RESUMEN
The human insulin receptor signalling system plays a critical role in glucose homeostasis. Insulin binding brings about extensive conformational change in the receptor extracellular region that in turn effects trans-activation of the intracellular tyrosine kinase domains and downstream signalling. Of particular therapeutic interest is whether insulin receptor signalling can be replicated by molecules other than insulin. Here, we present single-particle cryoEM structures that show how a 33-mer polypeptide unrelated to insulin can cross-link two sites on the receptor surface and direct the receptor into a signalling-active conformation. The 33-mer polypeptide engages the receptor by two helical binding motifs that are each potentially mimicable by small molecules. The resultant conformation of the receptor is distinct from-but related to-those in extant three-dimensional structures of the insulin-complexed receptor. Our findings thus illuminate unexplored pathways for controlling the signalling of the insulin receptor as well as opportunities for development of insulin mimetics.
Asunto(s)
Insulina , Receptor de Insulina , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Fosforilación , Receptor de Insulina/metabolismo , Transducción de SeñalRESUMEN
Human type 1 insulin-like growth factor receptor is a homodimeric receptor tyrosine kinase that signals into pathways directing normal cellular growth, differentiation and proliferation, with aberrant signalling implicated in cancer. Insulin-like growth factor binding is understood to relax conformational restraints within the homodimer, initiating transphosphorylation of the tyrosine kinase domains. However, no three-dimensional structures exist for the receptor ectodomain to inform atomic-level understanding of these events. Here, we present crystal structures of the ectodomain in apo form and in complex with insulin-like growth factor I, the latter obtained by crystal soaking. These structures not only provide a wealth of detail of the growth factor interaction with the receptor's primary ligand-binding site but also indicate that ligand binding separates receptor domains by a mechanism of induced fit. Our findings are of importance to the design of agents targeting IGF-1R and its partner protein, the human insulin receptor.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/química , Receptores de Somatomedina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Cricetulus , Cristalografía por Rayos X , Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Cinética , Ligandos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMEN
BACKGROUND: Basal insulin peglispro (BIL) is a novel, PEGylated insulin lispro that has a large hydrodynamic size compared with insulin lispro. It has a prolonged duration of action, which is related to a delay in insulin absorption and a reduction in clearance. Given the different physical properties of BIL compared with native insulin and insulin lispro, it is important to assess the cellular internalization characteristics of the molecule. METHODS AND MATERIALS: Using immunofluorescent confocal imaging, we compared the cellular internalization and localization patterns of BIL, biosynthetic human insulin, and insulin lispro. We assessed the effects of BIL on internalization of the insulin receptor (IR) and studied cellular clearance of BIL. RESULTS: Co-localization studies using antibodies to either insulin or PEG, and the early endosomal marker EEA1 showed that the overall internalization and subcellular localization pattern of BIL was similar to that of human insulin and insulin lispro; all were rapidly internalized and co-localized with EEA1. During ligand washout for 4 h, concomitant loss of insulin, PEG methoxy group, and PEG backbone immunostaining was observed for BIL, similar to the loss of insulin immunostaining observed for insulin lispro and human insulin. Co-localization studies using an antibody to the lysosomal marker LAMP1 did not reveal evidence of lysosomal localization for insulin lispro, human insulin, BIL, or PEG using either insulin or PEG immunostaining reagents. BIL and human insulin both induced rapid phosphorylation and internalization of human IR. CONCLUSIONS: Our findings show that treatment of cells with BIL stimulates internalization and localization of IR to early endosomes. Both the insulin and PEG moieties of BIL undergo a dynamic cellular process of rapid internalization and transport to early endosomes followed by loss of cellular immunostaining in a manner similar to that of insulin lispro and human insulin. The rate of clearance for the insulin lispro portion of BIL was slower than the rate of clearance for human insulin. In contrast, the PEG moiety of BIL can recycle out of cells.
