Oligomers of ERBB3 Have Two Distinct Interfaces That Differ in Their Sensitivity to Disruption by Heregulin

Abstract

ErbB receptors associate in a ligand-dependent or -independent manner, and overexpression of epidermal growth factor receptor (ErbB1) or ErbB2 results in ligand-independent activation. Ligand-independent activation is poorly understood, and dimerization alone is not sufficient for activation. ErbB receptors also form higher order oligomers, but the mechanism of oligomer formation and their contribution to signaling are not known. The kinase-deficient ErbB3 as well as its extracellular domains are particularly prone to ligand-independent oligomerization, and oligomers are destabilized by binding of the ligand heregulin. In contrast, ligand binding facilitates heterodimerization with ErbB2 and is expected to stabilize an extended conformation of the ErbB3 extracellular domain (ECD) in which the dimerization interface is exposed. In the absence of ligand, ErbB3 can adopt a closed conformation that is held together by an intramolecular tether. We used a constitutively extended form of the ErbB3-ECD to analyze the conformation of the ECD in oligomers and the mechanism of oligomer disruption by heregulin. The extended conformation of the ECD forms oligomers more readily, suggesting the crystallographically defined dimer interface is one of the interfaces involved in oligomerization. Heregulin destabilizes oligomeric complexes but not dimers, which are neither stabilized nor disrupted by ligand binding, indicating a distinct second interface in oligomers of ErbB3. Cross-linking and activation studies on membrane-embedded ErbB3/ ErbB2 chimeras confirm this dual effect of heregulin. Most of the ErbB3-ECD on the cell surface is apparently kept in an open conformation through oligomerization, and the resulting oligomers adopt a conformation representing a state of reduced activity.The ErbB or EGFR 1 family of receptors in humans includes four members, EGFR (ErbB1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4), that are involved in a wide range of differentiation and growth control events. Overexpression, especially of EGFR or ErbB2, has been observed in a variety of tumors (1-5). Controlled activation of these receptors requires binding of a ligand of the EGF or heregulin family of growth and differentiation factors, resulting in cross-phosphorylation of the dimerized receptors at specific tyrosines in the cytoplasmic portion. However, under conditions of overexpression, tyrosine phosphorylation occurs constitutively, and cells expressing elevated levels of ErbB2 show a more aggressive growth behavior (5).Of the different pairs of receptor dimers that can form, the combination of ErbB2 and ErbB3 shows the strongest potency in terms of stimulating cell proliferation (6). Many tumors that show overexpression of ErbB2, especially those that are more prone to become resistant to conventional treatment, also show elevated levels of ErbB3 (7). The ErbB2/ErbB3 dimer is unique in that ErbB2 has a potent cytoplasmic kinase domain, but its extracellular domains (ECD) fail to bind any known ligand directly (8, 9). On the other ha...

Electrospray–differential mobility analysis of bionanoparticles

“…proteasome [16], ErbB3 [60], hemoglobin (Hb), Hemopure (a crosslinked Hb) [49], and for correlating heart disease risks with the size of lipoproteins [61]. A brief review of some of these results is available elsewhere [49].…”

The ERBB3 receptor in cancer and cancer gene therapy

“…48,50,51 (4) The latter interpretation is supported by the observation that constitutively locked ERBB3 bound ligand as well as did the extended conformation. 52 (5) ERBB3 does not form stable, ligand-bound homo-dimers, 53 in contrast to EGFR. Part of the reason for this may be amino acid changes in loops adjacent to domain II dimerization arms; disulfide-bonded module 6 is utilized in EGFR dimerization, 54 whereas for ERBB2/ERBB3 heterodimer formation module 7 plays the key role.…”

“…53 By use of a constitutively extended form of ERBB3 it was shown that intermolecular complexes included two different types of interfaces, one involved in oligomerization that is sensitive to NRG disruption, and another for dimer formation that is not affected by NRG. 52 It was proposed that self-associated ERBB3 constitutes the catalytically inactive oligomeric state. Binding of the ligand releases the ERBB3 and may stabilize the extended form of the receptor to expose the dimerization interface for interaction with ERBB2.…”

“…Binding of the ligand releases the ERBB3 and may stabilize the extended form of the receptor to expose the dimerization interface for interaction with ERBB2. 52 (7) The extracellular domain of ERBB3 retains NRG ligand binding even at acidic endosomal pH (in both the extended and locked conformations), and the genetically engineered constitutively locked conformation even showed a strong association at minimum pH 5.5. 50 This was in contrast to the binding of TGFα or EGF to the EGFR extracellular domain, that is much reduced at low pH.…”

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

“…This biphasic property has been observed for HRG associations at the cell surface, even when only ErbB3 was expressed (8, 16). In vitro measurements using purified monomeric extracellular receptor domains revealed only a low-affinity association constant (10,11,14). Therefore, the biphasic association could not be attributed to the presence of two types of HRG receptors (ErbB3 and B4), but rather to the functional divergence of each receptor species on the cell surface.…”