血小板源生长因子受体与肿瘤

血小板源生长因子(platelet-derived growth factor,PDGF)经由其受体(platelet-derived growth fac tor receptor,PDGFR)表现细胞效应。PDGF和PDGFR涉及多种肿瘤的发病机制并在血管生成中起重要作用。PDGF在肿瘤中的自分泌刺激、PDGFR的过表达或过度活化或者刺激肿瘤内血管生成都会促进肿瘤生长;PDGFR的阻断可以降低实体瘤中组织间质液压而增强药物传送。这些机制可能提示在肿瘤治疗中PDGFR抑制剂单用、与化疗药物或者和其他靶点药物联合用药的可能性和可行性。随着PDGFR拮抗剂,如imatinib的上市,PDGFR作为抗肿瘤药物的靶点备受瞩目。     

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.     

Detection of exosomal tyrosine receptor kinase B as a potential biomarker in ovarian cancer

Ovarian cancer (OC) is a lethal disease diagnosed at advanced stages due to the lack of specific biomarkers. Tyrosine receptor kinase B (TrkB), which has recently been found to be related to OC progression, represents a promising potential biomarker for OC diagnosis and prognosis. The discovery of circulating exosomes as biomarkers for various diseases led us to explore exosomal TrkB in OC. Our previous study proved that the expression of TrkB was elevated in OC tissues. In this study, we focused on the detection of exosomal TrkB in OC. Exosomes were first gathered from three different OC cell lines’ conditioned medium, serum samples of patients with OC as well as xenograft mice serum by serial centrifugation method. Then, we identified exosomes by transmission electron microscopy, NanoSight analysis, and expression of typical exosomal protein markers. The existence of TrkB in exosomes was measured by Western blot analysis, and the expression was detected by enzyme-linked immunosorbent assay. In this study, we demonstrated that exosomes could derive from OC cell lines, serum from OC xenograft nude mice, and clinical patients. Our study shows that serum exosomal TrkB may be considered a minimally invasive biomarker for OC.     

Proteasomal inhibition alters the trafficking of the neurotrophin receptor TrkA

Neurotrophin receptors of the Trk family promote neuronal survival. The signal transduction of Trk receptors is regulated by endosomal trafficking. Monoubiquitination of receptor tyrosine kinases is an established signal for sorting of internalized receptors to late endosomes. The NGF receptor TrkA is sorted to late endosomes and undergoes ubiquitination, indicating a so far undefined regulatory role of proteasomal activity in the trafficking of TrkA. Surprisingly, we found that proteasomal inhibition alters the trafficking of TrkA from the late endosomal sorting pathway to the recycling pathway. Many neurodegenerative diseases are associated with impaired proteasomal activity. Thus, our study suggests that missorting of neurotrophic receptors might contribute to neuronal death in those neurodegenerative diseases that are known to be associated with impaired proteasomal function.     

Nerve growth factor receptor TrkA exists as a preformed, yet inactive, dimer in living cells

The tropomyosin-related kinase A (TrkA) receptor and its ligand, nerve growth factor (NGF), play crucial roles in the development and function of the nervous system. NGF is believed to activate TrkA by bridging two TrkA monomers, leading to TrkA transphosphorylation. However, here we show that the majority of TrkA receptors exist as preformed, yet inactive, homodimers prior to NGF binding by using three different approaches such as chemical crosslinking and enzyme fragment complementation assay. Furthermore, TrkA homodimers are formed in endoplasmic reticulum before newly synthesized receptors reach the cell surface. These findings shed light on molecular mechanisms underlying transmembrane signaling by TrkA.

Structured summary

TrkAphysically interacts with TrkA by protein complementation assay (View interaction)TrkAphysically interacts with TrkA by bimolecular fluorescence complementation (View interaction)TrkAphysically interacts with TrkA by cross-linking study (View interaction)     

The Xenopus platelet-derived growth factor α receptor: cDNA Cloning and demonstration that mesoderm induction establishes the lineage-specific pattern of ligand and receptor gene expression

We have cloned the Xenopus PDGF α receptor cDNA and have used this clone, along with cDNA encoding PDGF A, to examine their expression pattern in Xenopus embryos and to determine the factors responsible for lineage specificity. Recombinant Xenopus α receptor expressed in COS cells exhibits PDGF-A-dependent tyrosine kinase activity. We find that receptor mRNA is present in cultured marginal zone tissue explants and in animal cap tissue induced to form mesoderm either by grafting to vegetal tissue or by treatment with recombinant activin A. In contrast, PDGF A mRNA is expressed in cultured, untreated animal cap tissue and is suppressed by mesoderm induction. These results suggest that ectodermally produced PDGF A may act on the mesoderm during gastrulation and that mesoderm induction establishes the tissue pattern of ligand and receptor expression. © 1993Wiley-Liss, Inc.     

