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C F Ibáñez

Publications and source records attributed to C F Ibáñez.

At least 19 recordsLinked to original sources
PubMed2006

Self-association of the transmembrane domain of RET underlies oncogenic activation by MEN2A mutations.

In patients with medullary thyroid carcinoma (MTC) and type 2A multiple endocrine neoplasia (MEN2A), mutations of cysteine residues in the extracellular juxtamembrane region of the RET receptor tyrosine kinase cause the formation of covalent receptor dimers linked by intermolecular disulfide bonds between unpaired cysteines, followed by oncogenic activation of the RET kinase. The close proximity to the plasma membrane of the affected cysteine residues prompted us to investigate the possible role of the transmembrane (TM) domain of RET (RET-TM) in receptor-receptor interactions underlying dimer formation. Strong self-association of the RET-TM was observed in a biological membrane. Mutagenesis studies indicated the involvement of the evolutionary conserved residues Ser-649 and Ser-653 in RET-TM oligomerization. Unexpectedly, RET-TM interactions were also abrogated in the A639G/A641R double mutant, first identified in a sporadic case of MTC. In agreement with this, no transforming activity could be detected in full-length RET carrying the A639G and A641R mutations, which remained fully responsive to glial cell-line-derived neurotrophic factor (GDNF) stimulation. When introduced in the context of C634R - a cysteine replacement that is prevalent in MEN2A cases - the A639G/A641R mutations significantly reduced dimer formation and transforming activity in this otherwise highly oncogenic RET variant. These data suggest that a strong propensity to self-association in the RET-TM underlies - and may be required for - dimer formation and oncogenic activation of juxtamembrane cysteine mutants of RET, and explains the close proximity to the plasma membrane of cysteine residues implicated in MEN2A and MTC syndromes.

Alanine↗
PubMed2004

Recent evolutionary origin within the primate lineage of two pseudogenes with similarity to members of the transforming growth factor-beta superfamily.

Using a search engine called Motifer, we searched the public database of the human genome for genes matching a consensus pattern of cysteine residues derived from members of the transforming growth factor-beta (TGF-beta) superfamily. We identified two genes (named MDF451 and MDF628) that display sequence similarity to members of the TGF-beta superfamily in the arrangement of six conserved cysteine residues. Phylogenetic analyses revealed that MDF451 and MDF628 constitute a distinct subgroup within the TGF-beta superfamily, distantly related to the GDNF subfamily of ligands. Both genes could be identified in several primate species in addition to human, including chimpanzee, gorilla, guereza, and green and gray monkey, but not in rodents or other non-primate mammals, and appear not to be present in the genomes of mouse, rat or zebrafish. RNAs for MDF451 and MDF628 were expressed at low levels within distinct regions of the human central nervous system, including adult cerebellum, adult spinal cord and fetal brain. Despite expression at the RNA level, both genes presented a transcribed upstream stop codon that would prevent translation of the TGF-beta-like reading frame. The coding potential of alternative reading frames was not immediately apparent. The two genes may represent TGF-beta-like pseudogenes that have recently appeared in evolution in a common ancestor of the primate lineage by duplication from a GDNF/TGF-beta-like ancestral gene.

Amino Acid Sequence↗
PubMed2001

The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development.

Nodal proteins have crucial roles in mesendoderm formation and left-right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling.

Activin Receptors↗
PubMed2001

Molecular modeling of the extracellular domain of the RET receptor tyrosine kinase reveals multiple cadherin-like domains and a calcium-binding site.

Using bioinformatic tools, mutagenesis, and binding studies, we have investigated the structural organization of the extracellular region of the RET receptor tyrosine kinase, a functional receptor for glial cell line-derived neurotrophic factor (GDNF). Multiple sequence alignments of seven vertebrate sequences and one invertebrate RET sequence delineated four distinct N-terminal domains, each of about 110 residues, containing many of the consensus motifs of the cadherin fold. Based on these alignments and the crystal structures of epithelial and neural cadherins, we have generated molecular models of each of the four cadherin-like domains in the extracellular region of human RET. The modeled structures represent realistic models from both energetic and geometrical points of view and are consistent with previous observations gathered from biochemical analyses of the effects of Hirschsprung's disease mutations affecting the folding and stability of the RET molecule, as well as our own site-directed mutagenesis studies of RET cadherin-like domain 1. We have also investigated the role of Ca(2+) in ligand binding by RET and found that Ca(2+) ions are required for RET binding to GDNF but not for GDNF binding to the GFRalpha1 co-receptor. In agreement with these results, RET, but not GFRalpha1, was found to bind Ca(2+) directly. Our results indicate that the overall architecture of the extracellular region of RET is more closely related to cadherins than previously thought. The models of the cadherin-like domains of human RET represent valuable tools with which to guide future site-directed mutagenesis studies aimed at identifying residues involved in ligand binding and receptor activation.

