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.
Landgraf
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R
UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Molecular Biology Institute, Los Angeles, CA 90095-1570, USA.
Eisenberg
D
D
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004330:Drosophila; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D020890:Neuregulin-1 / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
D017931:DNA Primers; D020890:Neuregulin-1; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D011994:Recombinant Proteins