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1.
J Recept Signal Transduct Res ; 37(6): 590-599, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28854843

ABSTRACT

The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET2 technique to monitor the effect of angiotensin II on the interaction between Rluc8 tagged insulin receptor and GFP2 tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT1 receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET2 technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.


Subject(s)
Blood Pressure/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Receptor, Angiotensin, Type 1/metabolism , Receptor, Insulin/metabolism , Adaptor Proteins, Signal Transducing , Angiotensin II/administration & dosage , Angiotensin II/metabolism , Bioluminescence Resonance Energy Transfer Techniques , Cell Line , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Humans , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , MAP Kinase Signaling System/drug effects , Protein Domains , Protein Kinase C/genetics , Protein Kinase C/metabolism , Receptor, Angiotensin, Type 1/genetics , Receptor, Insulin/genetics , Src Homology 2 Domain-Containing, Transforming Protein 2/genetics , Src Homology 2 Domain-Containing, Transforming Protein 2/metabolism
2.
Mol Cell Endocrinol ; 382(2): 938-49, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24275181

ABSTRACT

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.


Subject(s)
Glucagon-Like Peptide 1/pharmacology , Islets of Langerhans/metabolism , Receptors, Glucagon/metabolism , Signal Transduction/drug effects , Animals , Cyclic AMP/metabolism , Exenatide , Glucagon-Like Peptide 1/analogs & derivatives , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor , HEK293 Cells , Humans , Incretins/metabolism , Incretins/pharmacology , Islets of Langerhans/drug effects , Islets of Langerhans/ultrastructure , Liraglutide , Mice , Mice, Inbred C57BL , Peptides/metabolism , Peptides/pharmacology , Protein Stability , Protein Transport , Proteolysis , Time-Lapse Imaging , Venoms/metabolism , Venoms/pharmacology
3.
J Recept Signal Transduct Res ; 32(2): 57-64, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22272819

ABSTRACT

The insulin receptor (IR) belongs to the receptor tyrosine kinase super family and plays an important role in glucose homeostasis. The receptor interacts with several large docking proteins that mediate signaling from the receptor, including the insulin receptor substrate (IRS) family and Src homology-2-containing proteins (Src). Here, we applied the bioluminescence resonance energy transfer 2 (BRET2) technique to study the IR signaling pathways. The interaction between the IR and the substrates IRS1, IRS4 and Shc was examined in response to ligands with different signaling properties. The association between IR and the interacting partners could successfully be monitored when co-expressing green fluorescent protein 2 (GFP2) tagged substrates with Renilla reniformis luciferase 8 (Rluc8) tagged IR. Through additional optimization steps, we developed a stable and flexible BRET2 assay for monitoring the interactions between the IR and its substrates. Furthermore, the insulin analogue X10 was characterized in the BRET2 assay and was found to be 10 times more potent with respect to IRS1, IRS4 and Shc recruitment compared to human insulin. This study demonstrates that the BRET2 technique can be applied to study IR signaling pathways, and that this assay can be used as a platform for screening and characterization of IR ligands.


Subject(s)
Green Fluorescent Proteins/analysis , Insulin Receptor Substrate Proteins/metabolism , Insulin/pharmacology , Luminescent Measurements , Receptor, Insulin/metabolism , Shc Signaling Adaptor Proteins/metabolism , Cells, Cultured , Humans , Insulin/analogs & derivatives , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , Protein Binding , Protein Interaction Domains and Motifs/drug effects , Recombinant Fusion Proteins/metabolism , Src Homology 2 Domain-Containing, Transforming Protein 1
4.
Structure ; 19(2): 203-11, 2011 Feb 09.
Article in English | MEDLINE | ID: mdl-21300289

