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

Abstract

Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces of MCF-7 human breast cancer cells. The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al. (2006) , suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow. The dissociation rate constant from the liganded monomer was intermediate. The dynamic changes in the association and dissociation kinetics in relation to the dimerization of ErbB displayed negative cooperativity, which resulted in apparent low-and high-affinity sites of HRG association on the cell surface.ligand-receptor interaction | cell signaling | epidermal growth factors T he ErbB family includes four member proteins, which localize to the plasma membrane of various types of cells and mediate signal transduction involved in cell differentiation and proliferation (1, 2). Among the ErbB family members, ErbB3 and ErbB4 are receptors for several extracellular protein ligands, including heregulin (HRG, also called neuregulin). ErbB3 is involved in the development of the heart and nervous system, and ErbB4 plays a role in lactation (3, 4). The dysregulation of ErbB3 and B4 (referred to hereafter as "HRG receptors") is often related to malignancy in human cancers (2). The long-term exposure of MCF-7 cells, a cultured cell line derived from a human breast carcinoma, to HRG induces the production of differentiation markers, such as milk proteins or lipid droplets, whereas similar exposure to epidermal growth factor induces proliferation (5).ErbB molecules must dimerize to transduce signals across the plasma membrane (3,4,6). Single ErbB molecules associate with single ligand molecules, such as HRG, and two liganded receptor molecules form a doubly liganded dimer on the plasma membrane (7). Dimerization is essential for the activation of ErbB molecules. This activation involves the phosphorylation of multiple sites along the cytoplasmic tail domain, which is catalyzed by the cytoplasmic kinase domain of the dimerization partner. In addition to homodimerization, some combinations of heterodimerization between members of the ErbB family have been described (1, 2). Several forms of unliganded and liganded homoand heterodi...

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“…The local apparent density was determined to be 300 × 10 −6 m , which may explain the lack of competition between HRG–nanorods and soluble HRG in these experiments. Considering the (hetero) dimerization of ErbB receptors and their relative HRG dissociation rates, it appears that the very high local concentration of HRG at nanorod tips results in the formation of strong bonds with ErbB2/3 dimers.…”

Transcriptionally inducible Pleckstrin homology-like domain, family A, member 1, attenuates ErbB receptor activity by inhibiting receptor oligomerization

“…To experimentally test the model-driven hypothesis that PHLDA1 inhibits oligomerization of ErbB receptors, we examined the association of fluorescent-labeled HRG (carboxytetramethylrhodamine (TMR)-HRG) with ErbB receptors on the apical surface of living MCF-7 cells using oblique illumination fluorescence microscopy (Fig. 6A) (35). A fluorescent spot emitted by a single TMR-HRG molecule detected in this experiment indicates the presence of either an HRG-bound ErbB3 monomer or a heterodimer between HRG-bound ErbB3 and an unliganded partner such as ErbB2 (Fig.…”

Total workflows of the single-molecule imaging analysis in living cells: a tutorial guidance to the measurement of the drug effects on a GPCR

“…Here we describe the detailed protocols to determine the molecular dynamics of GPCRs in living cells with the total internal reflection fluorescence microscopy (TIRFM). TIRFM is a common SMI method [7][8][9][10], in which the fluorophore-labeled molecules within a limited depth ~200 nm above the coverslip are illuminated by the evanescent field on the glass-water interface (Fig. 1).…”