Dual Receptor System Research Articles

Robust In Vitro Pharmacology of Tmod, a Synthetic Dual-Signal Integrator for Cancer Cell Therapy.

Progress toward improved solid-tumor treatment has long been hindered by the lack of truly tumor-specific targets. We have developed an approach to T cell therapy based on a dual-receptor system called Tmod™ that addresses this problem. The Tmod system exploits one of the few common genetic differences between tumor and normal cells: loss of heterozygosity (LOH). It utilizes the basic mechanistic logic that evolved in early vertebrates to mediate self vs. non-self discrimination, where an activation stimulus is blocked by self-ligands. Tmod constructs employ a chimeric antigen receptor (CAR) or T cell receptor (TCR) as activator component and a modified LIR-1 inhibitory receptor (blocker) to achieve high selectivity based on expression of the blocker antigen (Ag). Here we explore the in vitro pharmacology of a blocker directed at the HLA-A*02 Ag paired with either a mesothelin CAR or an HLA-A*11-restricted KRAS peptide TCR. While more sensitive to receptor expression changes on effector cells, we show that Tmod response is well-buffered against variations in Ag levels on target cells. In addition, the data reveal at least two distinguishable pharmacologic mechanisms of Tmod blocker function: (1) reducing activator sensitivity and (2) decreasing activation magnitude.

PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells.

Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.

Mesothelin-specific CAR-T cell therapy that incorporates an HLA-gated safety mechanism selectively kills tumor cells

BackgroundMesothelin (MSLN) is a classic tumor-associated antigen that is expressed in lung cancer and many other solid tumors. However, MSLN is also expressed in normal mesothelium which creates a significant...

Mechanism of Ganglioside Receptor Recognition by Botulinum Neurotoxin Serotype E.

The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228–1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides.

Analysis of a Naturally Occurring PAR4 Variant By Hydrogen/Deuterium Exchange Reveals Its Structural Rearrangement upon Activation By Thrombin and the Molecular Basis for Altered Reactivity

Protease activated receptors 1 and 4 (PAR1 and PAR4) are GPCRs that mediate thrombin signaling on human platelets. In this dual receptor system, PAR4 is responsible for sustained signaling required for stable platelet aggregation. In addition, common PAR4 polymorphisms affect its reactivity, which contributes to the variability in platelet response among individuals. The threonine residue 120 leads to a variant that is hyper-reactive to agonists and resistant to some antagonist compared to an alanine at this site. The molecular mechanisms by which this substitution influences PAR4 activation are not known. Further, the structural basis for PAR activation in general is also unknown. PARs are activated by enzymatic cleavage of the N-terminus exposing a tethered ligand that interacts with a binding pocket on the receptor's extracellular side. Since the ligand is attached to the receptor, there are likely substantial conformational changes that occur during PAR activation. The goal of this study was to determine the structural rearrangement of PAR4 upon activation by thrombin to test the hypothesis that polymorphisms at position 120 directly contribute to the ligand binding site and the subsequent PAR4 signaling cascades. Here, we used hydrogen-deuterium exchange mass spectrometry to map the conformational rearrangement of the hyper-reactive PAR4 variant (PAR4-120T). Full length human PAR4-120T was expressed in and purified from SF9 insect cells without additional stabilizing modifications. Our initial peptide mapping studies generated 93.5% (344/368) amino acid sequence coverage with multiple overlapping peptides. In the uncleaved (apo) state, the seven transmembrane domains of PAR4 show limited deuterium uptake as a result of being buried in detergent micelles. TM4 and TM5 displayed lower deuterium uptake than the other helices which potentially indicate the formation of dimerization interface, which is in agreement with our previous biochemical studies. Helix 8, a short α-helix between TM7 and C-terminus, showed reduced deuterium uptake. Together with other studies, this may indicate helix 8 plays a role in anchoring the receptor on lipid rafts. In comparison, the N- and C-termini displayed higher solvent accessibility based on the high deuterium uptake. Surprisingly, the tethered ligand sequence of PAR4 (GYPGQV) at the N-terminus has significantly lower solvent accessibility than neighboring regions in the apo state. This suggests it is less flexible and may be localized near the binding pocket even before thrombin cleavage. Comparison of deuterium uptake between apo and thrombin cleaved (active) states of PAR4-120T revealed reduced solvent accessibility at amino acids Cys149-Leu156 at TM3 and Ser329-Ser333 at TM7. This identifies the potential tethered ligand binding region of PAR4, which has not been previously described yet. Interestingly, in our molecular models Thr120 was located near this region spatially, which potentially explains the impact of this residue on PAR4 reactivity. We further observed an increase in deuterium uptake indicating a potential role for extracellular loop III and the connected regions of TM6 and TM7 in the receptor activation mechanism. In summary, our study on PAR4 is the first to elucidate the structural dynamics and molecular rearrangement of a protease activated receptor upon activation. Our results provide the overall molecular architecture of PAR4, uncovers the tethered ligand binding region, and further reveals the molecular basis for the inter-individual platelet reactivity based on the genotype of this key platelet receptor. Altogether, our studies may have significant clinical impact towards explaining the diversity of individual responses to existing and forthcoming therapies. DisclosuresNo relevant conflicts of interest to declare.

