The effect of cell-ECM adhesion on signalling via the ErbB family of growth factor receptors

Integrins and growth factor receptors of the ErbB family are involved in the regulation of cellular interactions with the extracellular microenvironment. Cross-talk between these two groups of transmembrane receptors is essential for cellular responses and can be regulated through the formation of multimolecular complexes. Tetraspanins as facilitators and building blocks of specialized microdomains may be involved in this process. In the present study, we demonstrated that, in contrast with previous reports, integrin-mediated adhesion did not stimulate ligand-independent activation of ErbB receptors in epithelial cells. However, integrin-dependent adhesion potentiated ligand-induced activation of EGFR (epidermal growth factor receptor) and ErbB2 and facilitated receptor homo- and hetero-dimerization. The actin cytoskeleton appeared to play a critical role in this phenomenon.     

Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements

The association of receptor tyrosine kinases is a key step in the initiation of growth factor-mediated signaling. Although the ligand-induced dimerization of inactive, monomeric receptors was the central dogma of receptor tyrosine kinase activation for decades, the existence of larger oligomers is now accepted. Both homoassociations and heteroassociations are of extreme importance in the epidermal growth factor (EGF) receptor family, leading to diverse and robust signaling. We present a statistically reliable, flow-cytometric homo-fluorescence resonance energy transfer method for the quantitative characterization of large-scale receptor clusters. We assumed that a fraction of a certain protein species is monomeric, whereas the rest are present in homoclusters of N-mers. We measured fluorescence anisotropy as a function of the saturation of fluorescent antibody binding, and fitted the model to the anisotropy data yielding the fraction of monomers and the cluster size. We found that ErbB2 formed larger homoclusters than ErbB1. Stimulation with EGF and heregulin led to a decrease in ErbB2 homocluster size, whereas ErbB1 homoclusters became larger after EGF stimulation. The activation level of ErbB2 was inversely proportional to its homocluster size. We conclude that homoclusters of ErbB1 and ErbB2 behave in a fundamentally different way. Whereas huge ErbB2 clusters serve as a reservoir of inactive coreceptors and dissociate upon stimulation, small ErbB1 homoclusters form higher-order oligomers after ligand binding.     

Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.     

Epidermal growth factor receptor (EGFR) signaling in cancer

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.     

ErbB and HB-EGF signaling in heart development and function

The epidermal growth factor (EGF)-ErbB signaling network is composed of multiple ligands of the EGF family and four tyrosine kinase receptors of the ErbB family. In higher vertebrates, these four receptors bind a multitude of ligands. Ligand binding induces the formation of various homo- and heterodimers of ErbB, potentially providing for a high degree of signal diversity. ErbB receptors and their ligands are expressed in a variety of tissues throughout development. Recent advances in gene targeting strategies in mice have revealed that the EGF-ErbB signaling network has fundamental roles in development, proliferation, differentiation, and homeostasis in mammals. The heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR/ErbB1) and ErbB4. Recent studies using several mutant mice lacking HB-EGF expression have revealed that HB-EGF has a critical role in normal heart function and in normal cardiac valve formation in conjunction with ErbB receptors. HB-EGF signaling through ErbB2 is essential for the maintenance of homeostasis in the adult heart, whereas HB-EGF signaling through EGFR is required during cardiac valve development. In this review, we introduce and discuss the role of ErbB receptors in heart function and development, focusing on the physiological function of HB-EGF in these processes.     

Quantitation of the Effect of ErbB2 on Epidermal Growth Factor Receptor Binding and Dimerization

The epidermal growth factor (EGF) receptor is a member of the ErbB family of receptors that also includes ErbB2, ErbB3, and ErbB4. These receptors form homo- and heterodimers in response to ligand with ErbB2 being the preferred dimerization partner. Here we use (125)I-EGF binding to quantitate the interaction of the EGF receptor with ErbB2. We show that the EGFR/ErbB2 heterodimer binds EGF with a 7-fold higher affinity than the EGFR homodimer. Because it cannot bind a second ligand, the EGFR/ErbB2 heterodimer is not subject to ligand-induced dissociation caused by the negatively cooperative binding of EGF to the second site on the EGFR homodimer. This increases the stability of the heterodimer relative to the homodimer and is associated with enhanced and prolonged EGF receptor autophosphorylation. These effects are independent of the kinase activity of ErbB2 but require back-to-back dimerization of the EGF receptor with ErbB2. Back-to-back dimerization is also required for phosphorylation of ErbB2. These findings provide a molecular explanation for the apparent preference of the EGF receptor for dimerizing with ErbB2 and suggest that the phosphorylation of ErbB2 occurs largely in the context of the EGFR/ErbB2 heterodimer, rather than through lateral phosphorylation of isolated ErbB2 subunits.     

Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family receptors

ErbB is a family of epidermal growth factor receptors representing an important class of receptor tyrosine kinases that play a leading role in cellular growth, development, and differentiation. Transmembrane domains of these receptors transduce biochemical signals across the plasma membrane via lateral homo- and heterodimerization. The relatively small size of ErbB transmembrane domain complexes with detergents or lipids makes it possible to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe an efficient expression system and a purification procedure for preparative-scale production of transmembrane peptides from all four ErbB proteins—ErbB1, ErbB2, ErbB3, and ErbB4—for the purpose of structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS cells as N-terminal extensions of thioredoxin A. The fusion proteins were cleaved with the light chain of human enterokinase. Several (10–30) milligrams of purified isotope-labeled transmembrane peptides were isolated using a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in a lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering and CD and NMR spectroscopy. The data obtained indicate that purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.     

Interaction of antibodies with ErbB receptor extracellular regions

Antibodies to the extracellular region of the ErbB receptors have played key roles in the development of a mechanistic understanding of this family of receptor tyrosine kinases. An extensively studied class of such antibodies inhibits activation of ErbB receptors, and these antibodies have been the focus of intense development as anti-cancer agents. In this review we consider the properties of ErbB receptors antibodies in light of the current structure-based model for ErbB receptor homo- and hetero-dimerization and activation. Crystal structures of the Fab fragments from five different inhibitory antibodies in complex with the extracellular regions of EGFR and ErbB2 have been determined. These structures highlight several different modes of binding and mechanisms of receptor inhibition. Information about antibody interactions with the structurally well-characterized soluble extracellular regions of ErbB receptors can be combined with the rich knowledge of the effects of these antibodies in cultured cells, and in vivo, to provide insights into the conformation and activation of ErbB receptors at the cell surface.     

The ErbB4 receptor in fetal rat lung fibroblasts and epithelial type II cells

ErbB receptors are important regulators of fetal organ development, including the fetal lung. They exhibit diversity in signaling potential, acting through homo- and heterodimers to cause different biological responses. We hypothesized that ErbB receptors show cell-specific and stimuli-specific activation, heterodimerization, and cellular localization patterns in fetal lung. We investigated this using immunoblotting, co-immunoprecipitation, and confocal microscopy in primary isolated E19 fetal rat lung fibroblasts and epithelial type II cells, stimulated with epidermal growth factor, transforming growth factor alpha, neuregulin 1beta, or treated with conditioned medium (CM) from the respective other cell type. Fetal type II cells expressed significantly more ErbB1, ErbB2, and ErbB3 protein than fibroblasts. ErbB4 was consistently identified by co-immunoprecipitation of all other ErbB receptors in both cell types independent of the treatments. Downregulation of ErbB4 in fibroblasts initiated cell-cell communication that stimulated surfactant phospholipid synthesis in type II cells. Confocal microscopy in type II cells revealed nuclear localization of all receptors, most prominently for ErbB4. Neuregulin treatment resulted in relocation to the extra-nuclear cytoplasmic region, which was distinct from fibroblast CM treatment which led to nuclear localization of ErbB4 and ErbB2, inducing co-localization of both receptors. We speculate that ErbB4 plays a prominent role in fetal lung mesenchyme-epithelial communication.     

Two EGF molecules contribute additively to stabilization of the EGFR dimer.

