215 MET ECD shedding and use of targeted therapies Similar to other receptors having intrinsic kinase activity, MET is subjective to degradation avoiding overactivity and possibly tumorigenesis (24). Besides ligand-dependent internalization and subsequent degradation by the proteasome and lysosome (32), membranous MET is subjective to proteolytic cleavage independent of ligand stimulation (24). In the last context, MET can be downregulated by presenilin-regulated intramembrane proteolysis (PS-RIP) (33). During PS-RIP, the receptor is first cleaved within its juxtamembrane domain by membrane metalloproteases. This results in the shedding of a soluble MET N-terminal fragment (MET-NTF) into the extracellular space, a process referred to as ectodomain shedding. Notably, the residual 55 kDa C-terminal fragment (MET-CTF) remains anchored to the membrane (24, 34-36). MET-NTF shedding is driven by ADAM metalloproteases 10 and 17 (ADAM10 and ADAM17), as shown by its inhibition upon their genetic ablation (33, 37, 38). Ectodomain shedding is immediately followed by a second cleavage event of the MET-CTF by the γ-secretase complex. The resulting 50 kDa intracellular domain of MET (MET-ICD) is released into the cytosol and degraded by the proteasome (33). The MET-CTF fragments escaping γ-secretase cleavage undergo lysosomal degradation (13, 16, 34) (Figure 1B). Production of NH2-terminal (N-terminal) MET monoclonal antibodies (moAbs) (25, 39), directed against the extracellular domain of MET, allowed further characterization of shed NTFs. Using these antibodies and breast cancer cell lines, Athauda et al. (40) specified the sizes of NTFs present in the culture medium under reducing conditions, being 50β, 55β, 75β, 85β, and 100β kDa (Table 1). Since the goal of this investigation was to evaluate whether the soluble MET ectodomain is a relevant biomarker for overall tumor burden and malignant potential (details below), no further functional analyses were performed concerning the origin of these fragments. However 15 years earlier (1991), using stably transfected MET NIH-3T3 cells, Prat et al. (25) already showed that NTF p75β originates from posttranslational processing of MET protein products encoded by full-length MET transcripts. More specifically it was shown that NTF p75β is generated by proteolytic processing of membrane bound MET using wt MET overexpressing GTL-16 cells. Finally, treating GTL-16 cells with TPA, illustrated that generation of NTF p75β from membranous p190MET and p140MET, another C-terminal truncated transmembranous protein product of MET which is generated in the endoplasmatic reticulum (41), is mediated by protein kinase C. It was in 2010 that Schelter et al. (15) showed that DN30, one of the antibodies used by Prat et al. (25), induced ADAM10 mediated ectodomain shedding in A549 lung carcinoma cells. 7
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