Extracellular Ligand-binding Domain Research Articles

Interactions of acetylcholine binding site residues contributing to nicotinic acetylcholine receptor gating: Role of residues Y93, Y190, K145 and D200

The nicotinic acetylcholine receptor exhibits multiple conformational states, resting (channel closed), active (channel open) and desensitized (channel closed). The resting state may be distinguished from the active and desensitized states by the orientation of loop C in the extracellular ligand binding domain (LBD). Homology modeling was used to generate structures of the Torpedo californica α2βδγ nAChR that initially represent the resting state (loop C open) and the desensitized state (loop C closed). Molecular dynamics (MD) simulations were performed on the extracellular LBD on each nAChR conformational state, with and without the agonist anabaseine present in each binding site (the αγ and the αδ sites). Three MD simulations of 10ns each were performed for each of the four conditions. Comparison of dynamics revealed that in the presence of agonist, loop C was drawn inward and attains a more stable conformation. Examination of side-chain interactions revealed that residue αY190 exhibited hydrogen-bonding interactions either with residue αY93 in the ligand binding site or with residue αK145 proximal to the binding site. αK145 also exhibited side chain (salt bridge) interactions with αD200 and main chain interactions with αY93. Residues αW149, αY198, γY116/δT119, γL118/δL121 and γL108/δL111 appear to play the role of stabilizing ligand in the binding site. In MD simulations for the desensitized state, the effect of ligand upon the interactions among αK145, αY190, and αY93 as well as ligand-hydrogen-bonding to αW149 were more pronounced at the αγ interface than at the αδ interface. Differences in affinity for the desensitized state were determined experimentally to be 10-fold. The changes in side chain interactions observed for the two conformations and induced by ligand support a model wherein hydrogen bond interactions between αD200 and αY93 are broken and rearrange to form a salt-bridge between αK145 and αD200 and hydrogen bond interactions between αY93 and αY190 and between αK145 and αY190.

The Pathogenic A391E Mutation in FGFR3 Induces a Structural Change in the Transmembrane Domain Dimer

Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.

Approaching the 5-HT3 receptor heterogeneity by computational studies of the transmembrane and intracellular domains

5-hydroxytryptamine type-3 receptor (5-HT₃), an important target of many neuroactive drugs, is a cation selective transmembrane pentamer whose functional stoichiometries and subunit arrangements are still debated, due to the extreme complexity of the system. The three dimensional structure of the 5-HT₃R subunits has not been solved so far. Moreover, most of the available structural and functional data is related to the extracellular ligand-binding domain, whereas the transmembrane and the intracellular receptor domains are far less characterised, although they are crucial for receptor function. Here, for the first time, 3D homology models of the transmembrane and the intracellular receptor domains of all the known human 5-HT₃ subunits have been built and assembled into homopentameric (5-HT(3A)R, 5-HT(3B)R, 5-HT(3C)R, 5-HT(3D)R and 5-HT(3E)R) and heteropentameric receptors (5-HT(3AB), 5-HT(3AC), 5-HT(3AD) and 5-HT(3AE)), on the basis of the known three-dimensional structures of the nicotinic-acetylcholine receptor and of the ligand gated channel from Erwinia chrysanthemi. The comparative analyses of sequences, modelled structures, and computed electrostatic properties of the single subunits and of the assembled pentamers shed new light both on the stoichiometric composition and on the physicochemical requirements of the functional receptors. In particular, it emerges that a favourable environment for the crossing of the pore at the transmembrane and intracellular C terminus domain levels by Ca²⁺ ions is granted by the maximum presence of two B subunits in the 5-HT₃ pentamer.

