Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B

We have investigated the relevant protease activity in rat liver, which is responsible for most of the receptor-mediated epidermal growth factor (EGF) degradation in vivo. EGF was sequentially cleaved by endosomal proteases a t a limited number of sites, which were identified by high performance liqu id chromatography and mass spectrometry. EGF proteolysis is initiated by hy drolysis at the C-terminal Glu(51)-Leu(52) bond. Three additional minor cle avage sites were identified at positions Arg(48)-Trp(49), Trp(49)-Trp(50), and Trp(50)-Glu(51) after prolonged incubation. Using nondenaturating immun oprecipitation 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 (EG;FR) proteolysis was further evaluated by immunoblotting, Using endosomal fractions prepared fro m EGF-injected rats and incubated in vitro, the EGFR was lost with a time c ourse superimposable with the loss of phosphotyrosine content. The cathepsi n-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, theref ore, describe a unique pathway for the endosomal processing of internalized EGF receptor complexes, which involves the sequential function of cathepsi n-B through selective degradation of both the ligand and receptor.