A Spectroscopic Approach to Investigate the Molecular Interactions between the Newly Approved Irreversible ErbB blocker "Afatinib" and Bovine Serum Albumin.

The interaction of afatinib (AFB) with bovine serum albumin (BSA) was examined via fluorescence and UV-Vis spectroscopy. Spectrofluorimetric measurements revealed that AFB can strongly quench the BSA intrinsic fluorescence through producing a non-fluorescent complex. This quenching mechanism was thoroughly investigated with regard to the type of quenching, binding constant, number of binding locations and the fundamental thermodynamic parameters. Subsequently, the association constant of AFB with BSA was computed at three different temperatures and was found to range from 7.34 to 13.19 x10(5) L mol(-1). Thermodynamic parameters calculations demonstrated a positive ΔSƟ value with both negative ΔHÏ´ and ΔGÏ´ values for AFB-BSA complex, which in turn infers that a spontaneous binding is taking place with both electrostatic bonding and hydrophobic interactions participating in the binding of AFB and BSA. Similarly, the UV absorption spectra of AFB-BSA system were studied and confirmed the interaction. Conformational alteration of the protein upon binding to AFB was elaborated with the aid of three dimensional fluorescence measurements as well as synchronous fluorescence spectra.

Afatinib, an irreversible ErbB family blocker, with protracted temozolomide in recurrent glioblastoma: a case report.

There are few effective treatments for recurrent glioblastoma multiforme (GBM). We present a patient with recurrent GBM who achieved a prolonged response to treatment with afatinib, an irreversible ErbB family blocker, plus temozolomide. A 58-year-old female patient was diagnosed with multifocal primary GBM. After surgical resection, first-line therapy comprised radiotherapy and temozolomide. Following disease progression after 3 temozolomide cycles, the patient entered a phase I/II clinical trial of afatinib (20-40 mg daily for 28 days) plus temozolomide (50 mg/m2 every 21/28 days). Next-generation sequencing analysis of the brain tumor specimen was performed. At the last assessment, 63 treatment cycles had been completed and the patient had survived for ~5 years since recurrence. Significant disease regression was observed after 5 cycles and was maintained during long-term follow-up. Adverse events were consistent with the known tolerability profile of afatinib and were managed by treatment interruption/dose reduction. The patient had several epidermal growth factor receptor (EGFR) aberrations, including gene amplification and EGFRvIII positivity. Three somatic mutations were identified, including an unprecedented extracellular-domain substitution (D247Y). The patient has survived ~6-fold longer than normally expected in patients with recurrent GBM. The complex EGFR genotype may underlie sustained response to afatinib plus temozolomide.

Structure-function relationships of ErbB RTKs in the plasma membrane of living cells.

We review the states of the ErbB family of receptor tyrosine kinases (RTKs), primarily the EGF receptor (EGFR, ErbB1, HER1) and the orphan receptor ErbB2 as they exist in living mammalian cells, focusing on four main aspects: (1) aggregation state and distribution in the plasma membrane; (2) conformational features of the receptors situated in the plasma membrane, compared to the crystallographic structures of the isolated extracellular domains; (3) coupling of receptor disposition on filopodia with the transduction of signaling ligand gradients; and (4) ligand-independent receptor activation by application of a magnetic field.

Differential and synergistic effects of epidermal growth factor receptor antibodies on unliganded ErbB dimers and oligomers.

