Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency.
In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by the HER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amont of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185HER2 receptor.
Srinivas
U
U
Division of Experimental Oncology E, Istituto Nazionale Tumori, Milan, Italy.
Tagliabue
E
E
Campiglio
M
M
Ménard
S
S
Colnaghi
M I
MI
D000230:Adenocarcinoma / Q000378:metabolism*; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D001666:Binding Sites, Antibody; D002460:Cell Line; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011817:Rabbits; D018719:Erb-b2 Receptor Tyrosine Kinases; D014407:Tumor Cells, Cultured
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; C048844:anti-IgG