ADJACENT CARBOXYTERMINAL TYROSINE PHOSPHORYLATION EVENTS IDENTIFY FUNCTIONALLY DISTINCT ERBB2 RECEPTOR SUBSETS - IMPLICATIONS FOR MOLECULARDIAGNOSTICS

Site-directed mutagenesis can define the effects of altering one or mo re amino acids within a protein, but this technique may lack sensitivi ty when used to characterize proteins which differ conformationally or posttranslationally at multiple sites. A novel alternative approach i nvolves the direct characterization of wild-type protein isoforms iden tified by site-specific immunodetection. To this end we have developed antibodies which recognize ErbB2 subsets characterized by adjacent ty rosine phosphorylation events (Y-1222 and Y-1248) in the C-terminal ta il of the oncoprotein. Here we use these phosphoantibodies to demonstr ate the existence of tyrosine-phosphorylated ErbB2 subsets which diffe r in their patterns of heterooligomer formation, in vitro autophosphor ylation, and recruitment of SH2-containing substrates. Furthermore, Y- 1222 and/or Y-1248 phosphoantibody immunoreactivity is readily detecta ble in ErbB2-overexpressing human breast tumors, in which context thes e phosphorylation events exhibit significant discordance. These data c onfirm the value of site-specific immunodetection as a strategy for ch aracterizing phosphoprotein function in vitro and in vivo and suggest that multisite phosphotyping of human tumors may contribute novel clin icopathologic insights into the significance of the ErbB2 overexpressi on phenotype. (C) 1998 Academic Press.