Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling.
Eide FF
,
Vining ER
,
Eide BL
,
Zang K
,
Wang XY
,
Reichardt LF
.
???displayArticle.abstract??? trkB encodes a receptor tyrosine kinase activated by three neurotrophins--brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4/5. In vivo, three isoforms of the receptor are generated by differential splicing--gp145trkB or the full-length trkB receptor, and trkB.T1 and trkB.T2, two cytoplasmically truncated receptors that lack kinases, but contain unique C termini. Although the truncated receptors appear to be precisely regulated during nervous system development and regeneration, their role in neurotrophin signaling has not been directly tested. In this paper, we studied the signaling properties and interactions of gp145trkB, trkB.T1, and trkB.T2 by expressing the receptors in a Xenopus oocyte microinjection assay. We found that oocytes expressing gp145trkB, but not trkB.T1 or trkB.T2, were capable of eliciting 45Ca efflux responses (a phospholipase C-gamma-mediated mechanism) after stimulation by BDNF. When trkB.T1 and trkB.T2 were coexpressed with gp145trkB, they acted as dominant negative receptors, inhibiting the BDNF signal by forming nonfunctional heterodimers with the full-length receptors. An ATP-binding mutant of gp145trkB had similar dominant inhibitory effects. Our data suggest that naturally occurring truncated trkB receptors function as inhibitory modulators of neurotrophin responsiveness. Furthermore, the homodimerization of gp145trkB appears to be an essential step in activation of the BDNF signaling cascade.
Fig. 1.
Dominant inhibitory effect of naturally occurring truncated trkB receptors in 45Ca efflux assay. Oocytes were injected with cRNA (2 ng/oocyte) and were then loaded with45Ca as described in Materials and Methods. After45Ca efflux levels stabilized, 250 ng/ml BDNF was added to the medium (indicated by arrow). Mean values from four determinations ± SD are shown. A, Comparison of BDNF-induced 45Ca efflux in oocytes expressing the full-length trkB receptor (closed squares) trkB.T1 (closed circles), trkB.T2 (closed triangles), or water control (open triangles). B, Dominant inhibitory effect of trkB.T1. Oocytes were injected with trkB cRNA (2 ng/oocyte) + varying quantities of trkB.T1 (0, 2, 8, or 18 ng/oocyte).C, Dominant inhibitory effect of trkB.T2. Oocytes were injected with 2 ng/oocyte trkB cRNA + varying quantities of trkB.T2 (0, 2, 8, or 18 ng/oocyte). D, Dominant inhibitory effect of trkB.T1 and trkB.T2 correlates with loss of trkB homodimers. A binomial model of dimer association (see Results) was used to predict the number of trkB homodimers (open squares). Peak45Ca efflux values for trkB.T1-injected (closed circles) and trkB.T2-injected (closed triangles) oocytes are plotted as a function of truncated:full-length trkB cRNA.
Fig. 2.
ATP-binding mutant of trkB inhibits BDNF signaling at the level of receptor tyrosine phosphorylation. A, ATP-binding site mutation abolishes BDNF-induced45Ca efflux response. Oocytes were injected with cRNA (2 ng/oocyte) encoding the full-length trkB receptor (closed squares) or its ATP-binding mutant. After45Ca efflux had stabilized, BDNF was added to the medium (250 ng/ml, arrow). B, Dominant inhibitory effect of the ATP-binding mutant. Oocytes were injected with 2 ng/oocyte trkB cRNA + varying quantities of ATP-binding mutant (0, 2, 8, and 18 ng/oocyte). C, Decrease in maximal45Ca efflux responses and trkB tyrosine phosphorylation correlate with a loss of trkB homodimers. A binomial model (see Results) was used to predict the number of trkB homodimers (open squares). Percent maximum trkB tyrosine phosphorylation was determined by labeling trkB ± ATP-binding mutant-expressing oocytes with 35S, stimulating with BDNF, immunoprecipitating lysates with APT antibody, separating on SDS-PAGE gels, and then quantitating bands by PhosphorImager analysis (see Materials and Methods). Data are displayed graphically as percent maximum tyrosine phosphorylation (closed circles). Finally, peak 45Ca efflux responses for ATP-binding mutant-expressing oocytes are plotted as a function of the ratio of truncated:full-length trkB cRNA (closed squares).