Asunto(s)
Endocitosis , Insulina Lispro/metabolismo , Línea Celular , Membrana Celular/metabolismo , Endosomas/metabolismo , Humanos , Ligandos , Lisosomas/metabolismo , Fosforilación , Receptor de Insulina/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE(-/-) mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Insulina/metabolismo , Insulisina/antagonistas & inhibidores , Animales , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacocinética , Células HEK293 , Humanos , Insulisina/química , Modelos Moleculares , ProteolisisRESUMEN
Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B'29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1-10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints ((3)H-thymidine incorporation), and not on metabolic endpoints ((14)C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity ((3)H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B'29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.
Asunto(s)
Insulina/metabolismo , Péptidos/farmacología , Receptor de Insulina/agonistas , Receptor de Insulina/antagonistas & inhibidores , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células CHO , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Femenino , Glucosa/metabolismo , Humanos , Insulina/química , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
More than 20 years after the description of the two IR (insulin receptor) isoforms, designated IR-A (lacking exon 11) and IR-B (with exon 11), nearly every functional aspect of the alternative splicing both in vitro and in vivo remains controversial. In particular, there is no consensus on the precise ligand-binding properties of the isoforms. Increased affinity and dissociation kinetics have been reported for IR-A in comparison with IR-B, but the opposite results have also been reported. These are not trivial issues considering the reported possible increased mitogenic potency of IR-A, and the reported link between slower dissociation and increased mitogenesis. We have re-examined the ligand-binding properties of the two isoforms using a novel rigorous mathematical analysis based on the concept of a harmonic oscillator. We found that insulin has 1.5-fold higher apparent affinity towards IR-A and a 2-fold higher overall dissociation rate. Analysis based on the model showed increased association (3-fold) and dissociation (2-fold) rate constants for binding site 1 of IR in comparison with IR-B. We also provide a structural interpretation of these findings on the basis of the structure of the IR ectodomain and the proximity of the sequence encoded by exon 11 to the C-terminal peptide that is a critical trans-component of site 1.
Asunto(s)
Insulina/farmacología , Receptor de Insulina/agonistas , Receptor de Insulina/metabolismo , Empalme Alternativo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Células CHO , Cricetinae , Humanos , Isoenzimas , Cinética , Modelos Moleculares , Unión Proteica , Receptor de Insulina/química , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ß-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ß-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.
Asunto(s)
Secuencia Conservada , Drosophila melanogaster , Evolución Molecular , Insulina/química , Insulina/metabolismo , Proteínas/química , Proteínas/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Secuencia de Aminoácidos , Animales , Glucemia/metabolismo , Cristalografía por Rayos X , Femenino , Humanos , Insulina/farmacología , Radioisótopos de Yodo , Lipogénesis/efectos de los fármacos , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas/farmacología , Ratas , Receptor de Insulina/metabolismo , Trehalosa/metabolismoAsunto(s)
Moléculas de Adhesión de Célula Nerviosa/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transducción de Señal/fisiologíaRESUMEN
The insulin from the Atlantic hagfish (Myxine glutinosa) has been one of the most studied insulins from both a structural and a biological viewpoint; however, some aspects of its biology remain controversial, and there has been no satisfying structural explanation for its low biological potency. We have re-examined the receptor binding kinetics, as well as the metabolic and mitogenic properties, of this phylogenetically ancient insulin, as well as that from another extant representative of the ancient chordates, the river lamprey (Lampetra fluviatilis). Both insulins share unusual binding kinetics and biological properties with insulin analogues that have single mutations at residues that contribute to the hexamerization surface. We propose and demonstrate by reciprocal amino acid substitutions between hagfish and human insulins that the reduced biological activity of hagfish insulin results from unfavorable substitutions, namely, A10 (Ile to Arg), B4 (Glu to Gly), B13 (Glu to Asn), and B21 (Glu to Val). We likewise suggest that the altered biological activity of lamprey insulin may reflect substitutions at A10 (Ile to Lys), B4 (Glu to Thr), and B17 (Leu to Val). The substitution of Asp at residue B10 in hagfish insulin and of His at residue A8 in both hagfish and lamprey insulins may help compensate for unfavorable changes in other regions of the molecules. The data support the concept that the set of unusual properties of insulins bearing certain mutations in the hexamerization surface may reflect those of the insulins evolutionarily closer to the ancestral insulin gene product.