ABSTRACT

The ectodomain of olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM) consists of five immunoglobulin (Ig) domains (IgI-V), followed by two fibronectin-type 3 (Fn3) domains (Fn3I-II). A complete structural model of the entire ectodomain of human OCAM has been assembled from crystal structures of six recombinant proteins corresponding to different regions of the ectodomain. The model is the longest experimentally based composite structural model of an entire IgCAM ectodomain. It displays an essentially linear arrangement of IgI-V, followed by bends between IgV and Fn3I and between Fn3I and Fn3II. Proteins containing IgI-IgII domains formed stable homodimers in solution and in crystals. Dimerization could be disrupted in vitro by mutations in the dimer interface region. In conjunction with the bent ectodomain conformation, which can position IgI-V parallel with the cell surface, the IgI-IgII dimerization enables OCAM-mediated trans-interactions with an intercellular distance of about 20 nm, which is consistent with that observed in synapses.


Subject(s)
Fibronectins/metabolism , Immunoglobulins/metabolism , Neural Cell Adhesion Molecules/metabolism , Recombinant Proteins/metabolism , Amino Acid Sequence , Binding Sites , Cell Membrane/metabolism , Crystallography, X-Ray , Dimerization , Fibronectins/chemistry , Fibronectins/genetics , Gene Expression , Humans , Immunoglobulins/chemistry , Immunoglobulins/genetics , Models, Structural , Molecular Dynamics Simulation , Molecular Sequence Data , Neural Cell Adhesion Molecules/chemistry , Neural Cell Adhesion Molecules/genetics , Pichia , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/genetics , Synapses/metabolism
5.
J Biol Chem ; 286(1): 661-73, 2011 Jan 07.
Article in English | MEDLINE | ID: mdl-20974844

ABSTRACT

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.


Subject(s)
Conserved Sequence , Drosophila melanogaster , Evolution, Molecular , Insulin/chemistry , Insulin/metabolism , Proteins/chemistry , Proteins/metabolism , Adipocytes/drug effects , Adipocytes/metabolism , Amino Acid Sequence , Animals , Blood Glucose/metabolism , Crystallography, X-Ray , Female , Humans , Insulin/pharmacology , Iodine Radioisotopes , Lipogenesis/drug effects , Male , Mice , Models, Molecular , Molecular Sequence Data , Protein Conformation , Proteins/pharmacology , Rats , Receptor, Insulin/metabolism , Trehalose/metabolism
6.
FEBS Lett ; 585(1): 58-64, 2011 Jan 03.
Article in English | MEDLINE | ID: mdl-21115007

ABSTRACT

Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET(2)50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.


Subject(s)
Luminescent Measurements/methods , Neural Cell Adhesion Molecules/chemistry , Protein Multimerization , Animals , COS Cells , Chlorocebus aethiops , Energy Transfer , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Lipoylation , Neural Cell Adhesion Molecules/genetics , Neural Cell Adhesion Molecules/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection
9.
J Neurosci ; 29(36): 11360-76, 2009 Sep 09.
Article in English | MEDLINE | ID: mdl-19741142

ABSTRACT

The formation of appropriate neuronal circuits is an essential part of nervous system development and relies heavily on the outgrowth of axons and dendrites and their guidance to their respective targets. This process is governed by a large array of molecules, including glial cell line-derived neurotrophic factor (GDNF) and the neural cell adhesion molecule (NCAM), the interaction of which induce neurite outgrowth. In the present study the requirements for NCAM-mediated GDNF-induced neurite outgrowth were investigated in cultures of hippocampal neurons, which do not express Ret. We demonstrate that NCAM-mediated GDNF-induced signaling leading to neurite outgrowth is more complex than previously reported. It not only involves NCAM-140 and the Src family kinase Fyn but also uses NCAM-180 and the fibroblast growth factor receptor. We find that induction of neurite outgrowth by GDNF via NCAM or by trans-homophilic NCAM interactions are not mutually exclusive. However, whereas NCAM-induced neurite outgrowth primarily is mediated by NCAM-180, we demonstrate that GDNF-induced neurite outgrowth involves both NCAM-140 and NCAM-180. We also find that GDNF-induced neurite outgrowth via NCAM differs from NCAM-induced neurite outgrowth by being independent of NCAM polysialylation. Additionally, we investigated the structural basis for GDNF-NCAM interactions and find that NCAM Ig3 is necessary for GDNF binding. Furthermore, we identify within the heel region of GDNF a binding site for NCAM and demonstrate that a peptide encompassing this sequence mimics the effects of GDNF with regard to NCAM binding, activation of intracellular signaling, and induction of neurite outgrowth.


