Functional isolation of activated and unilaterally phosphorylated heterodimers of ERBB2 and ERBB3 as scaffolds in ligand-dependent signaling

Abstract

The EGFR (ERBB) family provides a model system for receptor signaling, oncogenesis, and the development of targeted therapeutics. Heterodimers of the ligand-binding-deficient ERBB2 (HER2) receptor and the kinase impaired ERBB3 (HER3) create a potent mitogenic signal, but the phosphorylation of ERBB2 in this context presents a challenge to established models of phosphorylation in trans. Higher order complexes of ERBB receptors have been observed biophysically and offer a theoretical route for ERBB2 phosphorylation, but it is not clear whether such complexes provide functionality beyond the constituent dimers. We now show that a previously selected inhibitory RNA aptamer that targets the extracellular domain (ECD) of ERBB3 acts by sterically disrupting these higher order interactions. Ligand binding, heterodimerization, phosphorylation of ERBB3, and AKT signaling are only minimally affected, whereas ERBB2 phosphorylation and MAPK signaling are selectively inhibited. The mapping of the binding site and creation of aptamer-resistant point mutants are consistent with a model of side-by-side oriented heterodimers to facilitate proxy phosphorylation, even at very low endogenous levels of receptors (below 10,000 receptors per cell). Additional modes of signaling with relevance to pathological ERBB expression states emerge at high receptor levels. Hence, higher order complexes of nonoverexpressed ERBB receptors are an integral and qualitatively distinct part of normal ERBB2/ERBB3 signaling. This mechanism of activation has implications for models of allosteric control, specificity of interactions, possible mechanisms of cross-talk, and approaches to therapeutic intervention that at present often generate experimental and clinical outcomes that do not reconcile with purely canonical, dimer-based models.oligomers | proxy activation B iochemical and structural analysis of the EGFR or ERBB (ErbB) family of receptor tyrosine kinases has provided a wealth of molecular details that have contributed significantly to our understanding of cell surface signaling and its deregulation in a broad range of diseases. The longstanding mechanistic model of receptor tyrosine phosphorylation in trans within ligand-activated dimers has undergone significant expansion in recent years. Beyond the regulation at the level of dimers, higher order clustering phenomena have been reported both for inactive and active receptor states (1-8). However, a critical and so far inaccessible question has been whether higher order complexes create qualitatively distinct signals that cannot emanate from dimers. The functional asymmetry of the closely related ERBB2/ERBB3 heterodimer presents an opportunity for experimental dissection but also a long-standing challenge to existing signaling models. ERBB2 is an orphan receptor that tyrosine phosphorylates its heterodimerization partners. ERBB3 is itself catalytically impaired but binds ligand, and its kinase domain allosterically activates its partners (9). This functional asymmetry is underscored by the fact tha...

Dynamic Analysis of the Epidermal Growth Factor (EGF) Receptor-ErbB2-ErbB3 Protein Network by Luciferase Fragment Complementation Imaging

“…However, it is difficult to explain how ErbB2 could be phosphorylated by the kinase-inactive ErbB3 in the context of an ErbB2/ErbB3 heterodimer. Indeed, recent work by Zhang et al (15) has suggested that the phosphorylation of ErbB2 occurs within ErbB2/ErbB3 tetramers in which the active ErbB2 from one dimer phosphorylates the ErbB2 in the other dimer. Thus, signaling by ErbB2/ ErbB3 heterodimers necessitates the formation of higher-order receptor oligomers not required by other ErbB pairings.…”

Pari passu dimers of dimers

“…In conclusion, the side-by-side model put forth by Zhang et al (9) provides insights on an enigma related to the mechanism of ErbB2-ErbB3 activation. Taken at face value, the report argues that higher-order complexes not only exist, but also are crucial for normal signaling.…”

“…Several limitations to the study of Zhang et al (9) should also be mentioned. First, no structural data are available to support the existence of the proposed tetrameric state.…”

“…In PNAS, Zhang et al (9) provide a possible answer to a longstanding, unanswered questioned that has annoyed the ErbB field: How does ErbB2 become activated in a simple heterodimeric model if ErbB3 is a dead or an impaired kinase? Several years ago, the same laboratory developed an RNA aptamer by using systematic evolution of ligands by exponential enrichment that selectively binds to ErbB3.…”

“…In the new study of Zhang et al (9), the authors use an insect cell-expressed version of ErbB3 ECD to map A30 binding to a segment near the junction of domains III and IV. This junction is accessible to A30 in the open and tethered conformations.…”