Amino Acid Sequence↗
PubMed2001

Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.

Neurotrophin-4 (NT-4) is produced by slow muscle fibers in an activity-dependent manner and promotes growth and remodeling of adult motorneuron innervation. However, both muscle fibers and motor neurons express NT-4 receptors, suggesting bidirectional NT-4 signaling at the neuromuscular junction. Mice lacking NT-4 displayed enlarged and fragmented neuromuscular junctions with disassembled postsynaptic acetylcholine receptor (AChR) clusters, reduced AChR binding, and acetylcholinesterase activity. Electromyographic responses, posttetanic potentiation, and action potential amplitude were also significantly reduced in muscle fibers from NT-4 knock-out mice. Slow-twitch soleus muscles from these mice fatigued twice as rapidly as those from wild-type mice during repeated tetanic stimulation. Thus, muscle-derived NT-4 is required for maintenance of postsynaptic AChR regions, normal muscular electrophysiological responses, and resistance to muscle fatigue. This neurotrophin may therefore be a key component of an activity-dependent feedback mechanism regulating maintenance of neuromuscular connections and muscular performance.

Acetylcholinesterase↗
PubMed2001

Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts.

Although both c-Ret and GFRalpha1 are required for responsiveness to GDNF, GFRalpha1 is widely expressed in the absence of c-Ret, suggesting alternative roles for "ectopic" sites of GFRalpha1 expression. We show that GFRalpha1 is released by neuronal cells, Schwann cells, and injured sciatic nerve. c-Ret stimulation in trans by soluble or immobilized GFRalpha1 potentiates downstream signaling, neurite outgrowth, and neuronal survival, and elicits dramatic localized expansions of axons and growth cones. Soluble GFRalpha1 mediates robust recruitment of c-Ret to lipid rafts via a novel mechanism requiring the c-Ret tyrosine kinase. Activated c-Ret associates with different adaptor proteins inside and outside lipid rafts. These results provide an explanation for the tissue distribution of GFRalpha1, supporting the physiological importance of c-Ret activation in trans as a novel mechanism to potentiate and diversify the biological responses to GDNF.

Animals↗
PubMed2000

Mammalian GFRalpha -4, a divergent member of the GFRalpha family of coreceptors for glial cell line-derived neurotrophic factor family ligands, is a receptor for the neurotrophic factor persephin.

Four members of the glial cell line-derived neurotrophic factor family have been identified (GDNF, neurturin, persephin, and enovin/artemin). They bind to a specific membrane-anchored GDNF family receptor as follows: GFRalpha-1 for GDNF, GFRalpha-2 for neurturin, GFRalpha-3 for enovin/artemin, and (chicken) GFRalpha-4 for persephin. Subsequent signaling occurs through activation of a common transmembrane tyrosine kinase, cRET. GFRalpha-4, the coreceptor for persephin, was previously identified in chicken only. We describe the cloning and characterization of a mammalian persephin receptor GFRalpha-4. The novel GFRalpha receptor is substantially different in sequence from all known GFRalphas, including chicken GFRalpha-4, and lacks the first cysteine-rich domain present in all previously characterized GFRalphas. At least two different GFRalpha-4 splice variants exist in rat tissues, differing at their respective COOH termini. GFRalpha-4 mRNA is expressed at low levels in different brain areas in the adult as well as in some peripheral tissues including testis and heart. Recombinant rat GFRalpha-4 variants were expressed in mammalian cells and shown to be at least partially secreted from the cells. Persephin binds specifically and with high affinity (K(D) = 6 nm) to the rat GFRalpha-4 receptor, but no cRET activation could be demonstrated. Although the newly characterized mammalian GFRalpha-4 receptor is structurally divergent from previously characterized GFRalpha family members, we suggest that it is a mammalian orthologue of the chicken persephin receptor. This discovery will allow a more detailed investigation of the biological targets of persephin action and its potential involvement in diseases of the nervous system.

Alternative Splicing↗
PubMed2000

Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase.

