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First published online 30 March 2005
doi: 10.1242/dev.01803

Development 132, 2191-2202 (2005)
Published by The Company of Biologists 2005
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The single AmphiTrk receptor highlights increased complexity of neurotrophin signalling in vertebrates and suggests an early role in developing sensory neuroepidermal cells

Èlia Benito-Gutiérrez1, Christian Nake2, Marta Llovera2, Joan X. Comella2,* and Jordi Garcia-Fernàndez1,*,{dagger}

1 Departament de Genética, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal, 645, E-08028, Barcelona, Spain
2 Departament de Ciéncies Médiques Bàsiques, Facultat de Medicina, Universitat de Lleida, Avinguda Rovira Roure 44, E-25198, Lleida, Spain



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  		Fig. 1. (A) Domain structure comparison of AmphiTrk, vertebrate Trk receptors (TrkA, TrkB and TrkC) and representative invertebrate Trk-like proteins from Lymnaea stagnalis (LTrk) and Drosophila melanogaster (DTrk). Modules are colour coded: yellow, Cys-rich clusters; green, leucine-rich domains; blue, type C2 IgG domains; grey, transmembrane domain; red, tyrosine-kinase domain; purple, N-terminal extension; turquoise; type V2 IgG domains. (B) AmphiTrk amino acid sequence. Amino acid positions are numbered on the left. The putative signal sequence cleavage site is indicated by an arrowhead. Leucine-rich motifs are in italics and flanking cysteine clusters in bold. Both Ig-like domains are underlined; conserved asparagines with structural roles for ligand binding are indicated by dots. The transmembrane region is underlined by a dotted line. The first phosphorylation site by cAMP/cGMP-dependent kinase proteins, KIS, is shown with a black background, as is the tyrosine responsible for Shc recruitment. Within the tyrosine kinase domain (boxed), the lysine responsible for ATP binding and the second phosphorylation site by cAMP/cGMP-dependent kinase proteins, RKFT, are shown by a black background. The autophosphorylation sequence (DIYSTDYYR) is highlighted in grey and the autophosphorylated tyrosines are shown by a black background. Glutamine located in the same position as the vertebrate docking site for PLC{gamma} is shown by a black background. AmphiTrk sequences have been deposited in the GeneBank under Accession Numbers AY902361-AY902364

 


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  		Fig. 2. Southern blot of a Branchiostoma floridae individual genomic DNA, digested with EcoRI (E) or XbaI (X). Hybridisations with probes derived from exons 4/5 or exon 10 show that AmphiTrk is a single copy gene.

 


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  		Fig. 3. Comparison of the genomic structure of AmphiTrk and human Trk genes. (A) Exonic domain distribution of human TRKA, TRKB and TRKC. Newly generated introns in human Trk receptors are indicated by arrows. (B) Exonic domain distribution of AmphiTrk. Domains are colour coded: purple, signal peptide; green, leucine-rich domains; yellow, cys-rich clusters; blue, type C2 Ig-like domains; orange, transmembrane surrounding regions; brown, transmembrane domain; red, tyrosine-kinase domain.

 


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  		Fig. 4. Phylogenetic analysis of AmphiTrk versus vertebrate Trk receptors and invertebrate Trk-related peptide sequences. (A) Phylogenetic tree generated with full-length sequences, (B) with isolated extracellular domains or (C) with tyrosine kinase domains only. In all cases, cladograms were calculated by the neighbour-joining method and bootstrap percentages over 1000 replicas are shown for every branch. Human and mouse ROR1 tyrosine kinase domains were used as outgroups in C.

 


