Negative Regulation of Epidermal Growth Factor Signaling by Selective Proteolytic Mechanisms in the Endosome Mediated by Cathepsin B

Abstract

We have investigated the relevant protease activity in rat liver, which is responsible for most of the receptormediated epidermal growth factor (EGF) degradation in vivo. EGF was sequentially cleaved by endosomal proteases at a limited number of sites, which were identified by high performance liquid chromatography and mass spectrometry. EGF proteolysis is initiated by hydrolysis at the C-terminal Glu 51 -Leu 52 bond. Three additional minor cleavage sites were identified at positions Arg 48 -Trp 49 , Trp 49 -Trp 50 , and Trp 50 -Glu 51 after prolonged incubation. Using nondenaturating immunoprecipitation and cross-linking procedures, the major proteolytic activity was identified as that of the cysteine protease cathepsin-B. The effect of injected EGF on subsequent endosomal EGF receptor (EGFR) proteolysis was further evaluated by immunoblotting. Using endosomal fractions prepared from EGF-injected rats and incubated in vitro, the EGFR was lost with a time course superimposable with the loss of phosphotyrosine content. The cathepsin-B proinhibitor CA074-Me inhibited both in vivo and in vitro the endosomal degradation of the EGFR and increased the tyrosine phosphorylation states of the EGFR protein and the molecule SHC within endosomes. The data, therefore, describe a unique pathway for the endosomal processing of internalized EGF receptor complexes, which involves the sequential function of cathepsin-B through selective degradation of both the ligand and receptor.Ligand-induced desensitization and down-regulation mechanisms are important factors in the regulation of transmembrane receptors (1), and within the receptor tyrosine kinase family, significant similarities and differences have emerged in attenuation mechanisms (2, 3). One area of research into the differing signaling outcomes of the receptor tyrosine kinases involves the intraendosomal processing of the internalized ligands and how this affects tyrosine phosphorylation of the receptor tyrosine kinases as well as their substrates (1). It has been proposed that the ligand degradation state within the endosomes contributes to both the location and specificity of downstream signaling (1,4). Suggestive evidence has come from the differences in the levels of endosomal degradation of EGF 1 and transforming growth factor ␣ (TGF␣) after internalization, which coincided with altered receptor trafficking (5) and was linked to the different biopotency observed for the two ligands. Recently, using the in situ liver model system for signal transduction, we have shown an alteration in insulin receptor phosphorylation and trafficking through the endosomal pathway in response to the insulin analog H2, a genetically engineered analog that displays a reduced rate of proteolysis in endosomes as compared with authentic insulin (4). Hence, the balance between net tyrosine phosphorylation and dephosphorylation in the endosome is regulated directly or indirectly by ligand proteolysis.EGF binding to its receptor rapidly induces receptor-mediated endocytosis through clathrin-coa...

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways

“…Moreover, NDRG1 depletion was found to result in abnormal endosomal sequestration of LDLR, suggesting that NDRG1 function is required for endosomal maturation (74). Similarly to LDLR, membrane EGFR is also degraded after internalization by receptor-mediated endocytosis (44). This observation suggests that the loss of NDRG1 in cancer cells might contribute to impaired EGFR degradation and a subsequent increase in EGFR activity.…”

Identification of differentially expressed proteins in human glioblastoma cell lines and tumors

“…Cystatin B was highly induced in U87MG⌬EGFR cells relative to U87MG cells, suggesting that aberrant signaling and/or processing of deleted EGFR may be involved in cystatin B regulation. EGFR processing involves a selective degradation of both the ligand and the receptor by cathepsin B (Authier et al, 1999). Cystatin B was highly and almost exclusively expressed in samples of glioblastomas.…”

Endosomal proteolysis of internalised [ArgA0]-human insulin at neutral pH generates the mature insulin peptide in rat liver in vivo

“…3a, lower panels, and c). Immunodepletion of ENs with antibodies to either cathepsin B [18], IDE [19,20], furin [21,22] or nardilysin failed to remove the arginyl-degrading activity (Fig. 3c).…”

“…Polyclonal antibody against growth factor receptor-bound protein 14 (GRB14) has been previously described [15]. Rabbit antimouse cathepsin D R291 [16,17], sheep anti-human cathepsin D M8147 [16,17] and rabbit anti-rat cathepsin B 7183 [18] were obtained from J. S. Mort (Shriners Hospital for Crippled Children, Montreal, Canada). Monoclonal antibody directed against insulin-degrading enzyme (IDE) [19,20] was obtained from R. A. Roth (Stanford University, Stanford, CA, USA).…”

“…Immunoblot analysis Electrophoresed samples were transferred as described previously [16][17][18][19][20][21][22] and the membranes were then incubated with primary antibody (antibodies against SHC diluted 1:5000, IR-β subunit diluted 1:1000, phospho-p44/42 MAP kinase diluted 1:350, p44/42 MAP kinase diluted 1:350, GRB14 diluted 1:500, IRS-1 diluted 1:1000, nardilysin diluted 1:1000, PY diluted 1:2500 or AP-B diluted 1:200). The bound immunoglobulin was detected using horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG.…”