Homo- and Heteroassociations Drive Activation of ErbB3

Tímea Váradi,Magdalena Schneider,Eva Sevcsik,Dominik Kiesenhofer,Florian Baumgart,Gyula Batta,Tamás Kovács,René Platzer,Johannes B Huppa,János Szöllősi,Gerhard J Schütz,Mario Brameshuber,Peter Nagy

doi:10.1016/j.bpj.2019.10.001

Tímea Váradi, Magdalena Schneider + Show 11 more

Open Access

https://doi.org/10.1016/j.bpj.2019.10.001

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Abstract

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.

Highlights

The four ErbB receptors (ErbB1–4) constitute a family of transmembrane proteins standing in the focus of interest of basic researchers and clinicians
Chinese hamster ovary (CHO) cells were seeded onto fibronectin-coated glass coverslips and allowed to adhere before they were labeled with a mixture of Alexa Fluor 488-coupled (AF488) and Alexa Fluor 647-coupled (AF647) Fab fragments against ErbB3 (AF488-ErbB3-Fab and AF647-ErbB3-Fab)
The high ErbB3 density at the plasma membrane prevented a direct assessment of ErbB3 oligomerization, so we utilized TOCCSL to stoichiometrically reduce the surface density down to a level in which single molecules can be observed as well-separated spots [25,31,34]

Summary

Introduction

The four ErbB receptors (ErbB1–4) constitute a family of transmembrane proteins standing in the focus of interest of basic researchers and clinicians. Upon ligand-induced, overexpression- or mutation-driven activation of their intracellular kinase domain phosphorylated tyrosine residues are generated in their C-terminal part, leading to the activation of the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and signal transducer and activator of transcription (STAT) signaling pathways [1]. In the case of Biophysical Journal 117, 1935–1947, November 19, 2019 1935 Varadi et al. ErbB1, known as epidermal growth factor (EGF) receptor, monomeric inactive receptors undergo ligand-induced dimerization accompanied by conformational changes in the extracellular, transmembrane, and intracellular kinase domains, culminating in the activation of the receptor [2,3,4]. ErbB1 and ErbB4 can be considered to be fullfledged receptors for EGF-like and heregulin (HRG)-type ligands, respectively, because they contain fully functional ligand binding and tyrosine kinase domains [1]. The major role of ErbB2 is to enhance the potency and durability of transmembrane signaling by serving as the preferred heterodimerization partner for all other ErbB proteins [8]

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