Adjacent Carboxyterminal Tyrosine Phosphorylation Events Identify Functionally Distinct ErbB2 Receptor Subsets: Implications for Molecular Diagnostics

Site-directed mutagenesis can define the effects of altering one or more amino acids within a protein, but this technique may lack sensitivity when used to characterize proteins which differ conformationally or posttranslationally at multiple sites. A novel alternative approach involves the direct characterization of wild-type protein isoforms identified by site-specific immunodetection. To this end we have developed antibodies which recognize ErbB2 subsets characterized by adjacent tyrosine phosphorylation events (Y¹²²²and Y¹²⁴⁸) in the C-terminal tail of the oncoprotein. Here we use these phosphoantibodies to demonstrate the existence of tyrosine-phosphorylated ErbB2 subsets which differ in their patterns of heterooligomer formation,in vitroautophosphorylation, and recruitment of SH2-containing substrates. Furthermore, Y¹²²²and/or Y¹²⁴⁸phosphoantibody immunoreactivity is readily detectable in ErbB2-overexpressing human breast tumors, in which context these phosphorylation events exhibit significant discordance. These data confirm the value of site-specific immunodetection as a strategy for characterizing phosphoprotein functionin vitroandin vivoand suggest that multisite phosphotyping of human tumors may contribute novel clinicopathologic insights into the significance of the ErbB2 overexpression phenotype.