Heregulin reverses the oligomerization of HER3.

We analyzed the propensity of the HER3 receptor and its extracellular domain (ECD) to undergo ligand-independent self-association. The HER3-ECD, purified from Drosophila S2 cells, binds the EGF-like domain of heregulin (hrg) with a K(d) of 1.9 nM as measured by surface plasmon resonance (SPR) studies. In a gel shift assay, the HER3-ECD self-associates into a uniform, slowly migrating species in a concentration-dependent manner, starting at concentrations of <10 nM. In contrast to the HER3-ECD, the ECD from the related HER2 receptor does not oligomerize under the same conditions. The direct interaction of HER3-ECDs was also demonstrated by pull-down assays and SPR measurements under physiological salt conditions. This self-association of the HER3-ECD was reversed by the addition of hrg but not by EGF. The apparent equilibrium dissociation constant for the HER3-ECD self-association is 15 nM, based on SPR measurements. In this analysis, hrg blocks HER3-ECD self-association, and the addition of hrg during the dissociation phase resulted in an accelerated off rate. This finding suggests that hrg can bind to and disrupt preexisting HER3-ECD oligomers. Full-length HER3 likewise exhibited self-association. Under conditions where co-immunoprecipitation and cross-linking of HER2 and HER3 were stimulated by hrg, HER3 self-association and cross-linking were disrupted by hrg. The implication is that the self-association of HER3-ECD favors the formation of catalytically inactive complexes of the HER3 receptor. Binding of hrg releases HER3 which may then form signaling-competent HER3-HER2 heterodimers.