Caveolin is an inhibitor of platelet-derived growth factor receptor signaling

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived growth factor (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of fibroblasts. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 fibroblast cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating factor of PDGF-mediated tyrosine kinase signaling.     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.     

Inhibition of autophosphorylation of epidermal growth factor receptor by a small peptide not employing an ATP-competitive mechanism

Previously we found that short peptides surrounding major autophosphorylation sites of EGFR (VPEY(1068)INQ, DY(1148)QQD, and ENAEY(1173)LR) suppress phosphorylation of purified EGFR to 30-50% at 4000 microM. In an attempt to improve potencies of the peptides, we modified the sequences by substituting various amino acids for tyrosine or by substituting Gln and Asn for Glu and Asp, respectively. Among the modified peptides, Asp/Asn- and Glu/Gln-substitution in DYQQD (NYQQN) and ENAEYLR (QNAQYLR), respectively, improved inhibitory potencies. The inhibitory potency of NYQQN was not affected by the concentration of ATP, while that of QNAQYLR was affected. Docking simulations showed different mechanisms of inhibition for the peptides: inhibition by binding to the ATP-binding site (QNAQYLR) and inhibition by binding to a region surrounded by alphaC, the activation loop, and the catalytic loop and interfering with the catalytic reaction (NYQQN). The inhibitory potency of NYQQN for insulin receptor drastically decreased, whereas QNAQYLR inhibited autophosphorylation of insulin receptor as well as EGFR. In conclusion, NYQQN is not an ATP-competitive inhibitor and the binding site of this peptide appears to be novel as a tyrosine kinase inhibitor. NYQQN could be a promising seed for the development of anti-cancer drugs having specificity for EGFR.     

Hrs regulates the endocytic sorting of the fibroblast growth factor receptor 2b

The keratinocyte growth factor receptor or fibroblast growth factor receptor 2b (KGFR/FGFR2b) is activated by the specific interaction with the keratinocyte growth factor (KGF/FGF7), which targets the receptor to the degradative pathway, and the fibroblast growth factor 10 (FGF10/KGF2), which drives the receptor to the juxtanuclear recycling route. Hrs plays a key role in the regulation of the endocytic degradative transport of ubiquitinated receptor tyrosine kinases, but the direct involvement of this protein in the regulation of FGFR endocytosis has not been investigated yet. We investigated here the possible role of Hrs in the alternative endocytic pathways of KGFR. Quantitative immunofluorescence microscopy and biochemical analysis showed that both overexpression and siRNA interference of Hrs inhibit the KGF-triggered KGFR degradation, blocking receptor transport to lysosomes and causing its rapid reapparance at the plasma membrane. In contrast, the FGF10-induced KGFR targeting to the recycling compartment is not affected by Hrs overexpression or depletion. Coimmunoprecipitation approaches indicated that Hrs is recruited to KGFR only after KGF treatment, although it is not tyrosine phosphorylated by the ligand. In conclusion, Hrs regulates the KGFR degradative pathway, but not its juxtanuclear recycling transport. In addition, the results suggest that Hrs recruitment to the receptor, but not its ligand-induced phosphorylation, could be required for its function.     

Identification of ptpro as a novel target gene of Wnt signaling and its potential role as a receptor for Wnt

Wnt/β-catenin signaling plays critical roles in embryonic development and tissue homeostasis in adults by controlling the expression of target genes. We found that expression of ptpro, which encodes a protein tyrosine phosphatase receptor type O (PTPRO), was induced by Wnt/β-catenin signaling in a T cell factor/lymphoid enhancer factor dependent manner. Biochemical assays found that PTPRO interacted with Wnt via its extracellular domain. In addition, ectopic expression of this extracellular domain inhibited Wnt-mediated reporter activity. These results suggest that ptpro is a target gene of Wnt/β-catenin signaling and that PTPRO may function as a novel receptor for Wnt.

Structured summary

MINT-7992076: Ptpro (uniprotkb:Q7TSY7) physically interacts (MI:0915) with Wnt3a (uniprotkb:P27467) by anti tag coimmunoprecipitation (MI:0007)MINT-7992094: Ptpro (uniprotkb:Q7TSY7) physically interacts (MI:0915) with Wnt-3a (uniprotkb:P27467) by cross-linking study (MI:0030)     

