Ectodomain shedding of the Notch ligand Jagged1 is mediated by ADAM17 but is not a lipid raft-associated event
Résumé
Notch signaling is an evolutionarily conserved pathway involved in cell fate specification. The initiating event in this pathway is the binding of a Notch receptor to a Delta/Serrate/Lag-2 (DSL) ligand on neighbouring cells triggering the proteolytic cleavage of Notch within its extracellular juxtamembrane region; a process known as proteolytic 'shedding' and catalyzed by members of the ADAM (a disintegrin and metalloproteinase) family of enzymes. Jagged1 is a Notch binding DSL ligand which is also shed by an ADAM-like activity raising the possibility of bi-directional cell-cell Notch signaling. In the current study we have unequivocally identified the sheddase responsible for shedding Jagged1 as ADAM17, the activity of which has previously been shown to be localized within specialized microdomains of the cell membrane known as 'lipid rafts'. However, we have shown that replacing the transmembrane and cytosolic regions of Jagged1 with a glycosylphosphatidylinositol (GPI) anchor, thereby targeting the protein to lipid rafts, did not enhance its shedding. Furthermore, the Jagged1 holoprotein, its ADAM-cleaved C-terminal fragment, and ADAM17 were not enriched in raft preparations devoid of contaminating non-raft proteins. We have also demonstrated that wild-type Jagged1 and a truncated polypeptide-anchored variant lacking the cytosolic domain were subject to similar constitutive and phorbol ester-regulated shedding. Collectively these data demonstrate that Jagged1 is shed by ADAM17 in a lipid raft-independent manner and that the cytosolic domain of the former protein is not a pre-requisite for either constitutive or regulated shedding.
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