Subject(s)
Glial Cell Line-Derived Neurotrophic Factor/physiology , Neural Cell Adhesion Molecules/physiology , Neurites/physiology , Amino Acid Sequence , Animals , Binding Sites/physiology , Cell Line , Cells, Cultured , Humans , Molecular Sequence Data , Rats , Rats, Wistar
10.
J Neurosci Res ; 87(8): 1806-12, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19185025

ABSTRACT

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.


Subject(s)
Cell Differentiation/physiology , Central Nervous System/embryology , Central Nervous System/metabolism , Neural Cell Adhesion Molecule L1/metabolism , Neurogenesis/physiology , Receptors, Fibroblast Growth Factor/metabolism , Amino Acid Motifs/physiology , Amino Acid Sequence/physiology , Animals , Cell Line , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Ligands , Neural Cell Adhesion Molecule L1/agonists , Neural Cell Adhesion Molecule L1/chemistry , Neurites/drug effects , Neurites/metabolism , Neurites/ultrastructure , Peptides/metabolism , Peptides/pharmacology , Phosphorylation/drug effects , Protein Binding/physiology , Rats , Receptors, Fibroblast Growth Factor/agonists , Up-Regulation/drug effects , Up-Regulation/physiology
11.
J Mol Biol ; 382(5): 1113-20, 2008 Oct 24.
Article in English | MEDLINE | ID: mdl-18706912

ABSTRACT

The crystal structure of the first immunoglobulin (Ig1) domain of neural cell adhesion molecule 2 (NCAM2/OCAM/RNCAM) is presented at a resolution of 2.7 A. NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). In the structure, two Ig domains interact by domain swapping, as the two N-terminal beta-strands are interchanged. beta-Strand swapping at the terminal domain is the accepted mechanism of homophilic interactions amongst the cadherins, another class of CAMs, but it has not been observed within the IgCAM superfamily. Gel-filtration chromatography demonstrated the ability of NCAM2 Ig1 to form dimers in solution. Taken together, these observations suggest that beta-strand swapping could have a role in the molecular mechanism of homophilic binding for NCAM2.


Subject(s)
Neural Cell Adhesion Molecule L1/chemistry , Cadherins/chemistry , Cell Adhesion/physiology , Chromatography, Gel , Crystallography, X-Ray , Dimerization , Humans , Immunoglobulins/chemistry , Models, Molecular , Neural Cell Adhesion Molecule L1/physiology , Neural Cell Adhesion Molecules/chemistry , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Solutions , Thermodynamics
12.
Neurochem Res ; 2008 Mar 27.
Article in English | MEDLINE | ID: mdl-18368488

ABSTRACT

Cell adhesion molecules (CAMs) constitute a large class of plasma membrane-anchored proteins that mediate attachment between neighboring cells and between cells and the surrounding extracellular matrix (ECM). However, CAMs are more than simple mediators of cell adhesion. The neural cell adhesion molecule (NCAM) is a well characterized, ubiquitously expressed CAM that is highly expressed in the nervous system. In addition to mediating cell adhesion, NCAM participates in a multitude of cellular events, including survival, migration, and differentiation of cells, outgrowth of neurites, and formation and plasticity of synapses. NCAM shares an overall sequence identity of approximately 44% with the neural cell adhesion molecule 2 (NCAM2), a protein also known as olfactory cell adhesion molecule (OCAM) and Rb-8 neural cell adhesion molecule (RNCAM), and the region-for-region sequence homology between the two proteins suggests that they are transcribed from paralogous genes. However, very little is known about the function of NCAM2, although it originally was described more than 20 years ago. In this review we summarize the known properties and functions of NCAM2 and describe some of the differences and similarities between NCAM and NCAM2.

