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 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.
Authier
F
F
INSERM U510, Faculté de Pharmacie Paris XI, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France. francois.authier@cep.u-psud.fr
Métioui
M
M
Bell
A W
AW
Mort
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002401:Cathepsin B / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D008099:Liver / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
D007457:Iodine Radioisotopes; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D002401:Cathepsin B