Requirements for the internalization of a murine monoclonal antibody directed against the HER-2/neu gene product c-erbB-2.
A murine monoclonal antibody, TA1, is directed against an epitope on the extracellular domain of the HER-2/neu (c-erbB-2) gene product. Requirements for TA1-induced internalization of c-erbB-2 have been studied using the SKBr3 human breast cancer cell line and several rat fibroblast cell lines that express either wild-type or mutant human c-erbB-2. Internalization of TA1 was monitored by assaying protease-resistant uptake of 125I-labeled TA1, by electron microscopy of gold-labeled TA1, and by inhibition of clonogenic growth of cells incubated with TA1 that had been conjugated with blocked ricin. Similar rates of internalization of TA1 were observed in SKBr3 and in rat fibroblasts that expressed human c-erbB-2. The route of endocytosis was the same as that observed with antibodies against other membrane receptors. Anti-c-erbB-2 and anti-transferrin receptor cointernalized through clathrin-coated pits, coated vesicles, endosomes, and multivesicular bodies. Products of mutant c-erbB-2 that lacked a portion of the tyrosine kinase domain or that lacked most of the cytoplasmic domain were endocytosed in the presence of TA1 as promptly as the wild-type c-erbB-2 product. Slightly more rapid internalization of TA1 was observed in rat cells that expressed c-erbB-2 with a single point mutation in the transmembrane domain. Taken together, our data suggest that neither the intracytoplasmic domain nor receptor phosphorylation is required for antibody-mediated endocytosis of c-erbB-2.
Department of Medicine, Duke Comprehensive Cancer Center, Duke University Medical Center, Durham, North Carolina 27710.
L A
LA
Maier
F J
FJ
Xu
S
S
Hester
C M
CM
Boyer
S
S
McKenzie
A M
AM
Bruskin
Y
Y
Argon
R C
RC
Bast
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000276:immunology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002874:Chromosome Mapping; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000276:immunology*; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D006046:Gold; D006801:Humans; D000922:Immunotoxins; D016253:Microscopy, Immunoelectron; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000276:immunology*; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases; D014162:Transfection
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D006046:Gold; D000922:Immunotoxins; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases