Promote Receptor Dimerization Research Articles

PDGFRA K385 mutants in myxoid glioneuronal tumors promote receptor dimerization and oncogenic signaling

Myxoid glioneuronal tumors (MGNT) are low-grade glioneuronal neoplasms composed of oligodendrocyte-like cells in a mucin-rich stroma. These tumors feature a unique dinucleotide change at codon 385 in the platelet-derived growth factor receptor α (encoded by the PDGFRA gene), resulting in the substitution of lysine 385 into leucine or isoleucine. The functional consequences of these mutations remain largely unexplored. Here, we demonstrated their oncogenic potential in fibroblast and Ba/F3 transformation assays. We showed that the K385I and K385L mutants activate STAT and AKT signaling in the absence of ligand. Co-immunoprecipitations and BRET experiments suggested that the mutations stabilized the active dimeric conformation of the receptor, pointing to a new mechanism of oncogenic PDGF receptor activation. Furthermore, we evaluated the sensitivity of these mutants to three FDA-approved tyrosine kinase inhibitors: imatinib, dasatinib, and avapritinib, which effectively suppressed the constitutive activity of the mutant receptors. Finally, K385 substitution into another hydrophobic amino acid also activated the receptor. Interestingly, K385M was reported in a few cases of brain tumors but not in MGNT. Our results provide valuable insights into the molecular mechanism underlying the activation of PDGFRα by the K385I/L mutations, highlighting their potential as actionable targets in the treatment of myxoid glioneuronal tumors.

A synthetic bivalent peptide ligand of EphB4 with potent agonistic activity

Interaction between ephrin receptor EphB4 and its ligand EFNB2 mediates bidirectional signaling important for cancer: forward EFNB2-to-EphB4 signaling that is tumor suppressive, and reverse EphB4-to-EFNB2 signaling that promotes angiogenesis important for tumor growth and metastasis. Molecular agents targeting these forward and reverse signals of EphB4-EFNB2 interaction can be used to probe the molecular mechanisms of these complex signaling pathways and develop new anticancer therapeutics. In this study, we applied a bivalent ligand design strategy to synthesize a novel dimeric peptide based on an antagonist TNYL-RAW. The dimeric peptide possessed higher EphB4 receptor binding affinity than the monomeric TNYL-RAW peptide. Interestingly, the dimerization of TNYL-RAW peptide converted a monomeric antagonist of EphB4 to a dimeric agonist. This dimeric agonist promoted EphB4 phosphorylation, internalization and degradation, reduced cancer cell motility, and inhibited tube formation of HUVEC. To investigate the mechanism of action of this bivalent dimeric peptide, FRET experiments and molecular dynamic simulation were conducted and suggested that this bivalent ligand recognizes two EphB4 simultaneously which may promote receptor dimerization and oligomerization. This was further supported by the study of this bivalent ligand containing deletion of critical residues on one of its monomers which impaired its simultaneous binding to two EphB4 and ability to cause EphB4 dimerization and phosphorylation. These results demonstrate the value of this novel bivalent agonist ligand of EphB4 as a probe of the bidirectional signaling of EphB4-EFNB2 and lead for cancer drug development.

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

CXCR4, a member of the family of chemokine-activated G protein–coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested that the receptor may exist as a dimer or an oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means, we confirm that CXCR4 can exist as a mixture of monomers, dimers, and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t—a small drug-like isothiourea derivative—rapidly destabilizes the oligomeric structure, whereas AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

Abstract 3758: Impact of ANXA7 I1 expression on PDGFRA and MET endosomal trafficking in glioblastoma