Fig. 3.
Intermolecular phosphorylation of ATP-binding mutant by wild-type gp145trkB.A, Schematic diagram of epitope-tagged wild-type and mutant trkB receptors. PCR was used to attach an IVH tag to the C terminus of the wild-type trkB receptor (trkBIVH) and myc tag to the C terminus of the ATP-binding mutant (ATPMutmyc) as described in Materials and Methods. Amino acid numbers are listed for the extracellular (EC) or transmembrane domains (TM), ATP-binding site (Lys or Met), and epitope tags (IVH or Myc). B, BDNF induces rapid tyrosine phosphorylation of trkBIVH receptors. COS cells were transfected with vector control (lanes 1, 2) or trkBIVH (lanes 3, 4). Cells in lanes 2 and 4 were stimulated for 5 min with 100 ng/ml BDNF. Lysates were immunoprecipitated with IVH Ab, separated by 6% SDS-PAGE, and then immunoblotted with the APT Ab 4G10. C, Intermolecular tyrosine phosphorylation of ATPMutmyc by trkBIVH. COS cells were transfected with vector alone (lanes 1, 6), ATPMutmyc (lane 2), ATPmutmyc + trkBIVH (lanes 3, 4), or trkBIVH alone (lane 5). Cells in lane 4 were pretreated with 50 mm sodium orthovanadate for 3 hr before lysis. Cells were stimulated with BDNF, then immunoprecipitated with either anti-myc (lanes 1–4) or anti-IVH Abs (lanes 5, 6). Proteins were separated by 6% SDS-PAGE and were then immunoblotted with APT Ab. The location of gp145trkB is indicated by anarrow.
ig. 4.
Epitope tags distinguish the ATP-binding mutant from the wild-type trkB receptor. A, COS cells transfected with either vector control (lane 1) or trkBIVH (lane 2). Lysates were immunoprecipitated with IVH Ab (12CA5), separated by 6% SDS-PAGE, then immunoblotted with IVH Ab. B, COS cells transfected with either vector control (lane 1) or ATPmutmyc (lane 2). Lysates were immunoprecipitated with anti-myc Ab (9E10), separated by 6% SDS-PAGE, and then blotted with anti-myc Abs.
Fig. 5.
Expression levels of wild-type and ATP-binding mutant reflect quantities of cRNA injected. A, IVH ECL blot of oocytes injected with trkBIVH ± ATPmutmyc. Oocytes were injected as described in Materials and Methods, lysed 36 hr later with RIPA buffer, separated on 7% SDS-PAGE gels, and then immunoblotted with IVH Ab (12CA5). Oocytes were injected with sterile water (lane 1), trkBIVH (1 ng; lane 2), or ATPMutmyc + trkBIVH at a ratio of 4:1 (4 ng/1 ng/oocyte; lane 3) or 9:1 (9 ng/1 ng/oocyte;lane 4). Lysates collected from 6 oocytes were loaded per lane. B, Myc ECL blot of oocytes co-injected with trkBIVH ± ATPmutmyc. Oocytes were injected and lysed as described above. Lysates from 2 oocytes were loaded per lane. Proteins were separated on 7% SDS-PAGE gels and then immunoblotted with myc Ab (9E10). Oocytes were injected with trkBIVH only (1 ng/oocyte; lane 1) or ATPMutmyc:trkBIVH at a ratio of 9:1 (9 ng/1 ng/oocyte; lane 2) or 4:1 (4 ng/1 ng/oocyte; lane 3).