Asunto(s)
Anguila Babosa , Insulina/química , Insulina/metabolismo , Lampreas , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Anguila Babosa/genética , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/metabolismo , Insulina/genética , Cinética , Lampreas/genética , Mitógenos , Modelos Moleculares , Mutación , Filogenia , Receptor de Insulina/genéticaRESUMEN
Fibroblast growth factor receptor (FGFR) signaling is pivotal in the regulation of neurogenesis, neuronal differentiation and survival, and synaptic plasticity both during development and in adulthood. In order to develop low molecular weight agonists of FGFR, seven peptides, termed hexafins, corresponding to the beta6-beta7 loop region of the FGF 1, 2, 3, 8, 9, 10, and 17, were synthesized. This region shares a homologous amino acid sequence with the FG-loop region of the second fibronectin Type III module of the neural cell adhesion molecule (NCAM) that binds to the FGFR. Hexafins were shown by surface plasmon resonance to bind to FGFR1-IIIc-Ig2-3 and FGFR2-IIIb-Ig2-3. The heparin analog sucrose octasulfate inhibited hexafin binding to FGFR1-IIIc-Ig2-3 indicating overlapping binding sites. Hexafin-binding to FGFR1-IIIc resulted in receptor phosphorylation, but inhibited FGF1-induced FGFR1 phosphorylation, indicating that hexafins act as partial agonists. Hexafin2, 3, 8, 10, and 17 (but not 1 or 9) induced neurite outgrowth from cerebellar granule neurons (CGNs), an effect that was abolished by two inhibitors of FGFR, SU5402 and inositol hexaphosphate (IP6) and a diacylglycerol lipase inhibitor, RHC-80267. The neuritogenic effects of selected hexafins could also be inhibited by FGF1 which by itself did not induce neurite outgrowth. Moreover, hexafin1, 3, 9, 10, and 17 (but not 2 or 8) promoted survival of CGNs induced to undergo apoptosis. Thus, selected hexafins induced neuronal differentiation and survival, making them promising pharmacological tools for the study of functional FGFR regulation in development of the nervous system.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/metabolismo , Neuronas/citología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Análisis de Varianza , Animales , Sitios de Unión , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ácido Fítico/farmacología , Pirroles/farmacología , Ratas , Ratas Wistar , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Grabación en VideoRESUMEN
The insulin/relaxin superfamily of peptide hormones comprises 10 members in humans. The three members of the insulin-related subgroup bind to receptor tyrosine kinases (RTKs), while four of the seven members of the relaxin-like subgroup are now known to bind to G-protein-coupled receptors (GPCRs), the so-called relaxin family peptide receptors (RXFPs). Both systems have a long evolutionary history and play a critical role in fundamental biological processes, such as metabolism, growth, survival and longevity, and reproduction. The structural biology and ligand-binding kinetics of the insulin and insulin-like growth factor I receptors have been studied in great detail, culminating in the recent crystal structure of the insulin receptor extracellular domain. Some of the fundamental properties of these receptors, including constitutive dimerization and negative cooperativity, have recently been shown to extend to other RTKs and GPCRs, including RXFPs, confirming kinetic observations made over 30 years ago.