Receptor dimerization is generally considered to be the primary signaling event upon binding of a growth factor to its receptor at the cell surface. Little, however, is known about the precise molecular details of ligand-induced receptor dimerization, except for studies of the human growth hormone (hGH) receptor. We have analyzed the binding of epidermal growth factor (EGF) to the extracellular domain of its receptor (sEGFR) using titration calorimetry, and the resulting dimerization of sEGFR using small-angle X-ray scattering. EGF induces the quantitative formation of sEGFR dimers that contain two EGF molecules. The data obtained from the two approaches suggest a model in which one EGF monomer binds to one sEGFR monomer, and that receptor dimerization involves subsequent association of two monomeric (1:1) EGF-sEGFR complexes. Dimerization may result from bivalent binding of both EGF molecules in the dimer and/or receptor-receptor interactions. The requirement for two (possibly bivalent) EGF monomers distinguishes EGF-induced sEGFR dimerization from the hGH and interferon-gamma receptors, where multivalent binding of a single ligand species (either monomeric or dimeric) drives receptor oligomerization. The proposed model of EGF-induced sEGFR dimerization suggests possible mechanisms for both ligand-induced homo- and heterodimerization of the EGFR (or erbB) family of receptors.     

Differential localization of ErbB receptor ensembles influences their signaling in hippocampal neurons

Our studies indicate that ErbB complexes participate in both survival and synaptic plasticity signals of hippocampal neurons but in a manner that depends on the subcellular localization of the receptor ensembles. Using dissociated hippocampal cultures, we found that neurons, rather than glial cells, are the primary targets of ErbB receptor ligands such as epidermal growth factor and heregulin. Further investigation demonstrated that ErbB receptors distribute differentially in hippocampal neurons with the epidermal growth factor receptor confined to neural cell bodies and the p185(c-neu) and ErbB4 receptors distributed to both neural soma and neurites. Activation of ErbB receptor and downstream signaling molecules were observed in neurites only after heregulin stimulation. The receptor complex which mediated neurite located signals was the p185(c-neu)/ErbB4 heterodimer. Colocalization of p185(c-neu), but not epidermal growth factor receptor, with postsynaptic density protein 95 suggests that the heregulin signaling contributes to synapse specific activities. However, the epidermal growth factor receptor complex mediates physiological survival signals, as neuronal survival was enhanced by epidermal growth factor, rather than heregulin. Collectively, these studies indicate that different ErbB ensembles localize to different locations on the neuron to mediate distinct signals and functions.     

Human betacellulin structure modeled from other members of EGF family

We have modeled betacellulin (BTC) to gain insight into the structural elements that can explain its properties. The epidermal growth factor (EGF) signal transduction pathway, a significant mediator of several cell functions, is based on four closely related tyrosine kinase receptors. The ErbB receptors are transmembrane glycoproteins and signal transduction is initiated by ligand binding that induces receptor homo- or heterodimerization to form a complex containing two molecules of ligand and two molecules of receptor. The EGF family of ligands can be divided into three groups based on their ability to bind and activate distinct ErbB receptor homo- and heterodimers. Each member of the group formed by BTC, heparin binding EGF (HB-EGF) and epiregulin (EP) can interact with both the EGF receptor (EGFR) and heregulin receptors (ErbB-3 and ErbB-4), and are hence called bispecific ligands. BTC and EP also present the distinctive feature that they activate all possible heterodimeric ErbB receptors. BTC has been modeled with the program MODELLER, using human EGF, human transforming growth factor alpha (hTGF), human HB-EGF and human heregulin one alpha (hHRG-1) as templates. The structure of the model as well as that of the templates were optimized and a simulation of 100 ps was run for all. The main structural properties of the model and the templates were compared and in conclusion the hBTC conformation was closely similar to that of hTGF. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0072-2.Electronic Supplementary Material available.     

Size and conformational features of ErbB2 and ErbB3 receptors: a TEM and DLS comparative study

ErbB2 and ErbB3 receptors belong to the epidermal growth factor receptor family. The members of this family are able to form homo- and heterodimers that trigger diverse downstream signalling concerned to multiple cellular events. In the absence of a ligand, ErbB3 adopts a characteristic tethered conformation, which differs from ErbB2 extended conformation. In this work, transmission electron microscopy (TEM) and dynamic light scattering (DLS) have been used to characterize the conformational features and the size of ErBb2 and ErbB3 receptors. Two main objectives are presented. The first one is to evaluate the use of TEM as a tool for structural studies for this family of receptors. The low molecular weight of these proteins represents a challenging purpose for TEM studies. The other one is to search for a relationship between the results obtained by TEM and those obtained for the hydrodynamic size measured by DLS. This comparison has allowed us to identify the conformational differences of the receptors and to anticipate the use of these experimental techniques for the study of the ligand activated heterodimerization, a process related to a significant number of human malignancies.     