Assembly of a π–π stack of ligands in the binding site of an acetylcholine-binding protein

Acetylcholine-binding protein is a water-soluble homologue of the extracellular ligand-binding domain of cys-loop receptors. It is used as a structurally accessible prototype for studying ligand binding to these pharmaceutically important pentameric ion channels, in particular to nicotinic acetylcholine receptors, due to conserved binding site residues present at the interface between two subunits. Here we report that an aromatic conjugated small molecule binds acetylcholine-binding protein in an ordered π–π stack of three identical molecules per binding site, two parallel and one antiparallel. Acetylcholine-binding protein stabilizes the assembly of the stack by aromatic contacts. Thanks to the plasticity of its ligand-binding site, acetylcholine-binding protein can accommodate the formation of aromatic stacks of different size by simple loop repositioning and minimal adjustment of the interactions. This type of supramolecular binding provides a novel paradigm in drug design.

Comprehensive survey and genomic characterization of Toll-Like Receptors (TLRs) in channel catfish, Ictalurus punctatus: Identification of novel fish TLRs

A comprehensive survey of channel catfish Toll-like receptors (TLRs) was undertaken following a genomic PCR approach based on degenerate primers. Twenty different TLRs were identified in channel catfish. Channel catfish TLR sequences were characterized by phylogenetic analysis based on their conserved Toll/interleukin-1 receptor domain and by in-depth analysis of leucine-rich repeat (LRR) motifs of the ligand binding extracellular domain (ECD). The catfish have representatives of all the TLR types defined in vertebrates with the exception of TLR6, TLR10, TLR11, TLR12, TLR13, TLR15, TLR23, and TLR24. Additionally, two new types were discovered: TLR25 and TLR26. TLR25 is also present in cyprinids, cichlids, plecoglossids, and adrianichthyids, suggesting its presence early in fish evolution. To date, TLR26 was found only in channel catfish. Like TLR18-23, TLR25 and TLR26 were not found in any other vertebrate classes and appear to be fish specific. Data mining using the catfish TLR sequences revealed that in addition to ictalurids and cyprinids, TLR4 is also present in salmonids. TLR19 and TLR20 were both found in ictalurids, cyprinids, and salmonids, demonstrating a wider range than previously known. The LRR structure within ECDs appeared generally well conserved. TLR7 demonstrated a very high identity to human TLR7 strongly suggesting that ligand specificity maybe conserved. Finally, expression profiling confirmed that most TLRs are widely expressed in a diversity of tissues and revealed marked differences of expression level.

Water-soluble LYNX1 Residues Important for Interaction with Muscle-type and/or Neuronal Nicotinic Receptors

Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and similar snake α-neurotoxins also targeting α7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.

Abstract LB-318: Reshaping the cancer proteome: therapeutic targeting of signaling and resistance in cancer by antisense manipulation of RNA processing.

Abstract Activation of the receptor tyrosine kinase (RTK) signaling pathways represents a key aspect of tumorigenesis in a broad range of human cancers. Thus, targeting of pathogenic RTKs, mainly with small molecule inhibitors and antibodies, has become an attractive therapeutic approach for cancer therapy. However, resistance by multiple mechanisms, including mutational re-activation of RTK signaling, activation of alternative RTK pathways, EMT transition and others, inevitably occurs over time. We recently identified a number of novel secreted soluble decoy RTKs isoforms (sdRTKs) generated by a U1-snRNP-dependent alternative intronic polyadenylation mechanism. We therefore developed a novel antisense-based method to effectively activate the expression of these sdRTKs, both in vitro and in vivo. This leads to the generation of endogenous dominant negative variants of RTKs, which can specifically affect the signaling of pathogenic RTKs and therefore provides a novel cancer therapy approach. Employing specific antisense compounds designed to activate intronic polyA sites, we are able to induce natural truncated EGFR variants that encode its extracellular ligand-binding domain but lack the intracellular signaling domain. These compounds thus can inhibit EGFR signaling by a three fold-mechanism: 1) removal of FL receptor, 2) sequestration of ligands and 3) non-productive dimerization with residual receptors. They are therefore designed to affect signaling both of targeted cells and of bystanders. Here we will show that, using this approach, we can target EGFR signaling both in vitro and in tumors. Importantly, with this method we can bypass the emergence of resistance typically seen with targeted drug treatments. Further development of this methodology will therefore provide the basis for an exciting novel approach to targeting and inhibiting oncogenic EGFR signaling and other pathways in a variety of cancers. Citation Format: Luca Cartegni, lee Spraggon. Reshaping the cancer proteome: therapeutic targeting of signaling and resistance in cancer by antisense manipulation of RNA processing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-318. doi:10.1158/1538-7445.AM2013-LB-318

