Tailoring Chimeric Ligands for Studying and Biasing ErbB Receptor Family Interactions (original) (raw)
Related papers
Engineered bivalent ligands to bias ErbB receptor-mediated signaling and phenotypes
The Journal of biological chemistry, 2011
The ErbB receptor family is dysregulated in many cancers, and its therapeutic manipulation by targeted antibodies and kinase inhibitors has resulted in effective chemotherapies. However, many malignancies remain refractory to current interventions. We describe a new approach that directs ErbB receptor interactions, resulting in biased signaling and phenotypes. Due to known receptor-ligand affinities and the necessity of ErbB receptors to dimerize to signal, bivalent ligands, formed by the synthetic linkage of two neuregulin-1β (NRG) moieties, two epidermal growth factor (EGF) moieties, or an EGF and a NRG moiety, can potentially drive homotypic receptor interactions and diminish formation of HER2-containing heterodimers, which are implicated in many malignancies and are a prevalent outcome of stimulation by native, monovalent EGF, or NRG. We demonstrate the therapeutic potential of this approach by showing that bivalent NRG (NN) can bias signaling in HER3-expressing cancer cells, re...
Journal of Biological Chemistry, 2003
EGF-like growth factors activate their ErbB receptors by promoting receptor-mediated homodimerization or, alternatively, by the formation of heterodimers with the orphan ErbB-2 through an as yet unknown mechanism. To investigate the selectivity in dimer formation by ligands, we have applied the phage display approach to obtain ligands with modified C-terminal residues that discriminate between ErbB-2 and ErbB-3 as dimerization partners. We used the epidermal growth factor/ transforming growth factor ␣ chimera T1E as the template molecule because it binds to ErbB-3 homodimers with low affinity and to ErbB-2/ErbB-3 heterodimers with high affinity. Many phage variants were selected with enhanced binding affinity for ErbB-3 homodimers, indicating that C-terminal residues contribute to the interaction with ErbB-3. These variants were also potent ligands for ErbB-2/ErbB-3 heterodimers despite negative selection for such heterodimers. In contrast, phage variants positively selected for binding to ErbB-2/ErbB-3 heterodimers but negatively selected for binding to ErbB-3 homodimers can be considered as "second best" ErbB-3 binders, which require ErbB-2 heterodimerization for stable complex formation. Our findings imply that epidermal growth factor-like ligands bind ErbB-3 through a multi-domain interaction involving at least both linear endings of the ligand. Apparently the ErbB-3 affinity of a ligand determines whether it can form only ErbB-2/ErbB-3 complexes or also ErbB-3 homodimers. Because no separate binding domain for ErbB-2 could be identified, our data support a model in which ErbB heterodimerization occurs through a receptor-mediated mechanism and not through bivalent ligands.
Negative Constraints Underlie the ErbB Specificity of Epidermal Growth Factor-like Ligands
Journal of Biological Chemistry, 2006
ErbB receptors in a highly selective manner, but the molecular basis for this specificity is poorly understood. We have previously shown that certain residues in human EGF (Ser 2 -Asp 3 ) and TGF␣ (Glu 26 ) are not essential for their binding to ErbB1 but prevent binding to ErbB3 and ErbB4. In the present study, we have used a phage display approach to affinity-optimize the C-terminal linear region of EGF-like growth factors for binding to each ErbB receptor and thereby shown that Arg 45 in EGF impairs binding to both ErbB3 and ErbB4. By omitting all these so-called negative constraints from EGF, we designed a ligand designated panerbin that binds ErbB1, ErbB3, and ErbB4 with similarly high affinity as their wild-type ligands. Homology models, based on the known crystal structure of TGF␣-bound ErbB1, showed that panerbin is able to bind ErbB1, ErbB3, and ErbB4 in a highly similar manner with respect to position and number of interaction sites. Upon in silico introduction of the experimentally known negative constraints into panerbin, we found that Arg 45 induced local charge repulsion and Glu 26 induced steric hindrance in a receptor-specific manner, whereas Ser 2 -Asp 3 impaired binding due to a disordered conformation. Furthermore, radiolabeled panerbin was used to quantify the level of all three receptors on human breast cancer cells in a single radioreceptor assay. It is concluded that the ErbB specificity of EGF-like growth factors primarily results from the presence of a limited number of residues that impair the unintended interaction with other ErbB receptors.