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  		Fig. 5. Interaction of AmphiTrk with mammalian neurotrophins. (A-D) Western blots of AmphiTrk induced phosphorylation of AKT and Erk1/2 following stimulation with mammalian neurotrophins in transfected cell cultures. Concentrations of neurotrophins are indicated above the blots (ng/ml) (E) Schematic representation of the chimaeric rat TrkA receptor containing the intracellular domain of AmphiTrk (HA-rTrkA-AmphiTrk). (F) Western blot of HA-rTrkA-AmphiTrk induced phosphorylation of AKT and Erk1/2 after stimulation with NGF. Detection of the HA epitope was carried out to confirm expression of the chimaeric receptor and the native rat TrkA, which was used as a positive control. (G) Detection of PLC{gamma} phosphorylation after stimulation with NGF (100 ng/ml) and immunoprecipitation with anti-PLC{gamma}. No phosphorylation of PLC{gamma} was detected through HA-rTrkA-AmphiTrk. An anti-PLC{gamma} antibody was used to control immunoprecipitation efficiency. Stimulation of cultures transfected with the empty vector (pCDNA3) was performed as a negative control in A-D,F,G. Tubulin detection was used to control the loading of the lanes in A-D,F,G. (H-K) Neurite outgrowth assays. Neurite outgrowth was induced by NGF stimulation and visualised by cotransfection with an enhanced yellow fluorescent protein (EYFP). (H) Empty vector (pCDNA3), (I) AmphiTrk, (J) chimaeric HA-rTrkA-AmphiTrk and (K) rTrkA. (L) Percentages of cells that developed neurites under the absence or presence of NGF stimulation.

 


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  		Fig. 6. Developmental expression of AmphiTrk. In all lateral views, the anterior is towards the left and the dorsal is towards the top. (A) Earliest AmphiTrk expression was detected in individual epidermal cells of the ventral midline of the early neurula. (B) The number of expressing cells began to increase at 14 hours of development. (C,D) Left- and right-hand views of the same neurula at 16 hours of development. (E-H) Cross-sections at different levels of the embryos shown in C and D (dotted lines). Positive cells are located within the epidermal layer (arrows) or just beneath it (arrowheads). (I) At 18 hours of development AmphiTrk-expressing cells in ventrolateral positions extend towards dorsal directions. (J) Magnified view of the area inside the rectangle in I, showing the fusiform shaped positive cells and the cytoplasmic distribution of AmphiTrk transcripts. (K) At late neurula stages, dorsalisation of the AmphiTrk signal was more evident. (L-O) In vivo DiI labelling assays. (L) Initial DiI crystal deposition in the ventral epidermis of the hatching neurula (arrows). (M) Cultured late neurula after DiI labelling shown in L. (N) Rhodamine filtered view of M, showing DiI labelled cells (arrows and arrowheads). (O) Merged image of M and N showing labelled cells that migrated individually towards dorsal positions (arrows). (P) In the early larval stage AmphiTrk was upregulated during Hatschek's pit formation (arrow) and weak expression was detected in a rostral patch of cells (arrowhead). (Q) At 26 hours of development, expression was just visible in the developing Hatschek's pit. (R) Magnified view of the area inside the dotted square in T, showing the dorsoposterior expression of AmphiTrk during Hatschek's pit formation. (S) Cross-section at the level of the dotted line in T, showing staining limited to the left-sided dorsolateral part of the Hatschek's pit primordia. (T,U) AmphiTrk expression was maintained in the Hatschek's pit at 28 hours (T) and 30 hours (U) of development. HP, Hatschek's pit; N, notochord; E, endostyle; CV, cerebral vesicle.

 


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  		Fig. 7. AmphiTrk expression in the adult central nervous system. Pictures are lettered from most anterior (A) to most posterior (E) section. (A) The anteriormost limit of AmphiTrk expression is located at the beginning of the proper nerve cord, where strong signal is visible around the ventral part of the central canal (arrows). Coincidentally, some cells are labelled in both sides of the dorsal part of the neural tube (arrowheads), at the level where the primary motor centre initiates. (B) Section including the posterior part of the Hatschek's pit, showing AmphiTrk expression in the most dorsal part (asterisk). Arrows and arrowhead indicate AphiTrk-expressing cells in the ventral and dorsal compartments, respectively. (C-E) AmphiTrk expression is maintained along the neural tube around the ventral side of the central canal (arrows). Iteratively, some cells simultaneously express AmphiTrk in the dorsal part of the neural tube, albeit with a slight right-left offset (arrowheads). (F) At some points along the neural tube, AmphiTrk-expressing cells extend ventrally, touching the surrounding membrane of the notochord (arrows). N, notochord; NT, neural tube; HP, Hatschek's pit; HE, Hesse eyecup; DR, dorsal root.

 

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© The Company of Biologists Ltd 2005