophin, a group of structurally related signaling proteins that are crucial for the survival, differentiation, and maintenance of specific neuronal populations (Snider, 1994). The effects of NGF are mediated by two different cell surface receptors: the receptor tyrosine kinase TrkA (Kaplan and Miller, 2000, Segal, 2003), and p75NTR (also called p75) (Dechant and Barde, 2002, Lee et al., 2001). Both receptors not only collaborate in NGF binding (Hempstead et al., 1991), internalization (Mahadeo et al., 1994), and signaling (Barker and Shooter, 1994), but also transduce signals independently of each other (Lad et al., 2003).

The subcellular localization of NGF receptors affects their signaling (Ginty and Segal, 2002, Neet and Campenot, 2001). Previous work has demonstrated that iodinated NGF shows not only strong surface binding to neurons but is also found within endosomal organelles (Claude et al., 1982, Ure and Campenot, 1997). Accordingly, TrkA signaling in neuronal cells may emerge from the cell surface as well as from endosomes (MacInnis and Campenot, 2002, Zhang et al., 2000). Given that there are different endosomal organelles, the question arises as to which endosomal organelles NGF receptors are sorted, and then whether certain organelles are restricted to eliciting specific signaling pathways.

Some previous studies started to examine the endosomal trafficking of the two NGF receptors. Regarding p75NTR, two studies agreed on the post-endosomal trafficking of this receptor to early endosomes (Bronfman et al., 2003, Delcroix et al., 2003). However, there have been two opposing views on the trafficking of p75NTR downstream of early endosomes. On one hand, internalized p75NTR ligands such as anti-p75NTR antibodies or iodinated NGF were sorted to the recycling pathway in neuronal cell lines (Bronfman et al., 2003, Eveleth and Bradshaw, 1992), indicating that p75NTR also recycles. On the other hand, it was demonstrated that iodinated NGF internalized by p75NTR expressing glia cells undergoes lysosomal degradation (Kahle et al., 1994), indicating that p75NTR might move to MVBs/late endosomes and lysosomes. Internalized antibodies or iodinated ligands might potentially dissociate from their receptors during endosomal acidification steps and subsequently follow separate trafficking routes (Yarden, 2001). If p75NTR ligands are more or less sensitive to endosomal pH decreases in different cell types, then this could provide an explanation for the dissimilar previous results.

Regarding TrkA, it has been convincingly established that NGF triggers the trafficking of this RTK via CCVs to early endosomes in neuronal cells (Delcroix et al., 2003, Howe et al., 2001). Thus, TrkA must subsequently be sorted to either recycling endosomes or to MVBs/late endosomes. One previous study using human TrkA-overexpressing Chinese hamster ovary (CHO) cells found re-release of internalized iodinated NGF (Zapf-Colby and Olefsky, 1998), indicating that TrkA would recycle instead of moving to MVBs/late endosomes and lysosomes. In contrast, the work of Jullien et al. showed that the degradation of endogenous TrkA in PC12 cells is blocked by lysosomal inhibitors (Jullien et al., 2002), indicating a sorting of TrkA from early endosomes to the degradative pathway.

Taken together, the trafficking of both NGF receptors downstream of early endosomes has remained unclear. Therefore, we compared the endocytic sorting of both endogenous p75NTR and TrkA in response to NGF in PC12 cells. Surprisingly, we revealed a differential sorting of both NGF receptors. Whereas p75NTR was sorted to recycling endosomes, TrkA was sorted to MVBs/late endosomes. The differential sorting of both NGF receptors could play a functional role in NGF signaling, since disruption of p75NTR recycling altered NGF signaling and phosphorylated TrkA was recovered in MVBs/late endosomes.

Section snippets

NGF induces the colocalization of p75NTR and TrkA with a marker of early endosomes

Many previous studies demonstrated a collaboration of p75NTR and TrkA in the formation of NGF high-affinity binding sites and in NGF signaling (Hempstead, 2002, Roux and Barker, 2002), as well as independent signaling functions of either NGF receptor (Lad et al., 2003). In addition, p75NTR and TrkA collaborate in the intracellular accumulation of NGF (Jullien et al., 2002, Mahadeo et al., 1994). Accordingly, it has been found that NGF induces the internalization of both receptors in PC12 cells (

Discussion

Growth factor receptor trafficking is an emerging focus of interest, since sorting and signaling of receptors are tightly linked (Di Fiore and De Camilli, 2001). NGF represents the prototypical growth factor in the nervous system. Because of the tantalizing morphological complexity of neurons, the elucidation of mechanisms linking signaling of neurotrophin receptors to their trafficking is a challenging task. So far, the endocytic sorting of the two NGF receptors is not well understood.

Conclusion

In summary, we propose a model for the endocytic trafficking of the two NGF receptors in response to NGF in PC12 cells, which could explain previous findings, for example, the data showing that PC12 cells not only degrade internalized iodinated NGF but also re-release intact NGF back into the culture medium (Buxser et al., 1990), or that NGF pretreatment differentially affected the cross-linking of iodinated NGF to its two surface receptor complexes in PC12 cells (Hosang and Shooter, 1987,

Reagents

Purified mouse 2.5 S NGF was a gift from Dr. W.C. Mobley (Stanford University) (Delcroix et al., 2003), whereas purified human NGF was a gift from Genentech (South San Francisco). All experiments presented here were performed using mouse NGF unless otherwise noted. Antibodies directed against the carboxyl-terminus of rat TrkA (TA-63) were raised in rabbits by immunization with the peptide CITQGRELERPRA (Eurogentec, Belgium), purified using a Protein G based kit from Pierce, and stored as a

Acknowledgments

We would like to thank Dr. L.F. Reichardt (UCSF) for the gift of REX antibodies, Dr. W.C. Mobley (Stanford University) and Genentech (South San Francisco) for the gift of purified NGF, Dr. J. Gruenberg (University of Geneva) for the gift of LBPA antibodies and stimulating discussions, and S. Rossaint for help with graphic art. This study was supported by a grant from the Swiss National Science Foundation.

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    Present address: Instituto de Investigaciones Biomédicas “Alberto Sols”, CSIC-UAM, E-28029, Madrid, Spain.

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