Insights into molecular interactions between the juxtamembrane and kinase subdomains of the Arabidopsis Crinkly-4 receptor-like kinase

Arabidopsis CRINKLY4 (ACR4), a receptor-like kinase required for plant growth and development, possesses an extracellular ligand binding domain, a transmembrane helix, and an intracellular domain (ICD). The ICD contains the juxtamembrane (JMD) and the C-terminal (CTD) subdomains, which flank the core kinase domain (KD), with at least 16 autophosphorylation sites. Phosphorylation sites are often docking sites for the modification-dependent recruitment of interacting proteins that orchestrate many downstream signaling events. In this context, we have specifically probed the role of the two phosphorylation sites Ser475 and Thr478 in the JMD using mutagenesis and phage-peptide screening techniques. Thus, naïve and phosphorylated 15-mer peptides derived from the JMD were panned against a 21-amino acid random phage peptide library. The phosphorylated peptide preferentially recognized the consensus sequence LxSLL. This sequence harbors the LxxLL motif, a known protein–protein interaction motif that is also present in the N-terminal lobe of the KD. We demonstrate the binding of JMD peptides to the KD and also show through kinetic analyses of mutants that phosphorylation of Ser475 and Thr478 in the JMD is necessary for optimal substrate phosphorylation in vitro. Our experiments suggest that an intramolecular interaction can occur between the JM and the N-terminal lobe of the KD.

Comprehensive survey and genomic characterization of Toll-like receptors (TLRs) in channel catfish, Ictalurus punctatus: identification of novel fish TLRs

A comprehensive survey of channel catfish Toll-like receptors (TLRs) was undertaken following a genomic PCR approach based on degenerate primers. Twenty different TLRs were identified in channel catfish. Channel catfish TLR sequences were characterized by phylogenetic analysis based on their conserved Toll/interleukin-1 receptor domain and by in-depth analysis of leucine-rich repeat (LRR) motifs of the ligand binding extracellular domain (ECD). The catfish have representatives of all the TLR types defined in vertebrates with the exception of TLR6, TLR10, TLR11, TLR12, TLR13, TLR15, TLR23, and TLR24. Additionally, two new types were discovered: TLR25 and TLR26. TLR25 is also present in cyprinids, cichlids, plecoglossids, and adrianichthyids, suggesting its presence early in fish evolution. To date, TLR26 was found only in channel catfish. Like TLR18-23, TLR25 and TLR26 were not found in any other vertebrate classes and appear to be fish specific. Data mining using the catfish TLR sequences revealed that in addition to ictalurids and cyprinids, TLR4 is also present in salmonids. TLR19 and TLR20 were both found in ictalurids, cyprinids, and salmonids, demonstrating a wider range than previously known. The LRR structure within ECDs appeared generally well conserved. TLR7 demonstrated a very high identity to human TLR7 strongly suggesting that ligand specificity maybe conserved. Finally, expression profiling confirmed that most TLRs are widely expressed in a diversity of tissues and revealed marked differences of expression level.

Increased Muscle Force Production and Bone Mineral Density in ActRIIB-Fc-Treated Mature Rodents

Myostatin is a highly conserved member of the transforming growth factor-β ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice. Significant muscle growth and enhanced muscle function were observed in adult mice treated for 3 days with ActRIIB-Fc. The ActRIIB-Fc-treated mice had enhanced fast fatigable muscle function, with only minor enhancement of fatigue-resistant fiber function. The ActRIIB-Fc-treated 18-month-old mice and orchidectomized mice showed significantly improved muscle function. Treatment with ActRIIB-Fc also increased bone mineral density and serum levels of a marker of bone formation. These observations highlight the potential of targeting ActRIIB receptor to treat age-related and hypogonadism-associated musculoskeletal degeneration.

Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors

In multimeric cell-surface receptors, the conformational changes of the extracellular ligand-binding domains (ECDs) associated with receptor activation remain largely unknown. This is the case for the dimeric metabotropic glutamate receptors even though a number of ECD structures have been solved. Here, using an innovative approach based on cell-surface labeling and FRET, we demonstrate that a reorientation of the ECDs is associated with receptor and G-protein activation. Our approach helps identify partial agonists and highlights allosteric interactions between the effector and binding domains. Any approach expected to stabilize the active conformation of the effector domain increased the agonist potency in stabilizing the active ECDs conformation. These data provide key information on the structural dynamics and drug action at metabotropic glutamate receptors and validate an approach for tackling such analysis on other receptors.

Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris

In the central nervous system (CNS), fast neuronal signals are facilitated by the oligodendrocyte-produced myelin sheath. Oligodendrocyte turnover or injury generates myelin debris that is usually promptly cleared by phagocytic cells. Failure to remove dying oligodendrocytes leads to accumulation of degraded myelin, which, if recognized by the immune system, may contribute to the development of autoimmunity in diseases such as multiple sclerosis. We recently identified low density lipoprotein receptor-related protein-1 (LRP1) as a novel phagocytic receptor for myelin debris. Here, we report characterization of the LRP1 interactome in CNS myelin. Fusion proteins were designed corresponding to the extracellular ligand-binding domains of LRP1. LRP1 partners were isolated by affinity purification and characterized by mass spectrometry. We report that LRP1 binds intracellular proteins via its extracellular domain and functions as a receptor for necrotic cells. Peptidyl arginine deiminase-2 and cyclic nucleotide phosphodiesterase are novel LRP1 ligands identified in our screen, which interact with full-length LRP1. Furthermore, the extracellular domain of LRP1 is a target of peptidyl arginine deiminase-2-mediated deimination in vitro. We propose that LRP1 functions as a receptor for endocytosis of intracellular components released during cellular damage and necrosis.

Transmembrane helix orientation influences membrane binding of the intracellular juxtamembrane domain in Neu receptor peptides

The transmembrane (TM) and juxtamembrane (JM) regions of the ErbB family receptor tyrosine kinases connect the extracellular ligand-binding domain to the intracellular kinase domain. Evidence for the role of these regions in the mechanism of receptor dimerization and activation is provided by TM-JM peptides corresponding to the Neu (or rat ErbB2) receptor. Solid-state NMR and fluorescence spectroscopy show that there are tight interactions of the JM sequence with negatively charged lipids, including phosphatidylinositol 4,5-bisphosphate, in TM-JM peptides corresponding to the wild-type receptor sequence. We observe a release of the JM sequence from the negatively charged membrane surface using peptides containing an activating V664E mutation within the TM domain or in peptides engineered to form TM helix dimers with Val664 in the interface. These results provide the basis of a mechanism for coupling ligand binding to kinase activation in the full-length receptor.

The Role of SmXXXSm Motifs in Wild-Type and Ala391Glu FGFR3 Transmembrane Domain Dimerization