Asunto(s)
Insulina/química , Insulina/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Relaxina/química , Relaxina/metabolismo , Regulación Alostérica , Humanos , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/metabolismo , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
C3, a synthetic peptide binding to the Ig1 module of the neural cell adhesion molecule (NCAM) has previously been identified and shown to inhibit NCAM homophilic binding and NCAM-mediated activation of the fibroblast growth factor (FGF) receptor (FGFR). However, C3 can also stimulate signalling on its own in a way similar to NCAM. Here we show that in the absence of NCAM, C3 can bind and activate FGFR, whereas in the presence of NCAM, C3 inhibits the NCAM-stimulated FGFR activation without activating FGFR on its own. Several competing models of FGFR activation by NCAM have been previously proposed. In one of them, the FGFR Ig2-Ig3 modules are involved in binding to NCAM, whereas in another - the FGFR "acid box" region mediates the interaction. The bi-modal effect of C3 can be explained in the context of the former model and is not consistent with the latter, thus providing evidence in support of the former model.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular , Drosophila , Factor 3 de Crecimiento de Fibroblastos/química , Factor 3 de Crecimiento de Fibroblastos/genética , Humanos , Ratones , Fosforilación , Unión Proteica , Ratas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
The insulin and insulin-like growth factor 1 receptors activate overlapping signalling pathways that are critical for growth, metabolism, survival and longevity. Their mechanism of ligand binding and activation displays complex allosteric properties, which no mathematical model has been able to account for. Modelling these receptors' binding and activation in terms of interactions between the molecular components is problematical due to many unknown biochemical and structural details. Moreover, substantial combinatorial complexity originating from multivalent ligand binding further complicates the problem. On the basis of the available structural and biochemical information, we develop a physically plausible model of the receptor binding and activation, which is based on the concept of a harmonic oscillator. Modelling a network of interactions among all possible receptor intermediaries arising in the context of the model (35, for the insulin receptor) accurately reproduces for the first time all the kinetic properties of the receptor, and provides unique and robust estimates of the kinetic parameters. The harmonic oscillator model may be adaptable for many other dimeric/dimerizing receptor tyrosine kinases, cytokine receptors and G-protein-coupled receptors where ligand crosslinking occurs.
Asunto(s)
Regulación Alostérica , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Humanos , Cinética , Modelos Biológicos , Modelos Moleculares , Unión ProteicaRESUMEN
The neural cell adhesion molecule L1 plays an important role in axon growth, neuronal survival, and synaptic plasticity. We recently demonstrated that the L1 fibronectin type III (FN3) modules interact directly with the fibroblast growth factor (FGF) receptor (FGFR). Sequence alignment of individual L1 FN3 modules with various FGFs suggested that four sequence motifs located in the third and fifth L1 FN3 modules might be involved in interactions with FGFR. The present study found that corresponding synthetic peptides, termed elcamins 1, 2, 3, and 4, bind and activate FGFR in the absence of FGF1. Conversely, in the presence of FGF1, elcamins inhibited receptor phosphorylation, indicating that the peptides are FGFR partial agonists. Elcamins 1, 3, and 4 dose dependently induced neurite outgrowth in cultured primary cerebellar neurons. The neuritogenic effect of elcamins was dependent on FGFR activation, insofar as the effect was abolished by the receptor inhibition. Thus, the identified peptides act as L1 mimetics with regard to activation of FGFR and induction of neurite outgrowth.
Asunto(s)
Diferenciación Celular/fisiología , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neurogénesis/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos/fisiología , Animales , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Molécula L1 de Adhesión de Célula Nerviosa/agonistas , Molécula L1 de Adhesión de Célula Nerviosa/química , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuritas/ultraestructura , Péptidos/metabolismo , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Fibroblast growth factor (FGF) receptor (FGFR) consists extracellularly of three immunoglobulin (Ig) modules (Ig1-3). Currently, there are two competing models (symmetric and asymmetric) of the FGF-FGFR-heparin complex based on crystal structures. Indirect evidence exists in support of both models. However, it is not clear which model is physiologically relevant. Our aim was to obtain direct, non-crystallographic evidence in support of them. We found by nuclear magnetic resonance that Ig2 could bind to FGF1 not only via the primary site (present in both models), but also via the secondary site (present only in the symmetric model). Thus, our data support the symmetric model.
Asunto(s)
Factor 1 de Crecimiento de Fibroblastos/química , Inmunoglobulinas/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Secuencia de Aminoácidos , Animales , Cristalografía , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Mapeo de Interacción de Proteínas , Resonancia por Plasmón de SuperficieRESUMEN
The fibroblast growth factor receptor (FGFR) can be activated through direct interaction with the neural cell adhesion molecule (NCAM). The extracellular part of the FGFR consists of three immunoglobulin-like (Ig) modules, and that of the NCAM consists of five Ig and two fibronectin type III (F3) modules. NCAM-FGFR interactions are mediated by the third FGFR Ig module and the second NCAM F3 module. Using surface plasmon resonance and nuclear magnetic resonance analyses, the present study demonstrates that the second Ig module of FGFR also is involved in binding to the NCAM. The second Ig module residues involved in binding were identified and shown to be localized on the "opposite sides" of the module, indicating that when NCAMs are clustered (e.g., due to homophilic binding), high-affinity FGFR binding sites may be formed by the neighboring NCAMs.
