Abstract
The activity of Notch ligands is tightly regulated by trafficking events occurring both before and after ligand-receptor interaction. In particular endocytosis and recycling have been shown to be required for full signaling activity of the ligands before they encounter the Notch receptor. However little is known about the precise endocytic processes that contribute to ligand internalization. Here we demonstrate that endocytosis contributes to Dll1 signaling activity by preserving the ligand from shedding and degradation. We further show that the glycosphingolipid-binding motif originally identified in Drosophila Notch ligands is conserved in mammals and is necessary for Dll1 internalization. Mutation of its conserved tryptophan residue results in a Dll1 molecule which is rapidly inactivated by shedding and degradation, does not recycle to the cell surface and does not activate Notch signaling. Finally, silencing in the signal-sending cells of glucosylceramide synthase, the enzyme implicated in the initial phase of glycosphingolipid synthesis, down-regulates Notch activation. Our data indicate that glycosphingolipids, by interacting with Dll1, may act as functional co-factors to promote its biological activity.
Highlights
Notch signaling is an evolutionary-conserved pathway involved in cell-cell communication [1]
We have previously shown that the Notch ligand Delta-like1 (Dll1) essentially localizes to these detergentresistant membranes (DRMs), contrary to non-active mutants [26], suggesting that these domains are involved in the regulation of Dll1 signaling activity
We have previously reported that wild type Dll1 can be detected in fractions containing detergent-resistant membranes (DRMs) after flotation in a sucrose gradient, while non-active mutants of Dll1 do not localize to these fractions [26]
Summary
Notch signaling is an evolutionary-conserved pathway involved in cell-cell communication [1]. Despite the apparent simplicity of this pathway, Notch activation is tightly regulated at multiple levels, both in the signal-emitting and signalreceiving cell [5,6]. Several studies have pointed to the importance of endocytosis and recycling of the ligand in signal-emitting cells [8,9]. Two possible non-exclusive models have been proposed to explain how ligand endocytosis could activate Notch signaling: i) prior to Notch binding, endocytosis and recycling would be required to generate an active surface-expressed ligand, and/or to maintain a certain level of ligand at the cell surface, ii) following interaction with the receptor, endocytosis of the ligand in the signal-sending cell would produce a mechanical force sufficient to induce structural changes in the receptor, allowing its proteolytic cleavage and subsequent activation of the pathway [11]
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