Abstract Purpose/Objective(s): Glioblastomas (GBM) are highly malignant tumors that arise from astrocytes. GBMs rely on signaling through receptor tyrosine kinases (RTK) to ensure tumorigenicity. EGFR, PDGFRA and MET are distinct RTKs; each is capable of binding a ligand, which promotes receptor dimerization and kinase activation. Annexin A7 (ANXA7), a protein involved in membrane binding and vesicle trafficking, is spliced into 2 isoforms - isoforms 1 (I1) and isoform 2 (I2). A normal adult brain will express both isoforms, however, in GBM, I1 expression is prevented. We determined that I1, but not I2, is tumor suppressive, in part, because it promotes the endocytic degradation of EGFR. Because I1 is typically not expressed in GBM, EGFR signaling persists unabated and tumors thrive. Recently, we extended these observations and determined that I1 also impacts additional RTKs, including PDGFRA and MET. By understanding how ANXA7 I1 impacts PDGFRA and MET signaling, we hope to target multiple tumorigenic RTKs and improve therapy for patients with GBM. Materials/Methods: To understand how and when I1 downregulates PDGFRα and MET, U251-malignant glioma (MG) cells, which only express I2 (P), were modified to express I1. Cells were serum starved overnight, and then stimulated with PDGFAA and HGF(20 ng/ml), which activate the aforementioned receptors respectively. As a positive control, cells were stimulated with EGF to activate EGFR. Total protein was then collected and analyzed by western blot analyses. Next, we sought to determine whether I1 was competent to down-regulate several RTKs coincidentally. To do this, P and I1 cells were serum starved, stimulated with EGF, PDGFAA or both, and total protein levels were evaluated. Results: The levels of activated EGFR, PDGFRα and MET are dramatically reduced in those cells that express I1 as compared to cells expressing I2 (P). Upon activation with both PDGFAA and EGF, in cells expressing I1 (I1), levels of PDGFRα and EGFR were markedly reduced 30 minutes post-stimulation as compared to cells expressing I2. Conclusion: Our results suggest I1 may be a master regulator of RTK endocytosis and degradation. Further investigations of the impact of I1 on endocytic trafficking patterns using immunofluorescence and confocal microscopy are underway. Collectively, understanding how I1 functions and targets multiple tumorigenic RTKs will provide the foundation for future selective therapies that restore I1 expression in GBM and reduce tumorigenicity. Citation Format: Zachary B. White, Centdrika Dates Hurt, Rajani Rajbhandari, Sindhu Nair, Susan Nozell, Markus Bredel. Impact of ANXA7 I1 expression on PDGFRA and MET endosomal trafficking in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3758.

The Contorsbody, an antibody format for agonism: Design, structure, and function

The careful design of the antibody architecture is becoming more and more important, especially when the purpose is agonism. We present the design of a novel antibody format that is able to promote receptor dimerization and induce signal transduction resulting in cell proliferation. Mono-specific bivalent Y-shape IgGs made of two light chains and two heavy chains are engineered into single chain dimers of two modified heavy chains, resulting in the fixation of the two Fab fragments along the Fc dimerizing moiety. By this, an antagonist of the Her-receptor family, Trastuzumab, is re-formatted into an agonist by simply incorporating the original binding motif into a different geometrically and sterically constrained conformation. This novel format, named Contorsbody, retains antigen binding properties of the parental IgGs and can be produced by standard technologies established for recombinant IgGs. Structural analyses using molecular dynamics and electron microscopy are described to guide the initial design and to confirm the Contorsbody as a very compact molecule, respectively. Contorsbodies show increased rigidity compared to IgGs and their Fab moieties are positioned parallel and adjacent to each other. This geometry has an increased potential to trigger cell surface antigen or receptor ‘cis’-dimerization without ‘trans’-bridging of cells or mere receptor blockade.

Impact of ANXA7 I1 Expression on PDGFRA and MET Endosomal Trafficking in Glioblastoma