Proximal signaling events and protein-protein interactions initiated after activation of the c-Ret receptor tyrosine kinase by its ligand, glial cell line-derived neurotrophic factor (GDNF), were investigated in cells carrying native and mutated forms of this receptor. Mutation of Tyr-1062 (Y1062F) in the cytoplasmic tail of c-Ret abolished receptor binding and phosphorylation of the adaptor Shc and eliminated activation of Ras by GDNF. Phosphorylation of Erk kinases was also greatly attenuated but not eliminated by this mutation. This residual wave of Erk phosphorylation was independent of the kinase activity of c-Ret. Mutation of Tyr-1096 (Y1096F), a binding site for the adaptor Grb2, had no effect on Erk activation by GDNF. Activation of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt was also reduced in the Y1062F mutant but not completely abolished unless Tyr-1096 was also mutated. Ligand stimulation of neuronal cells induced the assembly of a large protein complex containing c-Ret, Grb2, and tyrosine-phosphorylated forms of Shc, p85(PI3K), the adaptor Gab2, and the protein-tyrosine phosphatase SHP-2. In agreement with Ras-independent activation of PI3K by GDNF in neuronal cells, survival of sympathetic neurons induced by GDNF was dependent on PI3K but was not affected by microinjection of blocking anti-Ras antibodies, which did compromise neuronal survival by nerve growth factor, suggesting that Ras is not required for GDNF-induced survival of sympathetic neurons. These results indicate that upon ligand stimulation, at least two distinct protein complexes assemble on phosphorylated Tyr-1062 of c-Ret via Shc, one leading to activation of the Ras/Erk pathway through recruitment of Grb2/Sos and another to the PI3K/Akt pathway through recruitment of Grb2/Gab2 followed by p85(PI3K) and SHP-2. This latter complex can also assemble directly onto phosphorylated Tyr-1096, offering an alternative route to PI3K activation by GDNF.

3T3 Cells↗
PubMed2000

The orphan receptor serine/threonine kinase ALK7 signals arrest of proliferation and morphological differentiation in a neuronal cell line.

The signaling capabilities and biological functions of activin receptor-like kinase 7 (ALK7), a type I receptor serine/threonine kinase predominantly expressed in the nervous system, are unknown. We have constructed a cell line derived from the rat pheochromocytoma PC12 in which expression of a constitutively active mutant of ALK7 (T194D) is under the control of a tetracycline-inducible promoter. For comparison, another cell line was engineered with tetracycline-regulated expression of a constitutively active variant of the transforming growth factor-beta type I receptor ALK5. Expression of activated ALK7 in PC12 cells resulted in activation of Smad2 and Smad3, but not Smad1, as well as the mitogen-activated protein kinases extracellular signal-regulated kinase and c-Jun N-terminal kinase. Reporter assays demonstrated that ALK7 activation stimulates transcription from the Smad-binding element of the Jun-B gene, the plasminogen activator inhibitor-1 gene, and AP-1 elements. In addition, ALK7 activation induced expression of endogenous gene products, including Smad7, c-fos mRNA, and plasminogen activator inhibitor-1. Thymidine incorporation assays revealed an anti-proliferative effect of ALK7 activation in PC12 cells, which correlated with increased transcription from the promoters of cycline-dependent kinase inhibitors p15(INK4B) and p21. Unexpectedly, ALK7 signaling produced a remarkable change in cell morphology characterized by cell flattening and elaboration of blunt, short cell processes. Interestingly, no such changes were observed upon induction of activated ALK5. The alterations in cell morphology upon ALK7 activation were more pronounced in cultures grown in full serum, were accompanied by rearrangements of actin filaments, and were maintained for several days after withdrawal of treatment. PC12 cultures that had been "primed" in this way showed an accelerated and augmented differentiation response to nerve growth factor. These results indicate that ALK7 may participate in the control of proliferation of neuronal precursors and morphological differentiation of postmitotic neurons.

Actins↗
PubMed1999

Distinct structural elements in GDNF mediate binding to GFRalpha1 and activation of the GFRalpha1-c-Ret receptor complex.

