The structure of GFRalpha1 domain 3 reveals new insights into GDNF binding and RET activation

Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.     

Evidence of both extra- and intracellular cysteine targets of protein modification for activation of RET kinase

By use of a specifically sulfhydryl group-reactive chemical, 1,4-butanediyl-bismethanethiosulfonate (BMTS), we studied the localization of oxidative stress-responsive target cysteines for activation of a receptor-type protein tyrosine kinase, c-RET. The chemical, which reacted with RET proteins on the cell surface for sulfhydryl-linked aggregation, induced autophosphorylation and activation of RET kinase. When extracellular domain-deleted RET mutant (RET-PTC-1) cells were exposed to BMTS, neither the molecular status nor the activity of the enzyme was affected, suggesting that the target cysteines of BMTS to which cells were exposed for reaction are located in the cysteine-rich region of the extracellular domain of RET kinase. Despite this result, the exposure of a subcellular form of c-RET or RET-PTC-1 kinase isolated by immunoprecipitation to BMTS did induce activation of the enzyme. These results suggest that cysteines in both the extracellular and the intracellular domains of RET can work as target sites of accessible BMTS and possibly other oxidative elements for structural modification and activation of RET kinase.     

Platelet-derived growth factors and their receptors: Structural and functional perspectives

The four types of platelet-derived growth factors (PDGFs) and the two types of PDGF receptors (PDGFRs, which belong to class III receptor tyrosine kinases) have important functions in the development of connective tissue cells. Recent structural studies have revealed novel mechanisms of PDGFs in propeptide loading and receptor recognition/activation. The detailed structural understanding of PDGF–PDGFR signaling has provided a template that can aid therapeutic intervention to counteract the aberrant signaling of this normally silent pathway, especially in proliferative diseases such as cancer. This review summarizes the advances in the PDGF system with a focus on relating the structural and functional understandings, and discusses the basic aspects of PDGFs and PDGFRs, the mechanisms of activation, and the insights into the therapeutic antagonism of PDGFRs. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Hydrogen peroxide induces Src family tyrosine kinase-dependent activation of Xenopus eggs

Fertilization is accompanied by a rapid and transient calcium release in eggs, which is required for the onset of zygotic developmental program or 'egg activation'. Recently, it was found that Src family tyrosine kinase (SFK)-dependent phospholipase C (PLC) activity is necessary for the calcium transience in fertilized Xenopus eggs. The present study demonstrates that hydrogen peroxide (H2O2) stimulates protein-tyrosine phosphorylation in Xenopus eggs, which occurs primarily in the egg cortex of the animal hemisphere as revealed by indirect immunofluorescence study. Egg SFK was found to be upregulated by H2O2 while the SFK-specific inhibitor PP1 effectively blocked H2O2-induced tyrosine phosphorylation. As in fertilized eggs, PLCgamma, but not Shc, was tyrosine-phosphorylated in H2O2-treated eggs. H2O2 also caused inositol 1,4,5-trisphosphate (IP3) production and sustained calcium release. After limited application of H2O2, elevated SFK activity and tyrosine phosphorylation were quickly reversed. Under such conditions, eggs showed cortical contraction and dephosphorylation of p42 MAP kinase, both of which are indicative of egg activation. These egg activation events, as well as H2O2-induced IP3 production and calcium release, were sensitive to PP1 and PLC inhibitor U-73122. Together, the present study demonstrated that H2O2 can mimic, at least in part, early events of Xenopus egg activation that require an SFK-dependent PLC pathway.     

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing

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Lyso-GM3, its dimer,and multimer: their synthesis,and their effect on epidermal growth factor-induced receptor tyrosine kinase

Glycosphingolipids, particularly gangliosides, are known to modulate growth factor receptor tyrosine kinase. A well-documented example is the inhibitory effect of GM3 on kinase associated with epidermal growth factor receptor (EGFR) in human epidermoid carcinoma A431 cells. Lyso-GM3 was detected as a minor component in A431 cells, and may function as an auxiliary factor in GM3-dependent inhibition of EGFR. We studied the inhibitory effect of chemically synthesized GM3, lyso-GM3, and its derivatives, on EGFR function, based on their interaction in membrane microdomain, with the following major findings: (1) GM3, EGFR, and caveolin coexist, but tetraspanins CD9 and CD82 are essentially absent, within the same low-density membrane fraction, separated by sucrose density gradient ultracentrifugation. (2) Strong interaction between EGFR and GM3 was indicated by increasing binding of EGFR to GM3-coated polystyrene beads, in a GM3 dose-dependent manner. Confocal microscopy results suggested that three components in the microdomain (GM3, EGFR, and caveolin) are closely associated. (3) Lyso-GM3 or lyso-GM3 dimer strongly inhibited EGFR kinase activity, in a dose-dependent manner, while lyso-GM3 trimer and tetramer did not. >50 μM lyso-GM3 was cytolytic, while >50 μM lyso-GM3 dimer was not cytolytic, yet inhibited EGFR kinase strongly. Thus, lyso-GM3 and its dimer exert an auxiliary effect on GM3-induced inhibition of EGFR kinase and cell growth, and lyso-GM3 dimer may be a good candidate for pharmacological inhibitor of epidermal tumor growth.     

