LIGAND AND P185(C-NEU) DENSITY GOVERN RECEPTOR INTERACTIONS AND TYROSINE KINASE ACTIVATION

The neu protooncogene (also known as c-erbB2, NGL, and HER2) encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine kinase act ivity that resembles the receptor for epidermal growth factor, The p18 5 gene and protein were originally identified in the brain and are tho ught to play a critical role in neurogenesis. Aberrant c-erbB2 protein overexpression also occurs in several human adenocarcinomas. A ligand for p185, neu-activating factor (NAF), specifically binds to neu rece ptor and increases the p185(c-neu) tyrosine phosphorylation in vitro a nd in vivo in a dose-dependent manner. We now show that NAF specifical ly binds to purified p185 expressed in baculovirus. Direct binding ana lysis showed that NAF binds with high affinity (K-d = 1.3 nM). We have investigated changes in the structure and association state of baculo virus-produced neu holoreceptor that are induced by ligand binding. In this study, we used sucrose gradients to show that purified p185(c-ne u) exists mainly in the monomeric form at low concentrations, whereas at higher concentrations p185(c-neu) exists as dimers or multimers. At low concentrations, but in the presence of ligand, p185(c-neu) sedime nts as a dimeric or multimeric form. Monomer-oligomer interconversion is absolutely ligand dependent at low receptor concentrations. The hig h molecular weight form of the receptor is enzymatically more active, as a consequence of ligand-driven activation of the receptor kinase. O ncogenic p185(neu) receptors sediment predominantly as high molecular weight forms and have constitutively active kinases.