Molecular and structural basis of Echovirus 11 infection by using the dual-receptor system of CD55 and FcRn

Echovirus 11 (Echo 11) is a serotype of Enterovirus B (EV-B) belonging to the genus Enterovirus in the family Picornaviridae . Echo 11 infections are associated with viral encephalitis (VE), viral meningitis (VM), and viral meningo-encephalitis (VME). Echo 11 was reported to cause severe illnesses in neonates or infants, with high morbidity and mortality. In addition, Echo 11 has frequently been identified as the causative agent of outbreaks, greatly threatens public health. For example, in early 2019, an Echo 11 outbreak occurred in a hospital of Guangdong Province in China, leading to nineteen infections of newborns and five of them died. CD55 has been designated an attaching receptor of several echoviruses in the 1990s. But CD55 alone is unable to help the virus to complete its whole entry process. In our earlier work, we found that the human neonatal Fc receptor (FcRn) is the cellular uncoating receptor for Enterovirus B and illustrated the entry mechanism taking Echo 6 as an example. We proposed the coordination of attaching and uncoating “dual-receptor system” in the entry pathways. Here, we analyzed 10 cryo-electron microscopy (Cryo-EM) structures of Echo 11 viral particles in three different forms (free, “A-particle” and empty particle), and Echo 11 in complex with receptor CD55 or FcRn, under both neutral (pH 7.4) and acidic (pH 5.5) conditions. With atomic or near-atomic resolutions (2.32−3.4 A), we obtained detailed characterization of the molecular events during Echo 11 entry. The structures revealed that CD55 is the attaching receptor of Echo 11, which facilitates the viral attachment to cell surface. Similar to Echo 12 and Echo 7, Echo 11 uses its VP2 and VP3 domain to bind short consensus repeat (SCR) module SCR 3 and SCR4 domains of CD55. FcRn is the uncoating receptor, which uses the α2 domain of its heavy chain FCGRT to insert into and bind the “canyon” of Echo 11 VP1 domain. The binding induced the extrusion of “pocket factor” and resulted in virus uncoating and genome release. FcRn induced Echo 11 viral particle conformational change and “pocket factor” release started at neutral pH, which is different from Echo 6, there the “pocket factor” release occurs at low pH. This hints us that acidic treatment might not be an essential requirement for the uncoating of all Enterovirus B which uses FcRn as their uncoating receptor. The uncoating process is able to start in early endosomes. SPR (Surface plasmon resonance) analysis certified that, Echo 11 can bind to both receptors, but has a higher affinity with FcRn than to CD55. FcRn-mediated Echo 11 uncoating was further confirmed with an FcRn-decorated liposome model, which induced the structural transitions of Echo 11 into empty particles. This work provided a molecular and structural basis for our understanding of the mechanisms of Echo 11 entry. And it is an important progress in dissecting the mechanism of the “dual-receptor system” in non-enveloped virus entry.

Concerning the discovery of mechanism for enterovirus B infection published in Cell.

Concerning the discovery of mechanism for enterovirus B infection published in Cell.

Glucocorticoid Receptor-Tethered Mineralocorticoid Receptors Increase Glucocorticoid-Induced Transcriptional Responses.

