Membrane Interaction of Bound Ligands Contributes to the Negative Binding Cooperativity of the EGF Receptor

The epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, migration, and differentiation, and aberrant EGFR signaling is implicated in a variety of cancers. EGFR signaling is triggered by extracellular ligand binding, which promotes EGFR dimerization and activation. Ligand-binding measurements are consistent with a negatively cooperative model in which the ligand-binding affinity at either binding site in an EGFR dimer is weaker when the other site is occupied by a ligand. This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling. Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear. Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity. In particular, in our simulations the extracellular domains of an EGFR dimer spontaneously lay down on the membrane in an orientation in which favorable membrane contacts were made with one of the bound ligands, but could not be made with the other. Similar interactions were observed when EGFR was glycosylated, as it is in vivo

Mechanisms of Extracellular Oncogenic Dysregulation and Antibody Targeting of the Epidermal Growth Factor Receptor

Regulation of the Epidermal Growth Factor Receptor (EGFR) by its growth factor ligands is critical in many biological processes, including development and tissue maintenance and growth. Aberrant overexpression or activation by mutation of EGFR is associated with many human tumors. In these contexts, constitutive signaling can lead to cellular transformation and oncogenesis, thereby driving the cancer. The EGFR is the target of several existing or developing cancer therapies or immunotherapies, including monoclonal antibodies that prevent its activation. Activating mutations in cytoplasmic tyrosine kinase domain have been identified in many cancers, and have been the focus of mechanistic work. In this dissertation, I focus on the mode of oncogenic dysregulation by novel extracellular mechanisms. Extracellular oncogenic variants of EGFR include point mutations and alternative splice variants of EGFR. I find through biochemical analysis of the activating missense mutations in the extracellular region of EGFR that the soluble extracelluar region of EGFR (sEGFR) harboring these mutations bind ligands with elevated affinities. The dimerization energetics of these sEGFR mutants is not measurably altered, which suggests that additional interactions from the membrane and/or the intracellular region are important to this novel mode of extracellular oncogenic dysregulation of EGFR. I present preliminary progress towards the application of hydrogen/deuterium exchange coupled to mass spectrometry to analyze such allosteric (dys)regulation of the EGFR. In a second focus, I studied mechanisms of antibody targeting of EGFR. There are several monoclonal therapeutic antibodies that are in clinical development or use that target the EGFR/ErbB/HER family of receptor tyrosine kinases, including cetuximab/Erbitux™, panitumumab/Vectibix™, and necitumumab/Portrazza™, which all target EGFR, as well as trastuzumab/Herceptin™ and pertuzumab/Perjeta™, which target ErbB2/HER2. Unfortunately, as observed for most targeted therapies for cancer, resistance to these antibody therapies limits the duration of their effective treatment. Recent exome sequencing analyses of KRAS-WT colorectal cancer patients resistant to cetuximab treatment has identified epitope mutations as a mechanism of resistance. Whereas these mutated receptors bind cetuximab with dramatically decreased affinities, I report that they retain high affinity binding for necitumumab, a humanized IgG1 anti-EGFR antibody that shares the same epitope as cetuximab and panitumumab, and was recently FDA approved for squamous non-small cell lung carcinoma. I determined an X-ray crystal structure of the Fab fragment of necitumumab with the most commonly found resistance mutation—S492R (or S468R using the numbering scheme that starts at the beginning of the mature EGFR protein). This structure reveals a relatively hydrophobic cavity in the paratope of necitumumab that can accommodate the arginine at position S492/468 in the EGFR epitope. Further I find that other cetuximab and panitumumab resistance variants of EGFR are also permissive for necitumumab binding, suggesting significant plasticity in binding of necitumumab to EGFR. A survey of structures of therapeutic antibodies bound to their targets suggests that paratope shape may be an important property to consider in the selection of monoclonal antibodies in therapeutic strategies. Another mechanism of oncogenic dysregulation is the gene rearrangement of EGFR that results in EGFR variant III (EGFRvIII), an important target of many classes of immunotherapies for glioblastoma multiforme (GBM). I show in small angle X-ray scattering analyses of the ectodomain of EGFRvIII some evidence of structural flexibility in domain II that may be important for its documented transactivation of other receptor tyrosine kinases. I also report an X-ray crystal structure of the ectodomain of EGFRvIII in complex with the antigen binding or VHH domain of a camelid heavy-chain only antibody (HCAb), that has ~25-fold specificity for EGFRvIII compared to wild type EGFR. The structure reveals that the VHH gains specificity for EGFRvIII by targeting an epitope on domain IV that is sterically occluded in wild type EGFR by the intramolecular ‘tether’. This structure provides the direct evidence of dynamic uncoupling of the ‘tether’. My work corroborates the utility of the ‘tether’ as a source of antibody specificity for oncogenic EGFR, and is the first structural view of specific antibody targeting of an oncogenic EGFR variant

Novel Insights into the Allosteric Activation of the Epidermal Growth Factor Receptor