Molecular evolution of the genes encoding receptor tyrosine kinase with immunoglobulinlike domains

Receptor tyrosine kinases (RTK) with five, three, or seven immunoglobulinlike domains in their extracellular regions are classified as subclasses III, IV, and V, respectively. Conservation of the exon/intron structure of the downstream part of the human KIT, FMS, and FLT3 genes that encode RTK of subclass III together with the particular chromosomal localization of these genes suggests that RTKIII genes have evolved from a common ancestor by cis and trans duplications. To strengthen this model of evolution and to determine if it can be extended to RTKIV and V genes, we constructed a phylogenetic tree of RTKIII, IV, and V on the basis of a multiple alignment of their catalytic tyrosine kinase domain sequences and determined the exon/intron structure of PDGFRA (subclass III), FGFR4 (subclass IV), and FLT4 (subclass V) genes in their downstream part. Phylogenetic analyses with amino acid or nucleotide sequences both resulted in one most parsimonious tree. The phylogenetic trees obtained indicate that all three subclasses are well individuated and that RTKIII and RTKV are closer to each other than RTKIV. Furthermore, RTKIII and FLT4 (subclass V) genes possess the same exon/intron structure in their downstream part while the structure of the RTKIV genes is very similar to that of RTKIII and FLT4. Both approaches are in complete agreement and indicate that RTKIII, IV, and V genes most probably evolved from a common ancestor already in pieces by successive duplications involving entire genes.Correspondence to: F. Agnès     

Design,synthesis, and biological evaluation of novel 3-pyrrolo[b]cyclohexylene-2-dihydroindolinone derivatives as potent receptor tyrosine kinase inhibitors

A novel series of 3-pyrrolo[b]cyclohexylene-2-dihydroindolinone derivatives targeting VEGFR-2, PDGFR-β and c-Kit kinases were designed and synthesized. The molecular design was based on the SAR features of indolin-2-ones as kinase inhibitors. SAR study of the series allowed us to identify compounds possessing more potent inhibitory activities against the three kinases than sunitinb with IC₅₀ values in the low nanomolar range in vitro. Additionally, some compounds also showed favorable antiproliferative activities against a panel of cancer cell lines (BXPC-3, T24, BGC, HEPG2 and HT29).     

Selection and growth regulation of genetically modified cells with hapten‐specific antibody/receptor tyrosine kinase chimera

Although receptor tyrosine kinases (RTKs) play a pivotal role in the development and maintaining the homeostasis of the body, overexpression or mutation of RTKs often induces tumorigenesis or metastasis. To mimic the function of RTKs, we developed two fusion receptors consisting of anti‐fluorescein antibody single‐chain Fv, extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of epidermal growth factor receptor or c‐fms based on previously constructed antibody/cytokine receptor chimeras. The expression of these chimeric receptors in the hematopoietic cell line Ba/F3 and non‐hematopoietic cell line NIH/3T3 resulted in the activation of receptors themselves, downstream signaling molecules and cell proliferation in response to fluorescein‐conjugated BSA, leading to selective expansion of transduced cells up to almost 100%. These results indicate that the cognate antigen could activate the chimeric receptors even though the wild‐type extracellular domains were switched to the antibody fragment. This is the first study to show that our antigen‐mediated genetically modified cell amplification (AMEGA) system could be applied to non‐hematopoietic cells by utilizing antibody/RTK chimeras. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Design,synthesis and structure-activity relationship of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure as multi-target inhibitors against receptor tyrosine kinase

Inspired by that the multi-target inhibitors against receptor tyrosine kinases (RTKs) have significantly improved the effect of clinical treatment for cancer, and based on the chemical structure of Linifanib (ABT-869, Abbott), two series of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure were designed and synthesized as multi-target inhibitors against RTKs. The preliminary biological evaluation showed that several compounds exhibited comparable potency with Linifanib. Compound S21 was identified as the most potent inhibitor against Fms-like tyrosine kinase 3 (FLT-3), kinase insert domain containing receptor (KDR) and platelet-derived growth factor receptor β (PDGFR-β) with its IC₅₀ values were 4?nM, 3?nM and 8?nM respectively, it also showed potent inhibitory activities against several cancer cells.     

