Identification of pl85 neu Sequences Required for Monoclonal Antibody- or Ligand-Mediated Receptor Signal Attenuation

Anti-pl85^neu antibodies downmodulate constitutively active pl85^neu receptors from the cell surface, which is associated with a reduction in the transformed phenotype. We have analyzed a group of mutant pl85^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 antipl85^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 (TintΔ), or TintΔ 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-pl85^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, pl85^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 pl85^neu forms resulting from prolonged surface localization may result in enhanced cellular transformation and desensitization to MAbmediated downregulation and phenotypic reversion.