Xl. Qian et al., IDENTIFICATION OF P185(NEU) SEQUENCES REQUIRED FOR MONOCLONAL ANTIBODY-MEDIATED OR LIGAND-MEDIATED RECEPTOR SIGNAL ATTENUATION, DNA and cell biology, 16(12), 1997, pp. 1395-1405
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 degradatio
n were examined by flow cytometric analysis and pulse-chase assays fol
lowing 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 surfa
ce downmodulation and degradation of surface receptor, However, kinase
-active deletion mutants with elimination of the putative internalizat
ion sequence (Tint Delta), or Tint Delta mutants also containing a lar
ge C-terminal truncation, displayed markedly impaired receptor endocyt
osis in response to MAb treatment, Cells expressing endocytosis-defect
ive 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 cel
ls expressing wild-type EGFR/neu receptors, Taken together, these data
suggest that, in addition to kinase activity, p185(neu) receptor endo
cytosis requires a functional modular structure, i.e., an internalizat
ion sequence, possibly to serve as target for endocytotic adapter prot
eins, Unattenuated signaling from oncogenic p185(neu) forms resulting
from prolonged surface localization may result in enhanced cellular tr
ansformation and desensitization to MAb-mediated downregulation and ph
enotypic reversion.