Mineralocorticoid and glucocorticoid receptors (MRs and GRs) constitute a functionally important dual receptor system detecting and transmitting circulating corticosteroid signals. High expression of MRs and GRs occurs in the same cells in the limbic system, the primary site of glucocorticoid action on cognition, behavior, and mood; however, modes of interaction between the receptors are poorly characterized. We used chromatin immunoprecipitation with nucleotide resolution using exonuclease digestion, unique barcode, and single ligation (ChIP-nexus) for high-resolution genome-wide characterization of MR and GR DNA binding profiles in neuroblastoma cells and demonstrate recruitment to highly similar DNA binding sites. Expressed MR or GR showed differential regulation of endogenous gene targets, including Syt2 and Ddc, whereas coexpression produced augmented transcriptional responses even when MRs were unable to bind DNA (MR-XDBD). ChIP confirmed that MR-XDBD could be tethered to chromatin by GR. Our data demonstrate that MR can interact at individual genomic DNA sites in multiple modes and suggest a role for MR in increasing the transcriptional response to glucocorticoids.

Dual Biomembrane Force Probe enables single-cell mechanical analysis of signal crosstalk between multiple molecular species

Conventional approaches for studying receptor-mediated cell signaling, such as the western blot and flow cytometry, are limited in three aspects: 1) The perturbing preparation procedures often alter the molecules from their native state on the cell; 2) Long processing time before the final readout makes it difficult to capture transient signaling events (<1 min); 3) The experimental environments are force-free, therefore unable to visualize mechanical signals in real time. In contrast to these methods in biochemistry and cell biology that are usually population-averaged and non-real-time, here we introduce a novel single-cell based nanotool termed dual biomembrane force probe (dBFP). The dBFP provides precise controls and quantitative readouts in both mechanical and chemical terms, which is particularly suited for juxtacrine signaling and mechanosensing studies. Specifically, the dBFP allows us to analyze dual receptor crosstalk by quantifying the spatiotemporal requirements and functional consequences of the up- and down-stream signaling events. In this work, the utility and power of the dBFP has been demonstrated in four important dual receptor systems that play key roles in immunological synapse formation, shear-dependent thrombus formation, and agonist-driven blood clotting.

Contributions of Protease-Activated Receptors PAR1 and PAR4 to Thrombin-Induced GPIIbIIIa Activation in Human Platelets.

Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target.

Systems biology insights into the meaning of the platelet's dual‐receptor thrombin signaling

Background Activation of human platelets with thrombin proceeds via two protease-activated receptors (PARs), PAR1 and PAR4, that have identical main intracellular signaling responses. Although there is evidence that they have different cleavage/inactivation kinetics (and some secondary variations in signaling), the reason for such redundancy is not clear. Methods We developed a multicompartmental stochastic computational systems biology model of dual-receptor thrombin signaling in platelets to gain insight into the mechanisms and roles of PAR1 and PAR4 functioning. Experiments employing continuous flow cytometry of washed human platelets were used to validate the model and test its predictions. Activity of PAR receptors in donors was evaluated by mRNA measurement and by polymorphism sequencing. Results Although PAR1 activation produced rapid and short-lived response, signaling via PAR4 developed slowly and propagated in time. Response of the dual-receptor system was both rapid and prolonged in time. Inclusion of PAR1/PAR4 heterodimer formation promoted PAR4 signaling in the medium range of thrombin concentration (about 10 nm), with little contribution at high and low thrombin. Different dynamics and dose-dependence of procoagulant platelet formation in healthy donors was associated with individual variations in PAR1 and PAR4 activities and particularly by the Ala120Thr polymorphism in the F2RL3 gene encoding PAR4. Conclusions The dual-receptor combination is critical to produce a response combining three critical advantages: sensitivity to thrombin concentration, rapid onset and steady propagation; specific features of the protease-activated receptors do not allow combination of all three in a single receptor.

Protease‐activated receptor 4 is more important than protease‐activated receptor 1 for the thrombin‐induced procoagulant effect on platelets

Protease-activated receptor 4 is more important than protease-activated receptor 1 for the thrombin-induced procoagulant effect on platelets

A Dual Receptor and Reporter for Multi-Modal Cell Surface Engineering.

