Regulated Proteolysis of NOTCH2 and NOTCH3 Receptors by ADAM10 and Presenilins

Abstract

cIn mammals, there are four NOTCH receptors and five Delta-Jagged-type ligands regulating many aspects of embryonic development and adult tissue homeostasis. NOTCH proteins are type I transmembrane receptors that interact with ligands on adjacent cells and are activated by regulated intramembrane proteolysis (RIP). The activation mechanism of NOTCH1 receptors upon ligand binding is well understood and requires cleavage by ADAM10 metalloproteases prior to intramembranous cleavage by ␥-secretase. How the other human NOTCH receptor homologues are activated upon ligand binding is not known. Here, we dissect the proteolytic activation mechanism of the NOTCH2 and NOTCH3 receptors. We show that NOTCH2 and NOTCH3 signaling can be triggered by both Delta-Jagged-type ligands and requires ADAM10 and presenilin-1 or -2. Importantly, we did not find any role for the highly related ADAM17/TACE (tumor necrosis factor alpha-converting enzyme) protease in ligand-induced NOTCH2 or NOTCH3 signaling. These results demonstrate that canonical ligand-induced proteolysis of the NOTCH1, -2, and -3 receptors strictly depends on consecutive cleavage of these receptors by ADAM10 and the presenilin-containing ␥-secretase complex, leading to transcriptional activation.

Insights into Autoregulation of Notch3 from Structural and Functional Studies of Its Negative Regulatory Region

“…The HD is cleaved during normal receptor maturation by a furin-like protease at a site called S1 (Logeat et al, 1998), but the NRR is resistant to further proteolysis in the absence of ligand (Gordon et al, 2007; Sanchez-Irizarry et al, 2004). Ligand stimulation induces receptor sensitivity to metalloprotease cleavage at a site called S2 (Brou et al, 2000; Groot et al, 2014; Mumm et al, 2000), which lies near the C-terminal end of the HD (Figure 1A). After metalloprotease cleavage, the truncated receptor, called NEXT, is primed for intramembrane cleavage at site S3 and additional sites by gamma secretase, which releases the intracellular part of Notch (NICD) from the membrane.…”

Human NOTCH2 Is Resistant to Ligand-independent Activation by Metalloprotease Adam17

“…We recently demonstrated that ligand induced proteolysis of NOTCH proteins is similar and all require the Adam10 protease and presenilin1/2-dependent ␥-secretase activity (12). The mechanism by which leukemia associated NOTCH1 HD mutations contribute to ligand independency in T-ALL has also been amply studied and involves destabilization of the NRR (6,18).…”

“…Plasmids and Transfections-Full-length human NOTCH1 and NOTCH2 cDNAs used here were constructed as described previously (12). Full-length FLAG-tagged mouse Notch2 cDNA was a kind gift from Y. Hamada and was described previously (25).…”

“…Calcium Chelation-We recently reported that the ligand-dependent signaling cascade for NOTCH2 and NOTCH3 in mammalian cells follow the NOTCH1 paradigm of regulated intramembrane proteolysis and the requirement of Adam10 ectodomain cleavage (12). To investigate the ligand-independent activation mechanism of NOTCH2 receptors in a comparative manner, we used EDTA, a divalent calcium chelator.…”

“…by the Adam10 metalloprotease prior to intramembranous cleavage by presenilins after ligand binding, following the NOTCH1 activation paradigm (12).…”