Structure-activity relationship studies on a series of piperazinebenzylalcohols and their ketone and amine analogs as melanocortin-4 receptor ligands

A series of piperazinebenzylalcohols were prepared and studied to compare with their ketone and amine analogs as MC4R antagonists. Several benzylalcohols such as 14a and 14g displayed low nanomolar binding affinities (K_i < 10 nM), and high selectivities over other melanocortin receptor subtypes.     

Heat stress induces tyrosine phosphorylation/activation of kinase Fa/GSK-3α (a human carcinoma dedifferentiation modulator) in A431 cells

Exposure of A431 cells to a rapid temperature increase from 37° to 46°C could induce an increased expression (∼200% of control) and tyrosine phosphorylation/activation (∼300% of control) of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) in a time-dependent manner, as demonstrated by an anti-kinase FA/GSK-3α immunoprecipitate kinase assay and by immunoblotting analysis with anti-kinase FA/GSK-3α and anti-phosphotyrosine antibodies. The heat induction on the increased expression of kinase FA/GSK-3α could be blocked by actinomycin D but not by genistein. In contrast, the heat induction on tyrosine phosphorylation/activation of kinase FA/GSK-3α could be blocked by genistein or protein tyrosine phosphatase, indicating that heat stress induces a dual control mechanism, namely, protein expression and subsequent tyrosine phosphorylation to cause cellular activation of kinase FA/GSK-3α. Taken together, the results provide initial evidence that kinase FA/GSK-3α represents a newly described heat stress–inducible protein subjected to tyrosine phosphorylation/activation, representing a new mode of signal transduction for the regulation of this human carcinoma dedifferentiation modulator and a new mode of heat induction on cascade activation of a protein kinase. J. Cell. Biochem. 66:16–26, 1997. © 1997 Wiley-Liss, Inc.     

Reduced NMDA receptor tyrosine phosphorylation in PTPalpha-deficient mouse synaptosomes is accompanied by inhibition of four src family kinases and Pyk2: an upstream role for PTPalpha in NMDA receptor regulation

Mice lacking protein tyrosine phosphatase alpha (PTPalpha) exhibited defects in NMDA receptor (NMDAR)-associated processes such as learning and memory, hippocampal neuron migration, and CA1 hippocampal long-term potentiation (LTP). In vivo molecular effectors linking PTPalpha and the NMDAR have not been reported. Thus the involvement of PTPalpha as an upstream regulator of NMDAR tyrosine phosphorylation was investigated in synaptosomes of wild-type and PTPalpha-null mice. Tyrosine phosphorylation of the NMDAR NR2A and NR2B subunits was reduced upon PTPalpha ablation, indicating a positive effect of this phosphatase on NMDAR phosphorylation via intermediate molecules. The NMDAR is a substrate of src family tyrosine kinases, and reduced activity of src, fyn, yes and lck, but not lyn, was apparent in the absence of PTPalpha. In addition, autophosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a tyrosine kinase linked to NMDAR signaling, was also reduced in PTPalpha-deficient synaptosomes. Altered protein tyrosine phosphorylation was not accompanied by altered expression of the NMDAR or the above tyrosine kinases at any stage of PTPalpha-null mouse development examined. In a human embryonic kidney (HEK) 293 cell expression system, PTPalpha enhanced fyn-mediated NR2A and NR2B tyrosine phosphorylation by several-fold. Together, these findings provide evidence that aberrant NMDAR-associated functions in PTPalpha-null mice are due to impaired NMDAR tyrosine phosphorylation resulting from the reduced activity of probably more than one of the src family kinases src, fyn, yes and lck. Defective NMDAR activity in these mice may also be linked to the loss of PTPalpha as an upstream regulator of Pyk2.     

Crystal structure analysis and solution studies of human Lck-SH3; zinc-induced homodimerization competes with the binding of proline-rich motifs

In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 A. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn(2+) dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.     

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.     