The rapid development of new small molecule drugs, nanomaterials, and genetic tools to modulate cellular function through cell surface manipulation has revolutionized the diagnosis, study, and treatment of disorders in human health. Since the cell membrane is a selective gateway barrier that serves as the first line of defense/offense and communication to its environment, new approaches that molecularly engineer or tailor cell membrane surfaces would allow for a new era in therapeutic design, therapeutic delivery, complex coculture tissue construction, and in situ imaging probe tracking technologies. In order to develop the next generation of multimodal therapies, cell behavior studies, and biotechnologies that focus on cell membrane biology, new tools that intersect the fields of chemistry, biology, and engineering are required. Herein, we develop a liposome fusion and delivery strategy to present a novel dual receptor and reporter system at cell surfaces without the use of molecular biology or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal functional groups that can conjugate a range of ligands while simultaneously reporting the conjugation through the emission of fluorescence. We demonstrate this dual receptor and reporter system by conjugating and tracking various cell surface ligands for temporal control of cell fluorescent signaling, cell-cell interaction, and tissue assembly construction.

Corticosterone targets distinct steps of synaptic transmission via concentration specific activation of mineralocorticoid and glucocorticoid receptors

Hippocampal neurons are affected by chronic stress and have a high density of cytoplasmic mineralocorticoid and glucocorticoid receptors (MR and GR). Detailed studies on the genomic effects of the stress hormone corticosterone at physiologically relevant concentrations on different steps in synaptic transmission are limited. In this study, we tried to delineate how activation of MR and GR by different concentrations of corticosterone affects synaptic transmission at various levels. The effect of 3-h corticosterone (25, 50, and 100 nM) treatment on depolarization-mediated calcium influx, vesicular release and properties of miniature excitatory post-synaptic currents (mEPSCs) were studied in cultured hippocampal neurons. Activation of MR with 25 nM corticosterone treatment resulted in enhanced depolarization-mediated calcium influx via a transcription-dependent process and increased frequency of mEPSCs with larger amplitude. On the other hand, activation of GR upon 100 nM corticosterone treatment resulted in increase in the rate of vesicular release via the genomic actions of GR. Furthermore, GR activation led to significant increase in the frequency of mEPSCs with larger amplitude and faster decay. Our studies indicate that differential activation of the dual receptor system of MR and GR by corticosterone targets the steps in synaptic transmission differently.

Mechanism and Role of High Density Lipoprotein-induced Activation of AMP-activated Protein Kinase in Endothelial Cells

The upstream signaling pathway leading to the activation of AMP-activated protein kinase (AMPK) by high density lipoprotein (HDL) and the role of AMPK in HDL-induced antiatherogenic actions were investigated. Experiments using genetic and pharmacological tools showed that HDL-induced activation of AMPK is dependent on both sphingosine 1-phosphate receptors and scavenger receptor class B type I through calcium/calmodulin-dependent protein kinase kinase and, for scavenger receptor class B type I system, additionally serine-threonine kinase LKB1 in human umbilical vein endothelial cells. HDL-induced activation of Akt and endothelial NO synthase, stimulation of migration, and inhibition of monocyte adhesion and adhesion molecule expression were dependent on AMPK activation. The inhibitory role of AMPK in the adhesion molecule expression and monocyte adhesion on endothelium of mouse aorta was confirmed in vivo and ex vivo. On the other hand, stimulation of ERK and proliferation were hardly affected by AMPK knockdown but completely inhibited by an N17Ras, whereas the dominant-negative Ras was ineffective for AMPK activation. In conclusion, dual HDL receptor systems differentially regulate AMPK activity through calcium/calmodulin-dependent protein kinase kinase and/or LKB1. Several HDL-induced antiatherogenic actions are regulated by AMPK, but proliferation-related actions are regulated by Ras rather than AMPK.

N-Glycosylation Regulates Fibroblast Growth Factor Receptor/EGL-15 Activity in Caenorhabditis elegans in Vivo

The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor.