Antibodies directed against the epidermal growth factor receptor (EGFR) offer a potentially powerful therapeutic approach against cancers driven by the EGFR pathway. EGFR antibodies are believed to halt cell surface activation by blocking ligand-induced receptor tyrosine kinase activation, i.e., ligand binding, a change in conformation, or the monomer-dimer transition. In this work, we demonstrate that wild-type EGFR and the truncated de2-7-EGFR (tumor-associated mutant) formed unliganded homo-oligomers and examined the effects of two clinically relevant antibodies on the conformation and quaternary state of these ligand-free EGFR oligomers on the surface of cells. The EGFR antibodies were mAb528, a ligand-blocking antibody that binds domain III, and mAb806, a conformationally sensitive antibody that binds near the dimer interface in domain II. We used a model cellular system, BaF/3 cells, with GFP-tagged receptors in the absence of interference from secreted ligands or other erbB receptor members. Different antibody-mediated effects (conformational transition, receptor cross-linking, or receptor dissociation) were distinguished by combining two complementary biophysical techniques: image correlation spectroscopy (submicrometer scale clustering) and homo-Forster resonance energy transfer (association and/or conformation on a 1-10 nm scale). mAb528 cross-linked EGFR into an inactive EGFR dimer of dimers but had no effect when added to de2-7-EGFR oligomers. mAb806 had a minor effect on EGFR dimers as expected from its poor binding to a conformationally shielded epitope on wtEGFR but bound de2-7-EGFR oligomers, causing a conformational change in the intracellular C-terminal GFP-tagged tail. The combination of the two antibodies had synergistic effects, increasing the level of cross-linking of de2-7-EGFR, but did not lead to enhanced cross-linking of EGFR. The results reveal new modes of receptor-antibody interactions for EGFR and de2-7-EGFR.

Ligand-induced ErbB receptor dimerization.

Structural studies have provided important new insights into how ligand binding promotes homodimerization and activation of the EGF receptor and the other members of the ErbB family of receptor tyrosine kinases. These structures have also suggested possible explanations for the unique properties of ErbB2, which has no known ligand and can cause cell transformation (and tumorigenesis) by simple overexpression. In parallel with these advances, studies of the EGF receptor at the cell surface increasingly argue that the structural studies are missing key mechanistic components. This is particularly evident in the structural prediction that EGF binding linked to receptor dimerization should be positively cooperative, whereas cell-surface EGF-binding studies suggest negative cooperativity. In this review, I summarize studies of ErbB receptor extracellular regions in solution and of intact receptors at the cell surface, and attempt to reconcile the differences suggested by the two approaches. By combining results obtained with receptor 'parts', it is qualitatively possible to explain some models for the properties of the whole receptor. These considerations underline the need to consider the intact ErbB receptors as intact allosterically regulated enzymes, and to combine cellular and structural studies into a complete picture.

Molecular dynamics simulation approach for the prediction of transmembrane helix-helix heterodimers assembly.

Computational methods are useful to identify favorable structures of transmembrane (TM) helix oligomers when experimental data are not available or when they cannot help to interpret helix-helix association. We report here a global search method using molecular dynamics (MD) simulations to predict the structures of transmembrane homo and heterodimers. The present approach is based only on sequence information without any experimental data and is first applied to glycophorin A to validate the protocol and to the HER2-HER3 heterodimer receptor. The method successfully reproduces the experimental structures of the TM domain of glycophorin A (GpA(TM)) with a root mean square deviation of 1.5 A. The search protocol identifies three energetically stable models of the TM domain of HER2-HER3 receptor with favorable helix-helix arrangement, including right-handed and left-handed coiled-coils. The predicted TM structures exhibit the GxxxG-like motif at the dimer interface which is presumed to drive receptor oligomerization. We demonstrate that native structures of TM domain can be predicted without quantitative experimental data. This search protocol could help to predict structures of the TM domain of HER heterodimer family.

Depicting combinatorial complexity with the molecular interaction map notation.

To help us understand how bioregulatory networks operate, we need a standard notation for diagrams analogous to electronic circuit diagrams. Such diagrams must surmount the difficulties posed by complex patterns of protein modifications and multiprotein complexes. To meet that challenge, we have designed the molecular interaction map (MIM) notation (http://discover.nci.nih.gov/mim/). Here we show the advantages of the MIM notation for three important types of diagrams: (1) explicit diagrams that define specific pathway models for computer simulation; (2) heuristic maps that organize the available information about molecular interactions and encompass the possible processes or pathways; and (3) diagrams of combinatorially complex models. We focus on signaling from the epidermal growth factor receptor family (EGFR, ErbB), a network that reflects the major challenges of representing in a compact manner the combinatorial complexity of multimolecular complexes. By comparing MIMs with other diagrams of this network that have recently been published, we show the utility of the MIM notation. These comparisons may help cell and systems biologists adopt a graphical language that is unambiguous and generally understood.

