Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion.
Exosomes are small membrane vesicles derived from intracellular multivescicular bodies (MVBs) that can undergo constitutive and regulated secretion from cells. Exosomes can also secrete soluble proteins through metalloprotease-dependent ectodomain shedding. In this study, we sought to determine whether ErbB1 receptors are present within exosomes isolated from the human keratinocyte cell line, HaCaT, and whether exosome-associated ErbB1 receptors can undergo further proteolytic processing. We show that full-length transmembrane ErbB1 is secreted in HaCaT exosomes. EGF treatment and calcium flux stimulated the release of phosphorylated ErbB1 in exosomes but only ligand-stimulated release was blocked by the ErbB1 kinase inhibitor, AG1478, indicating that ligand-dependent ErbB1 receptor activation can initiate ErbB1 secretion into exosomes. In addition, other immunoreactive but truncated ErbB1 isoforms were detected in exosomes suggestive of additional proteolytic processing. We demonstrate that cellular and exosomal ErbB1 receptors can undergo ectodomain shedding to generate soluble N-terminal ectodomains and membrane-associated C-terminal remnant fragments (CTFs). ErbB1 shedding was activated by calcium flux and the metalloprotease activator APMA (4-aminophenylmercuric acetate) and was blocked by a metalloprotease inhibitor (GM6001). Soluble ErbB1 ectodomains shed into conditioned medium retained the ability to bind exogenous ligand. Our results provide new insights into the proteolysis, trafficking and fate of ErbB1 receptors and suggest that the novel ErbB1 isoforms may have functions distinct from the plasma membrane receptor.
Sanderson
Michael P
MP
Tumor Immunology Program, German Cancer Research Center (DKFZ), D010/TP3, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.
Keller
Sascha
S
Alonso
Angel
A
Riedle
Svenja
S
Dempsey
Peter J
PJ
Altevogt
Peter
P
D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification; D005089:Exocytosis; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D020033:Protein Isoforms / Q000096:biosynthesis / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011799:Quinazolines; D015398:Signal Transduction; D020032:Tyrphostins / Q000494:pharmacology
D004151:Dipeptides; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D020033:Protein Isoforms; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; C010024:4-aminophenylmercuriacetate; C512478:EGFR protein, human; D066246:ErbB Receptors; D045726:Metalloproteases; D010662:Phenylmercuric Acetate