DEMONSTRATION BY 2-COLOR FLOW-CYTOMETRY THAT TYROSINE KINASE-ACTIVITYIS REQUIRED FOR DOWN-MODULATION OF THE ONCOGENIC NEU RECEPTOR

Expression of rat oncogenic neu receptor, p185T-neu (a growth factor r eceptor with constitutive tyrosine kinase activity), causes cells to b ecome transformed. Treatment with anti-neu receptor monoclonal antibod ies reverts the transformed phenotype by down-modulation of p185T-neu. Monoclonal antibody treatment of cells expressing normal neu receptor , p185C-neu (which lacks constitutive tyrosine kinase activity), does not result in down-modulation of p185C-neu. To understand further the role the biochemical activity of p185T-neu plays in transformation and endocytosis, we created a series of mutations in p185T-neu. We found that fibroblasts expressing the tyrosine kinase-defective mutants cann ot form foci in culture. colonies in soft agar, or tumors in immunocom promised mice. To follow the antibody-induced endocytosis of neu recep tors expressed in these transfectants, we developed a novel two-color flow cytometric assay and confirmed receptor localization by electron microscopy. Cells were treated with mAb7.16.4 over time. After 4 hr of antibody treatment, less than 50% of full-length p185T-neu and of mut ant T691 remained on the cell surface, whereas internal expression of the neu receptors within these cells initially increased and then decr eased to the original internal receptor level. In contrast, the level of kinase-deficient mutated neu receptors remaining on the cell surfac e initially decreased by 35%, but, after 4 hr of antibody treatment, t he cell surface expression level returned to approximately the origina l level. Concurrently, fluctuations in expression levels were seen int ernally over time as well. These cell lines were also treated with gol d-conjugated mAb7.16.4. Using electron microscopy, we consistently fou nd the gold particles within multivesicular bodies of cell lines expre ssing full-length or mutated neu receptor. These data strongly suggest that the fate of the neu receptor, once internalized, is directed by its tyrosine kinase activity. When the kinase activity of the neu rece ptor is disrupted, the receptor is internalized but recycled to the ce ll surface, whereas neu receptors which have constitutive kinase activ ity are internalized and presumably degraded when engaged with anti-ne u receptor mAb. Understanding the regulation of receptor endocytosis, degradation, and recycling will contribute to the development of novel therapeutic protocols to combat human malignancies, particularly thos e associated with the overexpression of the human homologue of the neu receptor, c-erbB2.