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.     

Bivalence of EGF-like ligands drives the ErbB signaling network.

Signaling by epidermal growth factor (EGF)-like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand-induced dimerization is unknown. We contrasted two existing models: a conformation-driven activation of a receptor-intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)-induced heterodimer between ErbB-3 and ErbB-2 favors a bivalence model; the ligand simultaneously binds both ErbB-3 and ErbB-2, but, due to low-affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C-terminal low-affinity site has broad specificity, but it prefers interaction with ErbB-2, an oncogenic protein acting as a promiscuous low-affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo- and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB-2/HER2.     

ErbBs in mammary development

Members of the ErbB (epidermal growth factor receptor) family of receptor tyrosine kinases are important in etiology of human mammary carcinoma, and are rational targets for cancer therapy. The frequent selection of ErbB2/HER2/Neu, and, less often, the epidermal growth factor receptor, and ErbB3 for overexpression in breast cancer implies that these receptors have important functions in normal mammary development. Better understanding of ErbBs in mammary development may yield important dividends for development and deployment of cancer therapies. The roles of these receptors and their ligands in mammary development are discussed, with an emphasis on new insights from genetic analysis of the receptors in mice.     

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.     

Controlled Dimerization of ErbB Receptors Provides Evidence for Differential Signaling by Homo- and Heterodimers

The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.     

ErbB receptors and their ligands in the breast

ErbB receptor tyrosine kinases are membrane-bound receptors that possess intrinsic, ligand-activated, tyrosine kinase activity. Binding of growth factors to these receptors induces the formation of ErbB homo- and heterodimers and initiates a signalling cascade that traverses the cytoplasm to communicate with the nucleus and the cytoskeleton. The effect of this cascade is the regulation of cellular proliferation, differentiation, apoptosis, migration and adhesion. Although ErbB signalling is important for normal growth and development in the breast, a dysregulation of ErbB activity can lead to tumourigenesis. This review will focus on the role of ErbB signalling in both normal mammary gland development and breast cancer, with an emphasis on the mechanisms behind receptor activation and the therapeutic agents designed to inhibit ErbB activity.     

Preparation of pro-oncogenic mutant forms V659E and V659Q of the transmembrane domain of receptor protein kinase ErbB2 for structural studies

Receptor tyrosine kinases (RTKs) play an important role in intercellular signal transduction through the plasma membrane. RTKs are integral membrane proteins activated upon lateral homo- or heterodimerization involving their transmembrane domain. The polymorphism and mutations in RTK transmembrane (TM) domains are directly associated with a number of human diseases. The family of epidermal growth factor receptors, ErbB, is an important class of RTKs participating in human cell growth, development, and differentiation. In order to investigate the influence of pathogenic mutations in ErbB TM domains on the structural and dynamic properties of these receptors and on specific interactions of their TM domains, we have developed highly effective systems of bacterial expression and purification of recombinant transmembrane fragments ErbB2_641–684 with pro-oncogenic substitution of Val⁶⁵⁹ by Glu or Gln. Transmembrane fragments were obtained in Escherichia coli BL21 (DE3) pLysS as a fusion protein with thioredoxin A. The purification protocol includes immobilized metal ion affinity chromatography (IMAC) and cation-exchange chromatography. The application of the protease Thrombin for hybrid protein hydrolysis considerably reduces financial expenditure as compared to the analogous protocols. The described techniques allow obtaining the milligram quantities of ErbB2 transmembrane fragments and its ¹⁵N-/[¹⁵N, ¹³C]-isotope-labeled derivatives for the analysis of their spatial structure using high-resolution heteronuclear NMR spectroscopy in a membrane-mimicking milieu.