13.
Mol Cell Neurosci ; 37(3): 528-36, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18222703

ABSTRACT

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.


Subject(s)
Fibroblast Growth Factors/pharmacology , Fibronectins/metabolism , Neural Cell Adhesion Molecule L1/metabolism , Neurons/drug effects , Neurons/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Adenylyl Imidodiphosphate/pharmacology , Animals , Animals, Newborn , Cells, Cultured , Cerebellum/cytology , Guanosine Triphosphate/pharmacology , Humans , Magnetic Resonance Imaging/methods , Neurites/drug effects , Neurons/cytology , Phosphorylation/drug effects , Rats
14.
Article in English | MEDLINE | ID: mdl-17277441

ABSTRACT

Fibroblast growth factors (FGFs) constitute a family of 22 structurally related heparin-binding polypeptides that are involved in the regulation of cell growth, survival, differentiation and migration. Here, a 1.4 A resolution X-ray structure of rat FGF1 is presented. Two molecules are present in the asymmetric unit of the crystal and they coordinate a total of five sulfate ions. The structures of human, bovine and newt FGF1 have been published previously. Human and rat FGF1 are found to have very similar structures.


Subject(s)
Fibroblast Growth Factor 1/chemistry , Animals , Cattle , Crystallography, X-Ray , Humans , Models, Molecular , Protein Conformation , Rats , Species Specificity
15.
J Neurochem ; 95(1): 46-55, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16181411

ABSTRACT

The second Ig module (IgII) of the neural cell adhesion molecule (NCAM) is known to bind to the first Ig module (IgI) of NCAM (so-called homophilic binding) and to interact with heparan sulfate and chondroitin sulfate glycoconjugates. We here show by NMR that the heparin and chondroitin sulfate-binding sites (HBS and CBS, respectively) in IgII coincide, and that this site overlaps with the homophilic binding site. Using NMR and surface plasmon resonance (SPR) analyses we demonstrate that interaction between IgII and heparin indeed interferes with the homophilic interaction between IgI and IgII. Accordingly, we show that treatment of cerebellar granule neurons (CGNs) with heparin inhibits NCAM-mediated outgrowth. In contrast, treatment with heparinase III or chondroitinase ABC abrogates NCAM-mediated neurite outgrowth in CGNs emphasizing the importance of the presence of heparan/chondroitin sulfates for proper NCAM function. Finally, a peptide encompassing HBS in IgII, termed the heparin-binding peptide (HBP), is shown to promote neurite outgrowth in CGNs. These observations indicate that neuronal differentiation induced by homophilic NCAM interaction is modulated by interactions with heparan/chondroitin sulfates.


Subject(s)
Heparin/metabolism , Immunoglobulins/chemistry , Immunoglobulins/metabolism , Neural Cell Adhesion Molecules/chemistry , Neural Cell Adhesion Molecules/metabolism , Animals , Binding Sites , Cell Line , Cerebellum/drug effects , Cerebellum/physiology , Chondroitin Sulfates/metabolism , Coculture Techniques , Fibroblasts/metabolism , Heparin/pharmacology , Heparitin Sulfate/chemistry , Heparitin Sulfate/pharmacology , Magnetic Resonance Spectroscopy , Mice , Neurites/drug effects , Neurites/physiology , Neurons/drug effects , Neurons/physiology , Peptide Fragments/pharmacology , Protein Structure, Tertiary , Sucrose/analogs & derivatives , Sucrose/metabolism , Surface Plasmon Resonance
16.
Article in English | MEDLINE | ID: mdl-16511179

ABSTRACT

Four amino-terminal immunoglobulin (Ig) modules and three fibronectin type III (F3) modules of the mouse neural cell-adhesion molecule L1 have been expressed in Drosophila S2 cells. The Ig modules I-IV of L1 crystallized in a trigonal space group, with unit-cell parameters a = b = 239.6, c = 99.3 A, and the crystals diffracted X-rays to a resolution of about 3.5 A. The F3 modules I-III of L1 crystallized in a tetragonal space group, with unit-cell parameters a = b = 80.1, c = 131 A, and the crystals diffracted X-rays to 2.8 A resolution. This is a step towards the structure determination of the multimodular constructs of the neural cell-adhesion molecule L1 in order to understand the function of L1 on a structural basis.