Ligand-induced receptor oligomerization is a widely accepted mechanism for activation of cell-surface receptors. We investigated ligand-receptor interactions in the glial cell-line derived neurotrophic factor (GDNF) receptor complex, formed by the c-Ret receptor tyrosine kinase and the glycosylphosphatidylinositol (GPI)-anchored subunit GDNF family receptor alpha-1 (GFRalpha1). As only GFRalpha1 can bind GDNF directly, receptor complex formation is thought to be initiated by GDNF binding to this receptor. Here we identify an interface in GDNF formed by exposed acidic and hydrophobic residues that is critical for binding to GFRalpha1. Unexpectedly, several GDNF mutants deficient in GFRalpha1 binding retained the ability to bind and activate c-Ret at normal levels. Although impaired in binding GFRalpha1 efficiently, these mutants still required GFRalpha1 for c-Ret activation. These findings support a role for c-Ret in ligand binding and indicate that GDNF does not initiate receptor complex formation, but rather interacts with a pre-assembled GFRalpha1- c-Ret complex.

Animals↗
PubMed1999

Ret-dependent and -independent mechanisms of glial cell line-derived neurotrophic factor signaling in neuronal cells.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to signal through a multicomponent receptor complex consisting of the Ret receptor tyrosine kinase and a member of the GFRalpha family of glycosylphosphatidylinositol-anchored receptors. In the current model of GDNF signaling, Ret delivers the intracellular signal but cannot bind ligand on its own, while GFRalphas bind ligand but are thought not to signal in the absence of Ret. We have compared signaling pathways activated by GDNF in two neuronal cell lines expressing different complements of GDNF receptors. In a motorneuron-derived cell line expressing Ret and GFRalphas, GDNF stimulated sustained activation of the Ras/ERK and phosphatidylinositol 3-kinase/Akt pathways, cAMP response element-binding protein phosphorylation, and increased c-fos expression. Unexpectedly, GDNF also promoted biochemical and biological responses in a line of conditionally immortalized neuronal precursors that express high levels of GFRalphas but not Ret. GDNF treatment did not activate the Ras/ERK pathway in these cells, but stimulated a GFRalpha1-associated Src-like kinase activity in detergent-insoluble membrane compartments, rapid phosphorylation of cAMP response element-binding protein, up-regulation of c-fos mRNA, and cell survival. Together, these results offer new insights into the dynamics of GDNF signaling in neuronal cells, and indicate the existence of novel signaling mechanisms directly or indirectly mediated by GFRalpha receptors acting in a cell-autonomous manner independently of Ret.

Animals↗
PubMed1999

Role of brain-derived neurotrophic factor in target invasion in the gustatory system.

Brain-derived neurotrophic factor (BDNF) is a survival factor for different classes of neurons, including gustatory neurons. We have studied innervation and development of the gustatory system in transgenic mice overexpressing BDNF under the control of regulatory sequences from the nestin gene, an intermediate filament gene expressed in precursor cells of the developing nervous system and muscle. In transgenic mice, the number and size of gustatory papillae were decreased, circumvallate papillae had a deranged morphology, and there was also a severe loss of lingual taste buds. Paradoxically, similar deficits have been found in BDNF knock-out mice, which lack gustatory neurons. However, the number of neurons in gustatory ganglia was increased in BDNF-overproducing mice. Although gustatory fibers reached the tongue in normal numbers, the amount and density of nerve fibers in gustatory papillae were reduced in transgenic mice compared with wild-type littermates. Gustatory fibers appeared stalled at the base of the tongue, a site of ectopic BDNF expression, where they formed abnormal branches and sprouts. Interestingly, palatal taste buds, which are innervated by gustatory neurons whose afferents do not traverse sites of ectopic BDNF expression, appeared unaffected. We suggest that lingual gustatory deficits in BDNF overexpressing mice are a consequence of the failure of their BDNF-dependent afferents to reach their targets because of the effects of ectopically expressed BDNF on fiber growth. Our findings suggest that mammalian taste buds and gustatory papillae require proper BDNF-dependent gustatory innervation for development and that the correct spatial expression of BDNF in the tongue epithelium is crucial for appropriate target invasion and innervation.

Animals↗
PubMed1999

Novel, testis-specific mRNA transcripts encoding N-terminally truncated choline acetyltransferase.

Previous studies reported the presence of choline acetyltransferase (ChAT) mRNA and protein in the mammalian testis. We have now found that none of the ChAT mRNAs produced in the testis is capable of encoding a full-length ChAT protein. Two ChAT cDNAs were isolated from an adult rat testis cDNA library encoding N-terminally truncated ChAT proteins of 450 and 414 amino acids (aa), respectively, the former containing a novel N-terminal extension of 69 residues. Rapid Amplification of cDNA Ends (RACE) analysis revealed a complex pattern of 5' untranslated mRNA termini generated from the ChAT gene locus in the testis, all representing truncated versions of the ChAT enzyme. Two of these proteins were produced in transfected fibroblasts and found to lack ChAT activity. Neither did they show binding to the ChAT substrates, acetyl CoA and choline, in a competition assay. These results indicate that mammalian testis lacks a bona fide ChAT enzyme but expresses truncated ChAT proteins with a possible unique function to the testis.