Fibroblast growth factor receptor 3 (FGFR3) is a single pass membrane protein and a member of the receptor tyrosine kinase (RTK) family of proteins. FGFR3 is critically involved in cellular processes that regulate skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain, and the transmembrane domain (TMD). Previous work with the isolated FGFR3 transmembrane (TM) domain has shown that it has the ability to self-dimerize. Mutations in FGFR3 can lead to a variety of diseases, but more specifically, mutations in the TMD of the protein can cause achondroplasia (the most common form of dwarfism, thanatophoric (skeletal) dysplasia, Crouzon syndrome (cranial dysplasia) with acanthosis nigricans (hyperpigmentation of the skin), and bladder cancer. Though the structures of the extracellular and cytosolic domains of FGFR3 have been resolved, the structure of the TMD dimer and the effects of pathogenic mutations on the dimer structure are still unknown. Using the ToxR assay, structural studies were carried out by studying the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. The SmXXXSm motif has been shown to drive the dimerization of many transmembrane proteins. Results from the ToxR assay indicate that the SmXXXSm does not play a role in the dimerization of the WT FGFR3 TMD. In contrast, similar studies carried out with the A391E mutant TMD show that the SmXXXSm motif plays a role in TMD dimerization. These results lead to the conclusion that the pathogenic A391E TMD has a different dimer structure than the WT FGFR3 TMD.

Abstract P4-09-09: Circulating HER2 extracellular domain (ECD) levels are associated with progression-free survival in metastatic breast cancer patients

Abstract Background: The human epidermal growth factor (HER2/neu) oncoprotein is composed of an intracellular tyrosine kinase portion, a short transmembrane section, and an extracellular ligand-binding domain (ECD). The latter is frequently cleaved from the surface of breast cancer cells and the serum HER2 ECD levels can be detected by enzyme-linked immunosorbent assay (ELISA). Consistent with majority of reports, our prior study showed that high circulating levels of the HER2 ECD indicates poor prognosis in 207 patients with metastatic breast cancer. In the present study, we aimed to elucidate whether serum HER2 ECD levels would predict the clinical outcome of metastatic breast cancer patients. Methods: Till May 19, 2012, one hundred ninty metastatic breast cancer patients were followed up. Serum was available from 190 patients at baseline, 46 patients in the assessment of time. Serum HER2 ECD levels were measured by ELISA. Disease state was assessed using Response Evaluation Criteria in Solid Tumors (RECIST). Progression-free survival (PFS) was defined as the interval from the time of randomization until the first documentation of disease progression or death due to any cause. PFS and median PFS were generated by Kaplan-Meier spots. Results: Elevated baseline HER2 ECD levels (bECD) (≥15 ng/ml) were observed in 40% (40/100) of patients with HER2-amplified tumors and 21.8% (19/87) of patients with HER2-negative tumors. Patients with bECD-elevated disease had a median PFS of 356 days compared with 229 days for patients with bECD-low disease(P = 0.001). In HER2-amplified cohort, median PFS for patients with low bECD levels was longer than those with bECD-elevated disease (391 days v 226 days, P = 0.005). And patients with low bECD levels treated with Herceptin-based regimen (n = 36) had a trend of longer PFS than patients(n = 24) treated with non-Herceptin regimen(447 days v 298 days, P = 0.227). Moreover, bECD-low patients (n = 36) showed a significantly longer median PFS than bECD-elevated patients(n = 22) when treated with Herceptin-based scheme (447 days v 229 days, P = 0.004). While, in patients with increased bECD levels, there was no statistical difference between Herceptin-treated groups (n = 22) and non-Herceptin-treated groups (n = 14) (229 days v 173 days, P = 0.761). In the HER2-negative group, patients with bECD-low disease had a median PFS of 313 days compared with 206 days for patients with bECD-elevated disease (P = 0.02). In a cohort of 46 patients whose HER2 ECD levels were monitored during treatment, 29 patients remains low ECD levels, 12 patients convert from elevated to low, 2 patients convert from low to high, and 3 patients remains elevated HER2 ECD levels. The patients who remained low ECD levels or achieved low ECD leves have significantly longer PFS than those whose levels remained above 15ng/mL or converted from low to high regardless of treatment given (440 days v 192days, P = 0.019). Conclusion: Elevated HER2 bECD predicts poor median PFS irrespective of HER2 status. HER2-amplified patients with low-HER2 ECD levels may get more benefit from Herceptin-based scheme. Remained low-HER2 ECD levels or decreases in serum HER2 ECD levels during treatment were associated with longer median PFS in metastatic breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-09-09.