Subject(s)
Fibronectins/chemistry , Gene Expression , Immunoglobulins/chemistry , Neural Cell Adhesion Molecule L1/chemistry , Animals , Crystallization , Crystallography, X-Ray , Mice
17.
Acta Crystallogr D Biol Crystallogr ; 60(Pt 3): 591-3, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14993704

ABSTRACT

Recombinant proteins consisting of either the four or five amino-terminal immunoglobulin (Ig) modules of the rat neural cell-adhesion molecule NCAM or the whole extracellular part [six Ig and five fibronectin type III (F3) modules] of mouse L1 have been expressed in Drosophila S2 cells. The proteins have been purified and crystallized. The crystals of the recombinant protein containing the four amino-terminal Ig modules of NCAM diffract X-rays to approximately 4 A resolution and belong to space group P622 or P6(3)22, with unit-cell parameters a = b = 258.7, c = 182.4 A. No diffraction was observed for the other two protein constructs. This is a step towards determining the structure of multimodular constructs of cell-adhesion molecules that exhibit high structural flexibility.


Subject(s)
Neural Cell Adhesion Molecule L1/chemistry , Animals , Cell Line , Crystallization , Crystallography, X-Ray , Drosophila/cytology , Drosophila/genetics , Gene Expression , Mice , Neural Cell Adhesion Molecule L1/genetics , Protein Structure, Tertiary/genetics , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
18.
Synapse ; 51(4): 270-8, 2004 Mar 15.
Article in English | MEDLINE | ID: mdl-14696014

ABSTRACT

Rapid activation of synaptic receptor-channels evokes an ion current that flows through the narrow synaptic cleft; this exerts a significant voltage drop and therefore strong electric field (10(4) V/m range) directed towards the current sinks in the cleft. To what extent this field affects fast diffusion of charged neurotransmitter molecules is not known. We draw a theoretical framework for this complex electrodiffusion phenomenon and establish the basic relationships between the synaptic current and the time course of neurotransmitter in the cleft. The analyses predict that excitatory currents could significantly accelerate the dispersion of negatively charged molecules from the cleft while attracting the positively charged molecules towards the current sinks. This previously unrecognized mechanism should affect the kinetics of synaptic receptor currents, thus contributing to fast synaptic signaling in the brain.


Subject(s)
Models, Neurological , Neurotransmitter Agents/metabolism , Synapses/metabolism , Diffusion , Membrane Potentials/physiology
19.
Structure ; 11(6): 691-701, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12791257

ABSTRACT

The neural cell adhesion molecule (NCAM) promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). NCAM also has a little-understood ATPase activity. We here demonstrate for the first time a direct interaction between NCAM (fibronectin type III [F3] modules 1 and 2) and FGFR1 (Ig modules 2 and 3) by surface plasmon resonance (SPR) analysis. The structure of the NCAM F3 module 2 was determined by NMR and the module was shown by NMR to interact with the FGFR1 Ig module 3 and ATP. The NCAM sites binding to FGFR and ATP were found to overlap and ATP was shown by SPR to inhibit the NCAM-FGFR binding, indicating that ATP probably regulates the NCAM-FGFR interaction. Furthermore, we demonstrate that the NCAM module was able to induce activation (phosphorylation) of FGFR and to stimulate neurite outgrowth. In contrast, ATP inhibited neurite outgrowth induced by the module.


Subject(s)
Adenosine Triphosphate/metabolism , Neural Cell Adhesion Molecules/chemistry , Neural Cell Adhesion Molecules/metabolism , Protein Structure, Secondary , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Fibroblast Growth Factor/chemistry , Receptors, Fibroblast Growth Factor/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Humans , Mice , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Structure, Tertiary , Rats , Receptor, Fibroblast Growth Factor, Type 1
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