Identification of p185neu sequences required for monoclonal antibody- or ligand-mediated receptor signal attenuation

Anti-p185(neu) antibodies downmodulate constitutively active p185(neu) receptors from the cell surface, which is associated with a reduction in the transformed phenotype, We have analyzed a group of mutant p185(neu) forms with carboxyl (C)-terminal truncations and/or an internal deletion of amino acids 1008-1057. Receptor endocytosis and degradation were examined by flow cytometric analysis and pulse-chase assays following anti-p185(neu) monoclonal antibody (MAb) treatment. Deletion of a sequence within the distal carboxyl terminus, including three known autophosphorylation sites, did not affect MAb-mediated receptor surface downmodulation and degradation of surface receptor, However, kinase-active deletion mutants with elimination of the putative internalization sequence (Tint Delta), or Tint Delta mutants also containing a large C-terminal truncation, displayed markedly impaired receptor endocytosis in response to MAb treatment, Cells expressing endocytosis-defective mutant proteins became insensitive to anti-p185(neu) MAb-mediated inhibition of anchorage-independent growth and were more oncogenic in vivo, Cells expressing endocytosis-defective mutant EGFR/neu chimeric proteins were more transforming upon EGF addition when compared to cells expressing wild-type EGFR/neu receptors, Taken together, these data suggest that, in addition to kinase activity, p185(neu) receptor endocytosis requires a functional modular structure, i.e., an internalization sequence, possibly to serve as target for endocytotic adapter proteins, Unattenuated signaling from oncogenic p185(neu) forms resulting from prolonged surface localization may result in enhanced cellular transformation and desensitization to MAb-mediated downregulation and phenotypic reversion.