Bivalence of EGF-like ligands drives the ErbB signaling network
Embo Journal, 1997
Although ligand-induced dimerization was first reported 6 Corresponding author for the epidermal growth factor (EGF) receptor (Yarden and Schlessinger, 1987a,b), the exact mechanism by which E.Tzahar and R.Pinkas-Kramarski contributed equally to this work the monomeric EGF molecule promotes dimerization is still unknown. This is particularly important because the Signaling by epidermal growth factor (EGF)-like three-loop structure of the EGF domain is a common ligands is mediated by an interactive network of four protein motif found in a variety of extracellular proteins, ErbB receptor tyrosine kinases, whose mechanism of including not only growth factors but also adhesion ligand-induced dimerization is unknown. We conmolecules and coagulation factors (Groenen et al., 1994). trasted two existing models: a conformation-driven Moreover, the receptor for EGF (also called ErbB-1) activation of a receptor-intrinsic dimerization site and undergoes extensive heterodimerization with three related a ligand bivalence model. Analysis of a Neu differentimembrane proteins of its subfamily: the orphan receptor ation factor (NDF)-induced heterodimer between ErbB-2 (Goldman et al., 1990;
Inhibiting EGFR Dimerization Using Triazolyl-Bridged Dimerization Arm Mimics
PLOS ONE, 2015
The epidermal growth factor receptor (EGFR) is overexpressed in multiple carcinomas and is the focus of a variety of targeted therapies. Here we report the design of peptide-based compounds that mimic the EGFR dimerization arm and inhibit allosteric activation of EGFR. These peptides are modified to contain a triazolyl bridge between the peptide strands to constrain the EGFR dimerization arm β-loop. In this study, we demonstrate that these peptides have significantly improved proteolytic stability over the non-modified peptide sequence, and their inhibitory effects are dependent on the number of the methylene units and orientation of the introduced triazolyl bridge. We identified a peptide, EDA2, which downregulates receptor phosphorylation and dimerization and reduces cell viability. This is the first example of a biologically active triazolyl-bridged peptide targeting the EGFR dimerization interface that effectively downregulates EGFR activation.
Structure−Function Studies of Ligand-Induced Epidermal Growth Factor Receptor Dimerization †
Biochemistry, 1998
We present a novel 96-well assay which we have applied to a structure-function study of epidermal growth factor receptor dimerization. The basis of the assay lies in the increased probability of EGFRs being captured as dimers by a bivalent antibody when they are immobilized in the presence of a cognate ligand. Once immobilized, the antibody acts as a tether, retaining the receptor in its dimeric state with a resultant 5-7-fold increase in binding of a radiolabeled ligand probe. When the assay was applied to members of the EGF ligand family, murine EGF, transforming growth factor alpha, and heparinbinding EGF-like growth factor were comparable with human EGF (EC 50) 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In contrast, amphiregulin (AR 1-84), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 µM. We further probed the involvement of the C-tail and the conserved leucine residue in receptor dimerization by comparing the activities of two genetically modified EGFs (the chimera mEGF/TGFR 44-50 and the EGF point mutant L47A) and a C-terminally extended form of AR (AR 1-90) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGFR 44-50 > L47A) L15A. AR 1-90 > AR 1-84. Although AR was much worse than predicted from its affinity, this defect could be partially rectified by co-localization of the immobilizing antibody with heparin. Thus, it seems likely that AR cannot dimerize the EGFR unless other accessory molecules are present to stabilize its functional association with the EGFR.
Seminars in cell & developmental biology, 2010
Our work is concerned with the origins and therapy of human cancers. Members of the epidermal growth factor receptor (EGFR) family of tyrosine kinases, also known as erbB or HER receptors, are over expressed and/or activated in many types of human tumors and represent important therapeutic targets in cancer therapy. Studies from our laboratory identified targeted therapy as a way to treat cancer. Rational therapeutics targeting and disabling erbB receptors have been developed to reverse the malignant properties of tumors. Reversal of the malignant phenotype, best seen with disabling the HER2 receptors using monoclonal antibodies is a distinct process from that seen with blocking of ligand binding to cognate receptors as has been done for EGFr receptors. Here we review the mechanisms of action deduced from a number of approaches developed in our laboratory and elsewhere, including monoclonal antibodies, peptide mimetics, recombinant proteins and small molecules. The biochemical and b...
Ligand-Induced Structural Transitions in ErbB Receptor Extracellular Domains
Structure, 2007
Crystallographic studies showed that epidermal growth factor (EGF) receptor activation involves major domain rearrangements. Without bound ligand, the extracellular region of the receptor (sEGFR) adopts a ''tethered'' configuration with its dimerization site occluded by apparently autoinhibitory intramolecular interactions. Ligand binding causes the receptor to become ''extended,'' breaking the tether and exposing the dimerization site. Using smallangle X-ray scattering (SAXS), we confirm that the tethered and extended conformations are also adopted in solution, and we describe low-resolution molecular envelopes for an intact sEGFR dimer. We also use SAXS to monitor directly the transition from a tethered to extended configuration in the monomeric extracellular regions of ErbB3 and a dimerizationdefective EGFR mutant. Finally, we show that mutating every intramolecular tether interaction in sEGFR does not greatly alter its conformation. These findings explain why tether mutants fail to activate EGF receptor and provide new insight into regulation of ErbB receptor conformation.