5' Untranslated Regions↗
PubMed1998

Early evolutionary origin of the neurotrophin receptor family.

Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system, but to date no component of this signalling system has been found in invertebrates. We describe a molluscan Trk receptor, designated Ltrk, from the snail Lymnaea stagnalis. The full-length sequence of Ltrk reveals most of the characteristics typical of Trk receptors, including highly conserved transmembrane and intracellular tyrosine kinase domains, and a typical extracellular domain of leucine-rich motifs flanked by cysteine clusters. In addition, Ltrk has a unique N-terminal extension and lacks immunoglobulin-like domains. Ltrk is expressed during development in a stage-specific manner, and also in the adult, where its expression is confined to the central nervous system and its associated endocrine tissues. Ltrk has the highest sequence identity with the TrkC mammalian receptor and, when exogenously expressed in fibroblasts or COS cells, binds human NT-3, but not NGF or BDNF, with an affinity of 2.5 nM. These findings support an early evolutionary origin of the Trk family as neuronal receptor tyrosine kinases and suggest that Trk signalling mechanisms may be highly conserved between vertebrates and invertebrates.

Amino Acid Sequence↗
PubMed1998

Targeted expression of a multifunctional chimeric neurotrophin in the lesioned sciatic nerve accelerates regeneration of sensory and motor axons.

Peripheral nerve injury markedly regulates expression of neurotrophins and their receptors in the lesioned nerve. However, the role of endogenously produced neurotrophins in the process of nerve regeneration is unclear. Expression of a multifunctional neurotrophin, pan-neurotrophin-1 (PNT-1), was targeted to the peripheral nerves of transgenic mice by using a gene promoter that is specifically activated after nerve lesion but that is otherwise silent in all other tissues and during development. PNT-1 is a chimeric neurotrophin that combines the active sites of the neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 and binds and activates all known neurotrophin receptors. In adult transgenic mice, PNT-1 was highly expressed in transected but not in intact sciatic nerve. Morphometric analyses at the electron microscopy level showed increased and accelerated recovery of axon diameter of myelinated fibers in crushed peripheral nerves of transgenic mice compared with wild type. Examination of nerve bundles in target tissues indicated accelerated reinnervation of foot pad dermis and flexor plantaris muscle in transgenic mice. Moreover, transected sensory and motor axons of transgenic mice showed faster and increased return of neurophysiological responses, suggesting an accelerated rate of axonal elongation. Importantly, transgenic mice also showed a markedly ameliorated loss of skeletal muscle weight, indicating functional regeneration of motor axons. Together, these data provide evidence, at both the anatomical and functional levels, that neurotrophins endogenously produced by the lesioned nerve are capable of significantly accelerating the regeneration of both sensory and motor axons after peripheral nerve damage. In addition, our results indicate that exogenous PNT-1 administration may be an effective therapeutic treatment of peripheral nerve injuries.

Animals↗
PubMed1998

Neurotrophin-7: a novel member of the neurotrophin family from the zebrafish.

A novel member of the neurotrophin family, zebrafish neurotrophin-7 (zNT-7), was isolated from the zebrafish Danio rerio. The amino acid sequence of zNT-7 is more closely related to that of fish nerve growth factor (NGF) and neurotrophin-6 (NT-6) than to that of any other neurotrophin. zNT-7 is, however, equally related to fish NGF and NT-6 (65% and 63% amino acid sequence identity, respectively) indicating that it represents a distinct neurotrophin sequence. zNT-7 contains a 15 amino acid residue insertion in a beta-turn region in the middle of the mature protein. Recombinant zNT-7 was able to bind to the human p75 neurotrophin receptor and to induce tyrosine phosphorylation of the rat TrkA receptor tyrosine kinase, albeit less efficiently than rat NGF. zNT-7 did not interact with rat TrkB or TrkC, indicating a similar receptor specificity as NGF. We propose that a diversification of the NGF subfamily in the neurotrophin evolutionary tree occurred during the evolution of teleost fishes which resulted in the appearance of several additional members, such as zNT-7 and NT-6, structurally and functionally related to NGF.

Amino Acid Sequence↗