Controlling Thrombopoietin Receptor Dimerization, Orientation and Activation Via Tryptophan 515

AbstractAbstract 3449 Background and Aims.The thrombopoietin receptor (TpoR, c-Mpl) plays a dual role in hematopoiesis. Beside the fact that it is one of the main regulators of megacaryopoiesis and platelet formation, TpoR is also expressed in HSCs and maintains them quiescent. TpoR signals as a homodimer after ligand binding in a 1:2 (ligand to receptor) ratio. Similar to other single pass membrane receptor, it contains an extracellular ligand binding domain, one helical transmembrane domain (TMD) and a cytoplasmic part important for JAKs binding and to mediate signaling. Unlike other cytokine receptors, TpoR contains a unique amphipathic motif (RW515QFP) at the junction between the TMD and the cytoplasmic domain. Mutations within this motif, namely W515K or W515L, lead to activation of the receptor and are associated with JAK2 V617F negative myeloproliferative neoplasms, specifically essential thrombocytopenia and primary myelofibrosis. We asked i) how a tryptophan at position 515 prevents autonomous activation of TpoR and ii) exactly how mutants at W515 impact on TpoR function and lead to oncogenesis. Methods.A combination of site-directed mutagenesis, biochemical and signaling assays, and spectroscopy analysis were employed. We made several specific mutations at position 515 to see if Trp515 is regulatory because of it size and/or aromatic character. We then tested the effect of mutations at adjacent positions in the wild type or mutated W515 constructs. Data were recorded from transiently transfected g2A cells and the results were confirmed in stably transduced BaF3 cells using dual luciferase and growth assays in the presence or absence of ligand. Adoptive transfer in lethally irradiated mice was used for assessing in vivo effects of TpoR mutants.The oligomerization status of the full length TpoR and some of our mutants was tested by employing the Gaussia princeps luciferase complementation assay, where Gaussia princeps luciferase fragments were fused in frame to the carboxyl terminus of TpoR or TpoR mutants. Recombination of the luciferase fragments results in a positive signal and reveals dimerization. Deuterium magic angle spinning (MAS) NMR spectroscopy and analytical ultracentrifugation were used to assess dimerization of the region encompassing the TMD. The tilt angle of this segment was assessed by FTIR spectroscopy. Results.Tryptophan seems to be absolutely required at position 515 to maintain the receptor inactive in absence of ligand. We identified secondary mutations (to tryptophan) that can prevent W515L/K/A activation of TpoR. The doubly mutated receptors behave like wild type receptors exhibiting no activation in absence of TPO and a normal response to TPO. These data suggest that W515 mutants may be specifically targeted for therapy. Mutations at W515 induced an increase in full length TpoR dimerization, and promoted dimerization of the segment comprising the TMD. However, our secondary site mutations showed differences between the mechanisms by which K or L versus A activate at W515. Overall, W515 impairs TpoR dimerization in a conformation productive for JAK2 activation. Measurements of the dichroic ratio by FTIR indicated that W515 increases the tilt angle of the TMD, thus reducing the interface required for TMD dimerization. We further explored the role of W515 in receptor activation by using a model where coiled coils are used to induce distinct dimeric conformations when fused to the TMD-cytosolic domains. W515 was essential for imparting differential signaling to the different dimers, both in cell lines and in vivo, where phenotypes in reconstituted mice no longer differ between receptors that have different dimeric orientations. Conclusions.Tryptophan 515 prevents TpoR dimerization in a productive orientation for JAK2 activation. This effect is reversible when secondary site mutations to tryptophan are introduced at certain positions around W515. Dimerization of TMD induced by ligand rotates W515 so that it is displaced from the headgroup region of the membrane to the dimer interface. This is crucial for imparting specific signaling as a function of dimeric orientation. Our results indicate that the region around W515 is a major switch for receptor function and could be exploited therapeutically. Disclosures:Constantinescu:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees.