Glioblastomas (GBM) are highly malignant tumors that arise from astrocytes. GBMs rely on signaling through receptor tyrosine kinases (RTK) to ensure tumorigenicity. EGFR, PDGFRA and MET are distinct RTKs; each is capable of binding a ligand, which promotes receptor dimerization and kinase activation. Annexin A7 (ANXA7), a protein involved in membrane binding and vesicle trafficking, is spliced into 2 isoforms - isoforms 1 (I1) and isoform 2 (I2). A normal adult brain will express both isoforms, however, in GBM, I1 expression is prevented. We determined that I1, but not I2, is tumor suppressive, in part, because it promotes the endocytic degradation of EGFR. Because I1 is typically not expressed in GBM, EGFR signaling persists unabated and tumors thrive. Recently, we extended these observations and determined that I1 also impacts additional RTKs, including PDGFRA and MET. By understanding how ANXA7 I1 impacts PDGFRA and MET signaling, we hope to target multiple tumorigenic RTKs and improve therapy for patients with GBM. To understand how and when I1 downregulates PDGFRα and MET, U251-malignant glioma (MG) cells, which only express I2 (P), were modified to express I1. Cells were serum starved overnight, and then stimulated with PDGFAA and HGF(20 ng/ml), which activate the aforementioned receptors respectively. As a positive control, cells were stimulated with EGF to activate EGFR. Total protein was then collected and analyzed by western blot analyses. Next, we sought to determine whether I1 was competent to down-regulate several RTKs coincidentally. To do this, P and I1 cells were serum starved, stimulated with EGF, PDGFAA or both, and total protein levels were evaluated. The levels of activated EGFR, PDGFRα and MET are dramatically reduced in those cells that express I1 as compared to cells expressing I2 (P). Upon activation with both PDGFAA and EGF, in cells expressing I1 (I1), levels of PDGFRα and EGFR were markedly reduced 30 minutes post-stimulation as compared to cells expressing I2. Our results suggest I1 may be a master regulator of RTK endocytosis and degradation. Further investigations of the impact of I1 on endocytic trafficking patterns using immunofluorescence and confocal microscopy are underway. Collectively, understanding how I1 functions and targets multiple tumorigenic RTKs will provide the foundation for future selective therapies that restore I1 expression in GBM and reduce tumorigenicity.

Toll-like Receptor 9 Contains Two DNA Binding Sites that Function Cooperatively to Promote Receptor Dimerization and Activation

Toll-like receptor 9 (TLR9) recognizes DNA containing CpG motifs derived from bacteria and viruses and activates the innate immune response to eliminate them. TLR9 is known to bind to CpG DNA, and here, we identified another DNA binding site in TLR9 that binds DNA containing cytosine at the second position from the 5' end (5'-xCx DNA). 5'-xCx DNAs bound to TLR9 in the presence of CpG DNA and cooperatively promoted dimerization and activation of TLR9. Binding at both sites was important for efficient activation of TLR9. The 5'-xCx DNA bound the site corresponding to the nucleoside binding site in TLR7 and TLR8 as revealed by the structural analysis. This study revealed that TLR9 recognizes two types of DNA through its two binding sites for efficient activation. This information may contribute to the development of drugs that control the activity of TLR9.

Mechanisms of Autoinhibition and Dimerization of the EGF Receptor Family

The epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases initiates cell signaling in response to growth factors, thereby regulating cell growth, migration and division. Four members comprise this family (EGFR/HER1/ErbB1, ErbB2, ErbB3 and ErbB4), and activating mutations in each protein are associated with severe cancers of the lung, colon, head and neck.ErbB proteins consist of an extracellular ligand binding domain, a single transmembrane alpha-helix, and an intracellular kinase domain. Structural and functional studies of isolated domains of the EGFR family have revealed details of the complex mechanism of receptor regulation. Somewhat lacking, however, is our knowledge of the full-length receptor in its native membrane environment. Ligand binding is known to promote receptor dimerization and activation, but it is unclear how receptor behavior is altered by the two-dimensional environment of the plasma membrane. In particular, the mechanism by which receptor domains cooperate to transmit signals across the membrane is not well understood.Toward this end, we employed quantitative FRET microscopy to study dimerization of EGFR family proteins in plasma membrane-derived vesicles. This method measures FRET efficiency as a function of two-dimensional receptor concentration in the membrane. We investigated four receptor pairs: homodimers of ErbB1, ErbB2, and ErbB3, as well as heterodimers containing ErbB2 and ErbB3. We first observe that the isolated transmembrane (TM) domains have an intrinsic propensity to associate. Next, we demonstrate that the extracellular domains function to prevent TM domain interactions. Finally, we show that the intracellular domains alone are sufficient to drive receptor dimerization in the absence of ligand. Dimerization occurs in a concentration-dependent manner, and ligand shifts this threshold to low receptor concentration. Importantly, both of these dimerization events occur over a physiologically relevant concentration range.

Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β1-adrenergic (β1AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β1AR. Immunization with the β1AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β1AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β1AR to which the mAbs bind. The anti-β1AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β1AR agonism.

Oncogenic Mutations Counteract Intrinsic Disorder in the EGFR Kinase and Promote Receptor Dimerization

The mutation and overexpression of the epidermal growth factor receptor (EGFR) are associated with the development of a variety of cancers, making this prototypical dimerization-activated receptor tyrosine kinase a prominent target of cancer drugs. Using long-timescale molecular dynamics simulations, we find that the N lobe dimerization interface of the wild-type EGFR kinase domain is intrinsically disordered and that it becomes ordered only upon dimerization. Our simulations suggest, moreover, that some cancer-linked mutations distal to the dimerization interface, particularly the widespread L834R mutation (also referred to as L858R), facilitate EGFR dimerization by suppressing this local disorder. Corroborating these findings, our biophysical experiments and kinase enzymatic assays indicate that the L834R mutation causes abnormally high activity primarily by promoting EGFR dimerization rather than by allowing activation without dimerization. We also find that phosphorylation of EGFR kinase domain at Tyr845 may suppress the intrinsic disorder, suggesting a molecular mechanism for autonomous EGFR signaling.

Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization

AbstractVEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 Ig-homology domains; domains 2 and 3 (D23) represent the ligand-binding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation. In the present study, isothermal titration calorimetry showed that the Gibbs free energy of VEGF-A, VEGF-C, or VEGF-E binding to D23 or the full-length ECD of VEGFR-2 is dominated by favorable entropic contribution with enthalpic penalty. The free energy of VEGF binding to the ECD is 1.0-1.7 kcal/mol less favorable than for binding to D23. A model of the VEGF-E/VEGFR-2 ECD complex derived from small-angle scattering data provided evidence for homotypic interactions in D4-7. We also solved the crystal structures of complexes between VEGF-A or VEGF-E with D23, which revealed comparable binding surfaces and similar interactions between the ligands and the receptor, but showed variation in D23 twist angles. The energetically unfavorable homotypic interactions in D4-7 may be required for re-orientation of receptor monomers, and this mechanism might prevent ligand-independent activation of VEGFR-2 to evade the deleterious consequences for blood and lymph vessel homeostasis arising from inappropriate receptor activation.

Galectin-3 Protein Modulates Cell Surface Expression and Activation of Vascular Endothelial Growth Factor Receptor 2 in Human Endothelial Cells

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3(-/-) and Mgat5(-/-) mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2.

Structural determinants of growth factor binding and specificity by VEGF receptor 2

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance

The HER2-targeted therapy trastuzumab is widely used for the treatment of patients with metastatic breast tumors overexpressing HER2. However, an objective response is observed in only 12% to 24% of patients treated with trastuzumab as a single agent and initial responders regress in <6 months (1-3). The reason for the clinical failure of trastuzumab in this setting remains unclear. Here we show that local lymph node-positive disease progression in 89% of breast cancer patients with HER2-positive tumors involves the HER2 oncogenic variant HER2Delta16. We further show that ectopic expression of HER2Delta16, but not wild-type HER2, promotes receptor dimerization, cell invasion, and trastuzumab resistance of NIH3T3 and MCF-7 tumor cell lines. The potentiated metastatic and oncogenic properties of HER2Delta16 were mediated through direct coupling of HER2Delta16 to Src kinase. Cotargeting of HER2Delta16 and Src kinase with the single-agent tyrosine kinase inhibitor dasatinib resulted in Src inactivation, destabilization of HER2Delta16, and suppressed tumorigenicity. Activated Src kinase was also observed in 44% of HER2Delta16-expressing breast carcinomas underscoring the potential clinical implications of coupled HER2Delta16 and Src signaling. Our results suggest that HER2Delta16 expression is an important genetic event driving trastuzumab-refractory breast cancer. We propose that successful targeted therapeutics for intervention of aggressive HER2-positive breast cancers will require a strategy to suppress HER2Delta16 oncogenic signaling. One possibility involves a therapeutic strategy employing single-agent tyrosine kinase inhibitors to disengage the functionally coupled oncogenic HER2Delta16 and Src tyrosine kinase pathways.