Factor XI Binding to Platelets

Platelets are crucial in supporting coagulation reactions. The classical view is that platelets bind the vitamin K-dependent factors (VII, IX, X, II, protein C, S, and Z) via their gamma carboxyglutamic acid (gla) residues, and in this way localize coagulation factor complexes to the platelet membrane.1 For binding of gla-containing proteins to the platelet surface to occur, the platelet needs to be activated after which negatively charged phospholipids, including phosphatidylserine, are translocated from the inner to the outer leaflet of the platelet membrane. Exposure of negatively charged lipids enables a Ca2+-dependent interaction of gla-containing proteins with the negatively charged platelet membrane. See accompanying article on page 1602 In addition to the gla-containing coagulation factors, multiple coagulation factors lacking a gla-domain, including thrombin,2 factor XI(a),3 factor XII(a),4 and high-molecular-weight kininogen5 interact with platelets, and the interaction of all these proteins has been shown to be (in part) mediated by glycoprotein Ibα (GPIbα). Recent work has demonstrated that also some of the gla-containing coagulation factors, factor VII(a),6 factor IX(a),7 and both zymogen and activated protein C8 interact with GPIbα. These findings indicate that localization of coagulation factors to the platelet surface is much more complicated than anticipated by the model in which the interaction with negatively charged phospholipids was considered required and sufficient for gla-containing proteins to bind to platelets. Indeed, it has been demonstrated that the thrombin-generating capacity of activated platelets from different …

Neuroimmune crosstalk in asthma: Dual role of the neurotrophin receptor p75NTR

Neurotrophins have been implicated in the pathogenesis of asthma because of their ability to induce airway inflammation and to promote hyperreactivity of sensory neurons, which reflects an important mechanism in the pathogenesis of airway hyperreactivity. Neurotrophins use a dual-receptor system consisting of Trk-receptor tyrosine kinases and the structurally unrelated p75NTR. Previous studies revealed an important role of p75NTR in the pathogenesis of allergic asthma. The aim of the study was to investigate the precise mechanisms of neurotrophins in neuroimmune interaction, which can lead to both airway inflammation and sensory nerve hyperreactivity in vivo. Mice selectively expressing p75NTR in immune cells or nerves, respectively, were generated. After sensitization and allergen provocation, hyperreactivity of sensory nerves was tested in response to capsaicin. Airway inflammation was analyzed on the basis of differential cell counts and cytokine levels in bronchoalveolar lavage fluids. Allergic mice selectively expressing p75NTR in immune cells showed normal inflammation but no sensory nerve hyperreactivity, whereas mice selectively expressing p75NTR in nerve cells had a diminished inflammation and a distinct sensory nerve hyperreactivity. Our data indicate that p75NTR plays a dual role by promoting hyperreactivity of sensory nerves and airway inflammation. Additionally, our study provides experimental evidence that development of sensory nerve hyperreactivity depends on an established airway inflammation in asthma. In contrast, development of airway inflammation seems to be independent from sensory nerve hyperreactivity. Because of its dual function, antagonization of p75NTR-mediated signals might be a novel approach in asthma therapy.

N-Glycosylation of Fibroblast Growth Factor Receptor 1 Regulates Ligand and Heparan Sulfate Co-receptor Binding

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.

Pulmonary distribution, regulation, and functional role of Trk receptors in a murine model of asthma

Neurotrophins have been implicated in the pathogenesis of asthma because of their ability to promote hyperreactivity of sensory neurons and to induce airway inflammation. Hyperreactivity of sensory nerves is one key mechanism of airway hyperreactivity that is defined as an abnormal reactivity of the airways to unspecific stimuli, such as cold air and cigarette smoke. Neurotrophins use a dual-receptor system consisting of Trk receptor tyrosine kinases and the structurally unrelated p75 neurotrophin receptor. The aim of this study was to characterize the distribution, allergen-dependent regulation, and functional relevance of the Trk receptors in allergic asthma. BALB/c mice were sensitized to ovalbumin. After provocation with ovalbumin or vehicle aerosol, respectively, Trk receptor expression was analyzed in lung tissue by means of fluorescence microscopy and quantitative RT-PCR. To assess the functional relevance of Trk receptors in asthma, we tested the effects of the intranasally administered pan-Trk receptor decoy REN1826. Allergic airway inflammation was quantified and lung function was measured by using head-out body plethysmography. Trk receptors were expressed in neurons, airway smooth muscle cells, and cells of the inflammatory infiltrate surrounding the bronchi and upregulated after allergen challenge. Local application of REN1826 reduced IL-4 and IL-5 cytokine levels but had no effect on IL-13 levels or the cellular composition of bronchoalveolar lavage fluid cells. Furthermore, REN1826 decreased broncho-obstruction in response to sensory stimuli, indicating a diminished hyperreactivity of sensory nerves, but did not influence airway smooth muscle hyperreactivity in response to methacholine. These results emphasize the important role of Trk receptor signaling in the development of asthma. Our data indicate that blocking of Trk receptor signaling might reduce asthma symptoms.