Negative constraints underlie the ErbB specificity of epidermal growth factor-like ligands.

Epidermal growth factor (EGF)-like growth factors bind their 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 TGFalpha (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 TGFalpha-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.

An oncogenic epidermal growth factor receptor signals via a p21-activated kinase-caldesmon-myosin phosphotyrosine complex.

Many ligand-independent receptor tyrosine kinases are tumorigenic. The biochemical signals that mediate ligand-independent transformation of cells by these transmembrane receptors are poorly defined. In this report, we demonstrate that a constitutively activated mutant epidermal growth factor receptor (v-ErbB) induces the formation of a transformation-specific signaling module that complexes with myosin II. The components of this signaling complex include the signal adapter proteins Shc, Grb2, and Nck, and tyrosine-phosphorylated forms of p21-activated kinase (Pak), caldesmon, and myosin light chain kinase. Transformation-specific, tyrosine phosphorylation of Pak enhances the catalytic activity of this serine/threonine kinase. Furthermore, the tyrosine phosphorylation of Pak is Rho-, but not Ras-, Rac-, or Cdc42-dependent. These results demonstrate that a ligand-independent epidermal growth factor receptor mutant can transduce oncogenic signals that are distinct from ligand-dependent, mitogenic signals. In addition, these data provide evidence for the coupling of oncogenic receptor tyrosine kinases with the actomyosin molecular motor. This myosin-associated signaling module may mediate some of the biochemical changes of myosin found in v-ErbB- transformed fibroblasts, thereby contributing to the regulation of the mechanical forces governing cellular adhesion, cytoskeletal tension, and, hence, anchorage-independent cell growth.

Binding specificities and affinities of egf domains for ErbB receptors.

ErbB receptor activation is a complex process and is dependent upon the type and number of receptors expressed on a given cell. Previous studies with defined combinations of ErbB receptors expressed in mammalian cells have helped elucidate specific biological responses for many of the recognized gene products that serve as ligands for these receptors. However, no study has examined the binding of these ligands in a defined experimental system. To address this issue, the relative binding affinities of the egf domains of eleven ErbB ligands were measured on six ErbB receptor combinations using a soluble receptor-ligand binding format. The ErbB2/4 heterodimer was shown to bind all ligands tested with moderate to very high affinity. In contrast, ErbB3 showed much more restrictive ligand binding specificity and measurable binding was observed only with heregulin, neuregulin2beta, epiregulin and the synthetic heregulin/egf chimera, biregulin. These studies also revealed that ErbB2 preferentially enhances ligand binding to ErbB3 or ErbB4 and to a lesser degree to ErbB1.

A minor tyrosine phosphorylation site located within the CAIN domain plays a critical role in regulating tissue-specific transformation by erbB kinase.

Avian c-erbB encodes a protein that is homologous to the human epidermal growth factor receptor. Truncation of the amino-terminal, ligand-binding domain of this receptor results in an oncogene product which is a potent inducing agent for erythroleukemias but not fibrosarcomas in chickens. Here we show that mutation of a single tyrosine residue, p5, in the carboxyl terminus of the erbB oncogene product allows it to become sarcomagenic in vivo and to transform fibroblasts in vitro. Mutations of other autophosphorylation sites do not generate comparable effects. The increased transforming activity of the p5 mutant is accompanied by an elevated level of mitogen-activated protein kinase phosphorylation. By analogy to the human epidermal growth factor receptor, p5 is a minor autophosphorylation site and is located in a domain known to be involved in regulating calcium influx and receptor internalization (CAIN domain). This area of the erbB product has been found to be repeatedly deleted in various sarcomagenic avian erythroblastosis virus isolates. We precisely deleted the CAIN domain and also made point mutations of the acidic residues within the CAIN domain. In both cases, fibroblast-transforming potential is activated. We interpret these data to mean that p5 and its surrounding region negatively regulate fibroblast-transforming and sarcomagenic potential. To our knowledge, this represents the first point mutation of an autophosphorylation site that activates erbB oncogenicity.