A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist

Hepatocyte growth factor (HGF) activates the Met receptor tyrosine kinase by binding and promoting receptor dimerization. Here we describe a mechanistic basis for designing Met antagonists based on NK1, a natural variant of HGF containing the N-terminal and the first kringle domain. Through detailed biochemical and structural analyses, we demonstrate that both mouse and human NK1 induce Met dimerization via a conserved NK1 dimer interface. Mutations designed to alter the NK1 dimer interface abolish its ability to promote Met dimerization but retain full Met-binding activity. Importantly, these NK1 mutants act as Met antagonists by inhibiting HGF-mediated cell scattering, proliferation, branching, and invasion. The ability to separate the Met-binding activity of NK1 from its Met dimerization activity thus provides a rational basis for designing Met antagonists. This strategy of antagonist design may be applicable for other growth factor receptors by selectively abolishing the receptor activation ability but not the receptor binding of the growth factors.

The Mucin Muc4 Potentiates Neuregulin Signaling by Increasing the Cell-surface Populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.

ErbB/Her dimerization in breast and lung cancer

643 Background: Transmembrane receptor tyrosine kinases play an important role in the pathogenesis of many types of cancer. The EGFR family includes erbB1 (EGFR or Her1), erbB2 (Her2 or neu), erbB3 (Her3), and erbB4 (Her4). It is well established that ligands like EGF and HRG bind to the extracellular region of the EGFR monomers and promote receptor dimerization. Receptor dimerization leads to increased tyrosine kinase activity resulting in uncontrolled cell proliferation and inhibition of apoptosis. We have developed a method for the determination of Her dimerization patterns in cancer using eTag™ multiplexed assays. Materials and Method: We have analyzed a total of 73 snap-frozen human breast tissues and 37 lung tissues comprising of both tumor and normal samples. Using the proximity-based multiplexed eTag assays, we determined the dimerization profiles in these tissues. Results: ErbB/Her dimerization was detected only in tumor tissues but not in normal breast tissues, whether matched with the same donor or not. Out of the 31 breast tumor samples analyzed, Her1/2 dimers were detected in 20 tumor samples. Her-2/3 dimers were detected in 29 out of 43 tumor samples. We also found that all tumor samples had higher Her-2 levels compared to normal breast samples. In addition, we detected Her-2/2 homodimers in 27 out of 43 tumor samples. In lung tissues, seven out of 27 tumors had Her1/1 dimers while four out of 27 tumors had Her1/2 dimers, indicating that the predominant dimerization types in lung are Her1/1 and Her1/2. Our quantitative dimerization assays showed that the total receptor expression levels and dimerization profiles were different in different tumor samples both in lung and breast samples. Conclusion: eTag technology can offer a valuable prognostic and quantitative tool for stratifying cancer patients for targeted therapy. In addition, eTag technology can help in assessing the activation state of the receptor during the course of patient treatment. No significant financial relationships to disclose.

Comparison of proto-oncogenic and mutant forms of the transmembrane region of the Neu receptor in TFE

A single mutation within the transmembrane region of the Neu receptor (Val664→Glu) is known to enhance tyrosine kinase activity, by promoting receptor dimerization. In order to gain insight into potential structural changes that arise as a result of the mutation, peptides corresponding to the complete transmembrane domain of proto-oncogenic and mutant forms of Neu have been studied by 1H nuclear magnetic resonance in the solvent trifluoroethanol (TFE). The chemical shifts are similar for both forms of the peptide, with the exception of amide residues close to the mutation site. Both peptides adopt a helical conformation, with a distinct bend one turn downstream of the mutation site. This deformation gives rise to several nuclear Overhauser effects, the majority of which were detected in both peptides, that are atypical for a straight canonical α-helix. Our data in this solvent do not support a conformational change in the transmembrane domain of monomeric Neu as a result of the mutation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis indicates that proto-oncogenic Neu peptides have a higher propensity to oligomerize in the solvent TFE than the Glu664 oncogenic form.