Allendoerfer,
Regulation of neurotrophin receptors during the maturation of the mammalian visual system.
1994, Pubmed
Allendoerfer,
Regulation of neurotrophin receptors during the maturation of the mammalian visual system.
1994,
Pubmed
Amaya,
FGF signalling in the early specification of mesoderm in Xenopus.
1993,
Pubmed
,
Xenbase
Barde,
Purification of a new neurotrophic factor from mammalian brain.
1982,
Pubmed
Beck,
Induction of noncatalytic TrkB neurotrophin receptors during axonal sprouting in the adult hippocampus.
1993,
Pubmed
Eide,
Neurotrophins and their receptors--current concepts and implications for neurologic disease.
1993,
Pubmed
,
Xenbase
Evan,
Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
1985,
Pubmed
,
Xenbase
Hyman,
Overlapping and distinct actions of the neurotrophins BDNF, NT-3, and NT-4/5 on cultured dopaminergic and GABAergic neurons of the ventral mesencephalon.
1994,
Pubmed
Jing,
Nerve growth factor mediates signal transduction through trk homodimer receptors.
1992,
Pubmed
Johnson,
Diverse forms of a receptor for acidic and basic fibroblast growth factors.
1990,
Pubmed
,
Xenbase
Klein,
The trkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain.
1990,
Pubmed
Klein,
Expression of the tyrosine kinase receptor gene trkB is confined to the murine embryonic and adult nervous system.
1990,
Pubmed
Knüsel,
Regulated neurotrophin receptor responsiveness during neuronal migrationand early differentiation.
1994,
Pubmed
Kokaia,
Rapid increase of BDNF mRNA levels in cortical neurons following spreading depression: regulation by glutamatergic mechanisms independent of seizure activity.
1993,
Pubmed
Koliatsos,
Evidence that brain-derived neurotrophic factor is a trophic factor for motor neurons in vivo.
1993,
Pubmed
Krieg,
Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.
1984,
Pubmed
,
Xenbase
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Leibrock,
Molecular cloning and expression of brain-derived neurotrophic factor.
1989,
Pubmed
Lindefors,
Spatiotemporal selective effects on brain-derived neurotrophic factor and trkB messenger RNA in rat hippocampus by electroconvulsive shock.
1995,
Pubmed
Middlemas,
trkB, a neural receptor protein-tyrosine kinase: evidence for a full-length and two truncated receptors.
1991,
Pubmed
Middlemas,
Identification of TrkB autophosphorylation sites and evidence that phospholipase C-gamma 1 is a substrate of the TrkB receptor.
1994,
Pubmed
Morse,
Brain-derived neurotrophic factor (BDNF) prevents the degeneration of medial septal cholinergic neurons following fimbria transection.
1993,
Pubmed
Niman,
Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition.
1983,
Pubmed
Rudge,
Neurotrophic factor receptors and their signal transduction capabilities in rat astrocytes.
1994,
Pubmed
Soppet,
The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor.
1991,
Pubmed
Squinto,
trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor.
1991,
Pubmed
Ueno,
Inhibition of PDGF beta receptor signal transduction by coexpression of a truncated receptor.
1991,
Pubmed
Ueno,
Dominant-negative mutations of platelet-derived growth factor (PDGF) receptors. Inhibition of receptor function by ligand-dependent formation of heterodimers between PDGF alpha- and beta-receptors.
1993,
Pubmed
,
Xenbase
Werner,
Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation.
1993,
Pubmed
White,
Structure and function of tyrosine kinase receptors.
1991,
Pubmed
Widmer,
Rapid phosphorylation of phospholipase C gamma 1 by brain-derived neurotrophic factor and neurotrophin-3 in cultures of embryonic rat cortical neurons.
1993,
Pubmed
Williams,
Expression of receptors for cholecystokinin and other Ca2+-mobilizing hormones in Xenopus oocytes.
1988,
Pubmed
,
Xenbase