Asunto(s)
Modelos Moleculares , Moléculas de Adhesión de Célula Nerviosa/química , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Receptores de Factores de Crecimiento de Fibroblastos/química , Animales , Sitios de Unión , Ratones , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/químicaRESUMEN
The neural cell adhesion molecule (NCAM) directly interacts with the fibroblast growth factor receptor (FGFR). Both fibronectin type III (FN3) modules of NCAM are involved in this interaction. One of the NCAM-FGFR contact sites has been localized recently to the upper N-terminal part of the second NCAM FN3 module encompassing the F and G beta-strands and the interconnecting loop region. Here, we investigated whether any of the six putative strand-loop-strand regions in the first NCAM FN3 module are involved in FGFR interactions. Peptide sequences encompassing these regions, termed encamins, were synthesized and tested for their ability to bind and activate FGFR. Encamins localized to the N-terminal part of the first FN3 module did not interact with FGFR, whereas encamins localized to the C-terminal part, termed EncaminA, C and E, bound to and activated FGFR. The encamins induced FGFR-dependent neurite outgrowth, and EncaminC and E promoted neuronal survival and enhanced pre-synaptic function. In conclusion, the interaction between NCAM and FGFR probably involves multiple contact sites at an interface formed by the two NCAM FN3 modules and FGFR, and encamins could constitute important pharmacological tools for the study of specific functional aspects of NCAM, including neuroprotection and modulation of plasticity.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas/metabolismo , Péptidos/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/fisiología , Humanos , Ratones , Moléculas de Adhesión de Célula Nerviosa/química , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Péptidos/química , Estructura Terciaria de Proteína/fisiología , RatasRESUMEN
The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analyses been demonstrated to be involved in NCAM-FGFR interactions. An FGFR activation motif (FRM) in the first NCAM FN3 module also has been suggested to take part in NCAM interactions with FGFR. Here, we show for the first time that a peptide motif in the second NCAM FN3 module, different from the previously described FGL motif (NLIKQDDGGSPIRHY; termed BCL) binds and activates FGFR and induces FGFR-dependent neurite outgrowth in cultures of cerebellar granule neurons. Our results provide evidence that the BCL motif is one of the multiple FGFR binding sites in NCAM.
Asunto(s)
Secuencias de Aminoácidos , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Oligopéptidos/química , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/química , Fosforilación , Resonancia por Plasmón de SuperficieRESUMEN
In this review, the structural biology of interaction between the neural cell adhesion molecule (NCAM) and the fibroblast growth factor (FGF) receptor is described and a possible mechanism of the FGF-receptor activation by NCAM is discussed. Most of the FGF-receptor molecules are thought to be constantly involved in a transient interaction with NCAM. However, the FGF-receptor becomes activated only when NCAM is involved the trans-homophilic binding (mediating cell-cell adhesion). The trans-homophilic binding between the NCAM molecules is believed to result in formation of either one- or two-dimensional 'zipper'-like arrays of the NCAM molecules, which leads to NCAM clustering and as a result to clustering of the FGF-receptor, which in turn may lead to its activation through a direct receptor-receptor dimerization (and thus activation) due to an increase in the local concentration of the receptor.
RESUMEN
The neuronal cell adhesion molecule (CAM) L1 promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). The present study demonstrates a direct interaction between L1 fibronectin type III (FN3) modules I-V and FGFR1 immunoglobulin (Ig) modules II and III by surface plasmon resonance analysis. Binding of L1 to FGFR1 was enhanced by adenosine 5'-triphosphate (ATP), adenylylmethylenediphosphonate (AMP-PCP), and guanosine-5'-triphosphate (GTP), but not adenosine monophosphate (AMP). The L1-FN3 modules were capable of activating FGFR1, reflected by receptor phosphorylation, and this resulted in the induction of differentiation of primary neurons, reflected by neurite outgrowth. Furthermore, ATP modulated L1-induced neuronal differentiation and FGFR1 phosphorylation through regulation of the L1-FGFR1 interaction.