Dynamics of Circulating Ligands and Soluble Receptor for Advanced Glycation End-Product in Liver Transplantation

Background and aim: The ligand-receptor for advanced glycation end products (RAGE) pathway is involved in liver injury and animal testing have highlighted that the RAGE blockade attenuates the extent of the liver injury. A truncated isoform of the RAGE (endogenous secreted RAGE, esRAGE), consisting of the extracellular ligand-binding domain only, is detectable as circulating soluble form (sRAGE) in the blood. These latter, acting as decoy, can contribute to the removal of circulating RAGE- ligands. Objective of this study is understand the hepatic expression of both RAGE isoforms (full-length and truncated), and kinetics of the RAGE-ligands and sRAGE plasma levels in patients undergoing liver transplantation (LT) and compared them with healthy donors. Methods: We prospectively included twenty four adult LT recipients (52.7 ± 9.2 years) of primary whole-size grafts by deceased donors (60.4 ± 17.9 years). On both LT recipients and donors, we measured the transcriptional expression of hepatic tissue RAGE and esRAGE, the plasma levels of the RAGE-ligands - N(epsilon)-carboxymethyllysine (CML) and high-mobility group protein 1 (HMGB-1) - and circulating sRAGE. In LT recipient CML, HMGB-1 and sRAGE plasma levels were measured before LT, after graft reperfusion, 1, 7, 30 and 90 days after LT. Results: In LT recipients the hepatic RAGE and esRAGE mRNA levels were higher than in healthy donors (p< 0.01 and p=0.02 respectively). The tissue full-length RAGE inversely correlated with antithrombin III (β= - 0.58, p = 0.013) and cholinesterase plasma levels (β= -0.717, p= 0.0018) and tended to directly correlate with MELD score (β= 0.422, p= 0.063). Basal plasma levels CML, but not sRAGE and HMGB-1, were higher in LT recipients than in healthy donors (p< 0.05). At follow-up CML levels decreased after graft reperfusion (p = 0.0001) while returned progressively to basal values during the follow-p. Instead, HMGB-1, increased after graft reperfusion (p = 0.0001) and returned suddenly to basal values one day after LT. The basal HMGB-1 correlated directly with MELD score (β= 0.448, p= 0.05). The plasma levels of sRAGE did not change significantly soon after LT while decreased seven days after LT (p = 0.0001) and remained constantly low during follow-up. Conclusions: The high levels of RAGE and esRAGE tissue expression in the end-stage liver failure highlight the involvement of the RAGEligand axis in liver injury. The circulating CML accumulation and the rapid increase of HMGB-1 followed by a dramatic decline of the protective sRAGE could have deleterious consequences on graft survival and long term outcomes in LT recipients.

Structural Mechanism of N-Methyl-D-Aspartate Receptor Type 1 Partial Agonism

N-methyl-D-aspartate (NMDA) receptors belong to a family of ionotropic glutamate receptors that contribute to the signal transmission in the central nervous system. NMDA receptors are heterotetramers that usually consist of two GluN1 and GluN2 monomers. The extracellular ligand-binding domain (LBD) of a monomer is comprised of discontinuous segments that form the functional domains D1 and D2. While the binding of a full agonist glycine to LBD of GluN1 is linked to cleft closure and subsequent ion-channel opening, partial agonists are known to activate the receptor only sub-maximally. Although the crystal structures of the LBD of related GluA2 receptor explain the mechanism for the partial agonism, structures of GluN1-LBD cannot distinguish the difference between full and partial agonists. It is, however, probable that the partial agonists of GluN1 alter the structure of the LBD in order to result in a different pharmacological response than seen with full agonists. In this study, we used molecular dynamics simulations to reveal an intermediate closure-stage for GluN1, which is unseen in crystal structures. According to our calculations, this intermediate closure is not a transient stage but an energetically stable conformation. Our results demonstrate that the partial agonist cannot exert firm GluN1-LBD closure, especially if there is even a small force that disrupts the LBD closure. Accordingly, this result suggests the importance of forces from the ion channel for the relationship between pharmacological response and the structure of the LBD of members of this receptor family.