Structural basis for activation of fibroblast growth factor signaling by sucrose octasulfate.

Sucrose octasulfate (SOS) is believed to stimulate fibroblast growth factor (FGF) signaling by binding and stabilizing FGFs. In this report, we show that SOS induces FGF-dependent dimerization of FGF receptors (FGFRs). The crystal structure of the dimeric FGF2-FGFR1-SOS complex at 2.6-A resolution reveals a symmetric assemblage of two 1:1:1 FGF2-FGFR1-SOS ternary complexes. Within each ternary complex SOS binds to FGF and FGFR and thereby increases FGF-FGFR affinity. SOS also interacts with the adjoining FGFR and thereby promotes protein-protein interactions that stabilize dimerization. This structural finding is supported by the inability of selectively desulfated SOS molecules to promote receptor dimerization. Thus, we propose that SOS potentiates FGF signaling by imitating the dual role of heparin in increasing FGF-FGFR affinity and promoting receptor dimerization. Hence, the dimeric FGF-FGFR-SOS structure substantiates the recently proposed "two-end" model, by which heparin induces FGF-FGFR dimerization. Moreover, the FGF-FGFR-SOS structure provides an attractive template for the development of easily synthesized SOS-related heparin agonists and antagonists that may hold therapeutic potential.

FRETting about your receptors

Signaling through the epidermal growth factor (EGF) pathway plays an important role in many cellular and developmental processes. Activation of the ErbB1 receptor by EGF is traditionally thought to occur by ligand binding, which promotes receptor dimerization, allowing cross-phosphorylation of the cytoplasmic domains of the receptor. The activated receptors then recruit signal effectors such as SHC and Grb2, triggering the signalling cascade. Verveer et al.1xQuantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane. Verveer, P et al. Science. 2000; 290: 1567–1570CrossRef | PubMedSee all References1 now show that this might be an oversimplification.Using a series of microscopy techniques, the authors show that localized stimulation of a cell leads to receptor activation that rapidly spreads across the whole cell. Using fluorescence lifetime imaging microscopy (FLIM), they measured fluorescence resonance energy transfer (FRET) between green-fluorescent protein (GFP)-tagged ErbB1 receptor and an injected Cy3-labeled phosphospecific antibody that only binds to the receptor in its active state. This technique shows which receptors are active in real time in live cells 2xFluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Bastiaens, P et al. Trends Cell Biol. 1999; 9: 48–51Abstract | Full Text | Full Text PDF | PubMed | Scopus (351)See all References2.Using this system, the authors show that, when cells are stimulated locally using EGF-coated beads, ErbB1 receptors become activated and that this activation spreads rapidly across the plasma membrane. By photobleaching the region around the beads, they showed that the receptors bound to the ligand (and beads) are unable to move, suggesting strong binding of the ligand to its receptor. Furthermore, using a mixture of unlabelled receptor and labelled kinase-dead receptor, which can be activated but cannot activate other receptors, they show that a small portion of functional receptors could activate a much larger fraction of the kinase-dead receptors than would be expected if simple dimerization occurred. They also demonstrate that this lateral activation does not depend on cytoplasmic proteins and occurs more rapidly in the presence of a protein phosphatase inhibitor. This implies that a certain threshold of receptor activation is needed to overcome the basal phosphatase activity and that high localized stimulation – e.g. by contact with a neighbouring cell – can produce activation across the whole cell.This model also suggests that EGF is only required for the initial local activation and is not required for propagation of the signal across the rest of the cell. These results could lead to potential new drug targets to modulate ErbB signalling in cancer. The methods used here could also easily be applied to other signalling pathways.