EGF receptor activation requires both ligand-binding and receptor-mediated dimerization through receptor domain II. The relationship between these processes, however, remains unclear. We have decoupled these processes to examine the ligand-binding affinity and the structure of constitutively-monomeric and -dimeric forms of the EGF receptor, as well as EGF receptor that dimerizes upon ligand-binding. Surprisingly, monomeric receptor binds to the ligands EGF and TGFα with an affinity equivalent to that of dimerizing receptor but with a unique binding enthalpy. This shows that monomeric, ligated EGF receptor adopts a state that is distinct from that of EGF receptor within a homodimer, and this state may be relevant to heterodimeric ErbB signaling complexes. Constitutively-dimerized receptor binds ligand with elevated affinity; however, it still requires ligand to form the receptor domain II dimeric interface. In the absence of ligand, no ordered, receptor domain II-mediated dimer interface is formed. Thus, the affinity effect does not arise from any pre-organization or stabilization of the ligand-binding sites on the receptor, but rather through an entropic effect of enforcing dimerization. Thus, pre-formed human receptor dimers require allosteric activation by ligand in order to signal, and this allosteric mechanism is distinct from that we recently observed for the D. melanogaster EGF receptor. Our observations on the allosteric mechanism of EGF receptor activation prompted us to ask whether other EGF receptor ligands may exert unique allosteric effects. To this end, we investigated the allosteric effects of the ligands Amphiregulin, Epiregulin, and Epigen on EGF receptor. We report that Epiregulin and Epigen, in particular, exert unique allosteric regulation on the receptor, as evidenced by divergent effects of EGFR variants on ligand-binding. Finally, we have studied ligand-binding and dimerization of receptors bearing activating extracellular mutations that cause glioblastoma. We report that these mutations elevate ligand-binding affinity, but they do not drive receptor dimerization. Our findings inform a revised model of ligand-induced receptor activation, in which the dimerization interface is highly sensitive to the presence and the identity of the bound ligand, and the domain I/domain II interface plays a crucial auto-inhibitory role

Tyrosine kinase signalling in breast cancer

Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors to signalling molecules on neighbouring cells. Receptors of the tyrosine kinase family play an important role in the integration and interpretation of these external stimuli, allowing a cell to respond appropriately to its environment. The activation of receptor tyrosine kinases (RTKs) is tightly controlled, allowing a normal cell to correctly integrate its external environment with internal signal transduction pathways. In contrast, due to numerous molecular alterations arising during the course of malignancy, a tumour is characterized by an abnormal response to its environment, which allows cancer cells to evade the normal mechanisms controlling cellular proliferation. Alterations in the expression of various RTKs, in their activation, and in the signalling molecules lying downstream of the receptors play important roles in the development of cancer. This topic is the major focus of the thematic review section of this issue of Breast Cancer Research

Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The “Rotation Model”

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility, and differentiation. The dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an extracellular ligand-binding domain and an intracellular kinase domain separated by a single transmembrane domain. The receptor is activated by a variety of polypeptide ligands such as epidermal growth factor and transforming growth factor α. It has long been thought that EGFR is activated by ligand-induced dimerization of the receptor monomer, which brings intracellular kinase domains into close proximity for trans-autophosphorylation. An increasing number of diverse studies, however, demonstrate that EGFR is present as a pre-formed, yet inactive, dimer prior to ligand binding. Furthermore, recent progress in structural studies has provided insight into conformational changes during the activation of a pre-formed EGFR dimer. Upon ligand binding to the extracellular domain of EGFR, its transmembrane domains rotate or twist parallel to the plane of the cell membrane, resulting in the reorientation of the intracellular kinase domain dimer from a symmetric inactive configuration to an asymmetric active form (the “rotation model”). This model is also able to explain how oncogenic mutations activate the receptor in the absence of the ligand, without assuming that the mutations induce receptor dimerization. In this review, we discuss the mechanisms underlying the ligand-induced activation of the preformed EGFR dimer, as well as how oncogenic mutations constitutively activate the receptor dimer, based on the rotation model

Antibodies Directed against the Extracellular Region of the Epidermal Growth Factor Receptor Adopt Distinct Modes of Binding and Inhibition

The work described in this dissertation comprises two distinct projects. In the first, we describe the structural and functional characterization of a family of Golgi associated cytosolic proteins, represented by Vps74 in fungi and GOLPH3 in animals, by X-ray crystallography, biophysical assays, and cellular techniques. We find that Vps74 is required for the proper steady state localization of a subset of Golgi enzymes in yeast, and that disruption of vps74 results in incomplete protein glycosylation. We further describe the crystal structures of Vps74 and GOLPH3, identifying structural motifs required both for oligomer formation and protein function. Finally, we find that both Vps74 and GOLPH3 specifically bind the Golgi enriched phospholipid, PtdIns4P. These results suggest a role for Vps74 and GOLPH3 in retrograde trafficking of components to the Golgi apparatus. In a separate and unrelated project, we characterize several inhibitory antibodies directed against the extracellular region of the epidermal growth factor receptor (EGFR). Aberrant activation of EGFR occurs in large proportion of epithelial cancers. Consequently, this receptor is a target for anti-cancer therapeutics that inhibit its activation, including antibodies and antibody-derived molecules. We have biochemically characterized a panel of conventional inhibitory antibodies with unique properties, and have identified approximate epitopes for these antibodies on domain 3 of EGFR. Additionally, we describe the crystal structures of three unconventional single chain antibody fragments in complex with the EGFR extracellular region. These single chain antibodies bind to novel epitopes on the receptor but share key characteristics with conventional inhibitory antibodies. Our findings highlight the diversity of binding modes among anti-EGFR antibodies, and suggest opportunities for novel therapeutics

Epidermal growth factor-like ligands regulate dimer selection.