EJ-3 - Targeting the her Family in Head and Neck Cancer: A Road with Hurdles

ABSTRACT The epidermal growth factor receptor (EGFR) is playing a role in the multistep carcinogenesis of squamous cell carcinoma of the head and neck (SCCHN) and its signaling promotes SCCHN growth and progression. EGFR is over expressed in the vast majority (>90%) of H&N cases and a high expression is associated with an unfavorable prognosis. Therefore, EGFR is a rational therapeutic target. In practice, both monoclonal antibodies (MAbs), which bind to the outer domain, and small molecule oral tyrosine kinase inhibitors (TKIs) that target the intracellular catalytic domain of the EGFR are being used. Cetuximab (Cet) is the only targeted agent that got approval for the treatment of SCCHN from the regulatory agencies of Europe and the United States, both in locoregionally advanced disease in association with radiation (RT) and in recurrent/metastatic disease in combination with platinum/5-FU-based chemotherapy (in United States also as single agent in second line) and is still under active investigation in this disease. Unfortunately, several important questions are still unanswered (e.g. is RT + Cet equal in efficacy to RT + cisplatin (P)?, is maintenance therapy essential and for how long?, what are the adequate markers to predict response to Cet?, etc). Moreover, some results have been disappointing (e.g. PRT + Cet is not superior to PRT alone) and some applications are still investigational (e.g. Cet + induction chemotherapy and Cet in the postoperative setting). Interestingly, the results of trials involving other EGFR-directed MAbs, i.e. zalutumumab, panitumumab and nimotuzumab, so far have been consistent with the cetuximab data, but failed their primary end points. The results with EGFR-directed TKIs overall have been disappointing. Dual TKIs of both EGFR and HER/2 (the preferred dimerization partner of EGFR) have shown some promise, as well as newer irreversible HER-family blockers, such as afatinib and PF-299804. Both afatinib and PF-299804 have also demonstrated preclinical activity against tumor cells bearing a truncated EGFR protein, lacking the extracellular ligand-binding domain (EGFR variant III). Despite the hurdles, targeting the HER has a place in SCCHN treatment.

Identification of natural killer cell receptor genes in the genome of the marsupial Tasmanian devil (Sarcophilus harrisii)

Within the mammalian immune system, natural killer (NK) cells contribute to the first line of defence against infectious agents and tumours. Their activity is regulated, in part, by cell surface NK cell receptors. NK receptors can be divided into two unrelated, but functionally analogous superfamilies based on the structure of their extracellular ligand-binding domains. Receptors belonging to the C-type lectin superfamily are predominantly encoded in the natural killer complex (NKC), while receptors belonging to the immunoglobulin superfamily are predominantly encoded in the leukocyte receptor complex (LRC). Natural killer cell receptors are emerging as a rapidly evolving gene family which can display significant intra- and interspecific variation. To date, most studies have focused on eutherian mammals, with significantly less known about the evolution of these receptors in marsupials. Here, we describe the identification of 43 immunoglobulin domain-containing LRC genes in the genome of the Tasmanian devil (Sarcophilus harrisii), the largest remaining marsupial carnivore and only the second marsupial species to be studied. We also identify orthologs of NKC genes KLRK1, CD69, CLEC4E, CLEC1B, CLEC1A and an ortholog of an opossum NKC receptor. Characterisation of these regions in a second, distantly related marsupial provides new insights into the dynamic evolutionary histories of these receptors in mammals. Understanding the functional role of these genes is also important for the development of therapeutic agents against Devil Facial Tumour Disease, a contagious cancer that threatens the Tasmanian devil with extinction.