Exploring electrostatic interactions of relaxin family peptide receptor 3 and 4 with ligands using a NanoBiT-based binding assay

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely relaxin family peptide receptor 1-4 (RXFP1-4). We recently disclosed electrostatic interactions of the homologous RXFP3 and RXFP4 with some agonists based on activation complementation. However, this activation assay-based approach cannot be applied to antagonists that do not activate receptors. Herein, we propose a general approach suitable for both agonists and antagonists based on our newly-developed NanoBiT-based binding assay. We first validated the binding assay-based approach using the agonist relaxin-3, then applied it to the chimeric antagonist R3(ΔB23-27)R/I5. Three positively charged B-chain Arg residues of the agonist and antagonist were respectively replaced by a negatively charged Glu residue; meanwhile, the negatively charged Glu and Asp residue in the essential WxxExxxD motif of both receptors were respectively replaced by a positively charged Arg residue. Based on binding complementation of mutant ligands towards mutant receptors, we deduced possible electrostatic interactions of the agonist and antagonist with both RXFP3 and RXFP4: their B-chain C-terminal Arg residue interacts with the deeply buried Glu residue in the WxxExxxD motif of both receptors, and one or two of their B-chain central Arg residues interact with the shallowly buried Asp residue in the WxxExxxD motif of both receptors. Our present work shed new light on the interaction mechanism of RXFP3 and RXFP4 with agonists and antagonists, and also provided a novel approach for interaction studies of some plasma membrane receptors with their ligands.     

Interaction of Axl receptor tyrosine kinase with C1-TEN,a novel C1 domain-containing protein with homology to tensin

Axl receptor tyrosine kinase is implicated in several malignancies and is the receptor for the vitamin K-dependent growth factor Gas6. From a yeast two-hybrid screen of protein-protein interactions with the Axl cytoplasmic domain, we detected a previously uncharacterised SH2 domain-containing protein. We cloned two novel splice variants of this protein that give rise to 1409- and 1419-amino acid proteins, differing only in their N-terminal residues and yielding a 150-kDa protein product by in vitro translation. The Axl-interacting C-terminus contains a tandem SH2 and PTB domain combination homologous to the focal adhesion protein tensin. We detected interaction of Axl with both domains in mammalian cells by co-immunoprecipitation and two-hybrid analyses. In addition, the protein possesses an N-terminal putative phorbol ester-binding C1 domain as well as a central tyrosine phosphatase motif. Thus, we have named the protein C1 domain-containing phosphatase and TENsin homologue (C1-TEN). Northern blot analysis of C1-TEN in human tissues revealed highest expression in heart, kidney, and liver. In summary, we have identified a novel multi-domain intracellular protein that interacts with Axl and which may furthermore be involved in other signal transduction pathways.     

Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexes with the GluR2 ligand-binding core

Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid (Br-HIBO), as well as of a mutant thereof (S1S2J-Y702F) in complex with ACPA and Br-HIBO, have been determined. The structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined with the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists.     

In vivo evidence for a regulatory role of the kinase activity of the <Emphasis Type="Italic">linotte</Emphasis>/<Emphasis Type="Italic">derailed</Emphasis> receptor tyrosine kinase,a <Emphasis Type="Italic">Drosophila</Emphasis> Ryk ortholog

The RYK subfamily of receptor tyrosine kinases is characterised by unusual, but highly conserved, amino acid substitutions in the kinase domain. The linotte/derailed gene encodes a Drosophila RYK subfamily member involved in embryonic and adult central nervous system development. Previous studies have shown that the kinase activity of this receptor is not required in vivo for its embryonic function. In this study, we have investigated the role of the cytoplasmic domain and the kinase activity of the linotte/derailed receptor tyrosine kinase in adult brain development. Our results indicate that these domains are not essential for adult brain development but they are required for the proper regulation of the activity of this receptor. This sheds light on a regulatory role for the kinase activity of a RYK subfamily member.Edited by C DesplanEmmanuel Taillebourg and Caroline Moreau-Fauvarque contributed equally to this work     

Genomic organization of the extracellular coding region of the human FGFR4 and FLT4 genes: Evolution of the genes encoding receptor tyrosine kinases with immunoglobulin-like domains

Receptor tyrosine kinases with five, seven, and three Ig-like domains in their extracellular region are grouped in subclasses IIIa, IIIb, and IIIc, respectively. Here, we describe the genomic organization of the extracellular coding region of the human FGFR4 (IIIc) and FLT4 (IIIb) genes and compare it to that of the human FGFR1(IIIc), KIT, and FMS (IIIa). The results show that while genes belonging to the same subclass have an identical exon/intron structure in their extracellular coding region—as they do in their intracellular coding region—genes of related subclasses only have a similar exon/intron structure. These results strongly support the hypothesis that the genes of the three subclasses evolved from a common ancestor by duplications involving entire genes, already in pieces. Hypotheses on the origin of introns and on the difference in the number of extracellular Ig-like domains in the three gene subclasses are discussed. Received: 19 August 1996 / Accepted: 2 January 1997