SUMMARY Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Recent structural studies have shown that neurotrophins interact with dimers of the p75 neurotrophin receptor (p75NTR), but the actual mechanism of receptor activation has remained elusive. Here we show that p75NTR forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys257 in its transmembrane domain. Mutation of Cys257 abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75NTR/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75NTR. FRET experiments revealed a close association of p75NTR intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys257 did not alter the oligomeric state of p75NTR, the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75NTR by a novel mechanism involving rearrangement of disulphide-linked receptor subunits.

We have recently shown that oxytocin inhibits cell proliferation when the vast majority of oxytocin receptors are excluded from caveolin-1-enriched microdomains, and that, on the contrary, it has a mitogenic effect when the receptors are targeted to these plasma membrane domains. In this study, we investigated whether the receptors located inside and outside caveolar microdomains initiate different signalling pathways and how this may lead to opposite effects on cell proliferation. Our data indicate that, depending on their localization, oxytocin receptors transactivate EGFR and activate ERK1/2 using different signalling intermediates. The final outcome is a different temporal pattern of EGFR and ERK1/2 phosphorylation, which is more persistent when the receptors are located outside caveolar microdomains and inhibit cell growth, and very transient when they are located in caveolar microdomains and stimulate cell growth. Finally, only the activation of receptors located outside caveolar microdomains correlates with the activation of the cell cycle inhibitor p21 WAF1/CIP1 , thus suggesting that the antiproliferative OTR effects may, in this case, be achieved by a sustained activation of EGFR and MAPK leading to the induction of this cell cycle regulator.

The p75 neurotrophin receptor (p75 NTR ) plays multiple roles in neuronal physiology through interactions with many ligands and coreceptors. However, its intracellular neuronal trafficking prior to and after neurotrophin activation is still poorly characterized. We have previously shown that in response to nerve growth factor (NGF), p75 NTR is retrogradely transported along the axons of motor neurons (MNs) in carriers shared with NGF, brainderived neurotrophic factor and the tyrosine kinase receptor TrkB. Here, we report that NGF does not enhance the internalization or degradation of p75 NTR , which undergoes a rapid dynamin-dependent and clathrin-independent recycling process in MNs. Instead, incubation of cells with NGF leads to the redirection of a pool of plasma membrane p75 NTR into clathrin-coated pits. The subsequent internalization of p75 NTR via clathrin-mediated endocytosis, as well as the activity of Rab5, are essential for the sorting of the p75 NTR -containing endosomes to the axonal retrograde transport pathway and for the delivery of p75 NTR to the soma. Our findings suggest that the spatial regulation of p75 NTR signalling is controlled by these ligand-driven routes of endocytosis.