Protease-activated receptor 1 as a potential therapeutic target for COVID-19

Acute respiratory disease caused by a novel coronavirus (SARS-CoV-2) has spread all over the world, since its discovery in 2019, Wuhan, China. This disease is called COVID-19 and already killed over 1 million people worldwide. The clinical symptoms include fever, dry cough, dyspnea, headache, dizziness, generalized weakness, vomiting, and diarrhea. Unfortunately, so far, there is no validated vaccine, and its management consists mainly of supportive care. Venous thrombosis and pulmonary embolism are highly prevalent in patients suffering from severe COVID-19. In fact, a prothrombotic state seems to be present in most fatal cases of the disease. SARS-CoV-2 leads to the production of proinflammatory cytokines, causing immune-mediated tissue damage, disruption of the endothelial barrier, and uncontrolled thrombogenesis. Thrombin is the key regulator of coagulation and fibrin formation. In severe COVID-19, a dysfunctional of physiological anticoagulant mechanisms leads to a progressive increase of thrombin activity, which is associated with acute respiratory distress syndrome development and a poor prognosis. Protease-activated receptor type 1 (PAR1) is the main thrombin receptor and may represent an essential link between coagulation and inflammation in the pathophysiology of COVID-19. In this review, we discuss the potential role of PAR1 inhibition and regulation in COVID-19 treatment.     

Insulin and epidermal growth factor receptor family members share parallel activation mechanisms

Insulin receptor (IR) and the epidermal growth factor receptor (EGFR) were the first receptor tyrosine kinases (RTKs) to be studied in detail. Both are important clinical targets—in diabetes and cancer, respectively. They have unique extracellular domain compositions among RTKs, but share a common module with two ligand‐binding leucine‐rich‐repeat (LRR)‐like domains connected by a flexible cysteine‐rich (CR) domain (L1‐CR‐L2 in IR/domain, I‐II‐III in EGFR). This module is linked to the transmembrane region by three fibronectin type III domains in IR, and by a second CR in EGFR. Despite sharing this conserved ligand‐binding module, IR and EGFR family members are considered mechanistically distinct—in part because IR is a disulfide‐linked (αβ)₂ dimer regardless of ligand binding, whereas EGFR is a monomer that undergoes ligand‐induced dimerization. Recent cryo‐electron microscopy (cryo‐EM) structures suggest a way of unifying IR and EGFR activation mechanisms and origins of negative cooperativity. In EGFR, ligand engages both LRRs in the ligand‐binding module, “closing” this module to break intramolecular autoinhibitory interactions and expose new dimerization sites for receptor activation. How insulin binds the activated IR was less clear until now. Insulin was known to associate with one LRR (L1), but recent cryo‐EM structures suggest that it also engages the second LRR (albeit indirectly) to “close” the L1‐CR‐L2 module, paralleling EGFR. This transition simultaneously breaks autoinhibitory interactions and creates new receptor‐receptor contacts—remodeling the IR dimer (rather than inducing dimerization per se) to activate it. Here, we develop this view in detail, drawing mechanistic links between IR and EGFR.     

Dopamine D2 receptor stimulation of mitogen-activated protein kinases mediated by cell type-dependent transactivation of receptor tyrosine kinases

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.     

Growth factor receptors in helminth parasites: signalling and host-parasite relationships

Parasitic helminths remain major pathogens of both humans and animals throughout the world. The success of helminth infections depends on the capacity of the parasite to counteract host immune responses but also to exploit host-derived signal molecules for its development. Recent progress has been made in the characterization of growth factor receptors of various nematode and flatworm parasites with the demonstration that transforming growth factor beta (TGF-beta), epidermal growth factor (EGF) and insulin receptor signalling pathways are conserved in helminth parasites and potentially implicated in the host-parasite molecular dialogue and parasite development.     

Epidermal growth factor receptor (EGFR) involvement in epithelial‐derived cancers and its current antibody‐based immunotherapies

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that is part of the family of tyrosine kinase receptors. The binding of EGFR to its cognate ligands leads to its autophosphorylation and subsequent activation of the signal transduction pathways involved in regulating cellular proliferation, differentiation, and survival. Accordingly, this receptor carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. Correspondingly, the loss of EGFR regulation results in many human diseases, with the most notable cancer. This receptor is overexpressed and/or mutated in multiple epithelial‐derived tumors, and associated with poor prognosis and survival in cancer patients. Here, we discuss in detail the role of EGFR in specific epithelial‐derived cancer pathologies; these include lung cancer, colorectal cancer, and squamous cell carcinomas. The development of multiple anticancer agents against EGFR diminished the progression and metastasis of tumors. Some of the most versatile therapeutic anti‐EGFR agents include the monoclonal antibodies (mAbs), demonstrating success in clinical settings when used in combination with cytotoxic treatments, such as chemotherapy and/or radiation. We thus discuss the development and application of two of the most notable therapeutic mAbs, cetuximab, and panitumumab, currently utilized in various EGFR‐related epithelial cancers.