There are thirteen known endogenous EGF-like ligands. We previously reported that Betacellulin (BTC) increases ligand-mediated corneal wound healing more than Epidermal Growth Factor (EGF) [Peterson et al. (2014) IOVS 55(5):2870-80], although the molecular reason for this is unknown. Despite being better at promoting wound healing via enhanced cell migration, BTC has reduced receptor affinity and weaker induction of EGFR phosphorylation. These data indicate that BTC’s response is not due to enhanced affinity or EGFR-kinase activity. Receptor phosphorylation and proximity ligation assays indicate that BTC treatment significantly increases ErbB3 phosphorylation and EGFR:ErbB3 heterodimers. BTC traffics EGFR at a faster rate than EGF, without noticeable differences in effector signaling. Thus, we demonstrate that despite both ligands binding to the EGFR, BTC biases the EGFR to dimerize with ErbB3 and regulates the biological response through trafficking and unknown effectors

Structure and Dynamics of the EGF Receptor as Revealed by Experiments and Simulations and Its Relevance to Non-Small Cell Lung Cancer

The epidermal growth factor receptor (EGFR) is historically the prototypical receptor tyrosine kinase, being the first cloned and the first where the importance of ligand-induced dimer activation was ascertained. However, many years of structure determination has shown that EGFR is not completely understood. One challenge is that the many structure fragments stored at the PDB only provide a partial view because full-length proteins are flexible entities and dynamics play a key role in their functionality. Another challenge is the shortage of high-resolution data on functionally important higher-order complexes. Still, the interest in the structure/function relationships of EGFR remains unabated because of the crucial role played by oncogenic EGFR mutants in driving non-small cell lung cancer (NSCLC). Despite targeted therapies against EGFR setting a milestone in the treatment of this disease, ubiquitous drug resistance inevitably emerges after one year or so of treatment. The magnitude of the challenge has inspired novel strategies. Among these, the combination of multi-disciplinary experiments and molecular dynamic (MD) simulations have been pivotal in revealing the basic nature of EGFR monomers, dimers and multimers, and the structure-function relationships that underpin the mechanisms by which EGFR dysregulation contributes to the onset of NSCLC and resistance to treatment

An operational view of intercellular signaling pathways

Animal cells use a conserved repertoire of intercellular signaling pathways to communicate with one another. These pathways are well-studied from a molecular point of view. However, we often lack an “operational” understanding that would allow us to use these pathways to rationally control cellular behaviors. This requires knowing what dynamic input features each pathway perceives and how it processes those inputs to control downstream processes. To address these questions, researchers have begun to reconstitute signaling pathways in living cells, analyzing their dynamic responses to stimuli, and developing new functional representations of their behavior. Here we review important insights obtained through these new approaches, and discuss challenges and opportunities in understanding signaling pathways from an operational point of view

STUDYING EGFR SIGNALING THROUGH SINGLE MOLECULE IMAGING AND COMPUTATIONAL MODELING

Signaling through the Epidermal Growth Factor Receptor (EGFR) plays an important role in both physiological and cancer-related processes. In this work, single-molecule microscopy measurements and computational modeling were closely integrated to better understand the mechanisms that regulate EGFR signaling. Technical improvements were made over the previously described Single-Molecule Pull-down (SiMPull) assay to facilitate direct detection of the phosphorylation state of thousands of individual receptors, and thereby estimate both the fraction of receptors phosphorylated at specific tyrosine residues and the frequency of multisite phosphorylation. These improvements enabled the first direct detection of multisite phosphorylation on full-length Epidermal Growth Factor Receptor (EGFR), and revealed that the extent of phosphorylation varied by tyrosine residue (biased phosphorylation). To help in understanding the underlying processes giving rise to these observations, a rule-based model for EGFR signaling was developed. The model suggested that biased phosphorylation could be explained by variations in adaptor protein abundances. This prediction arises from the structure of the model, in which a phospho-site that is bound by an adaptor protein is sterically protected from the action of phosphatases. Testing model predictions confirmed that overexpression of the adaptor protein Grb2 leads to phosphorylation levels enhanced specifically at a site where this protein binds. Finally, this model was extended to explore the possible mechanisms leading to differential signaling induced by EGFR ligands. Model results suggest that ligand-dependent differences in dimer lifetimes lead to differential multisite phosphorylation and ubiquitination, which in turn could influence signaling kinetics and cellular outcomes