Hisashi Umemori

Receptor Specificity of the Fibroblast Growth Factor Family THE COMPLETE MAMMALIAN FGF FAMILY

In mammals, fibroblast growth factors (FGFs) are encoded by 22 genes. FGFs bind and activate alternatively spliced forms of four tyrosine kinase FGF receptors (FGFRs 1–4). The spatial and temporal expression patterns of FGFs and FGFRs and the ability of specific ligand-receptor pairs to actively signal are important factors regulating FGF activity in a variety of biological processes. FGF signaling activity is regulated by the binding specificity of ligands and receptors and is modulated by extrinsic cofactors such as heparan sulfate proteoglycans. In previous studies, we have engineered BaF3 cell lines to express the seven principal FGFRs and used these cell lines to determine the receptor binding specificity of FGFs 1–9 by using relative mitogenic activity as the readout. Here we have extended these semiquantitative studies to assess the receptor binding specificity of the remaining FGFs 10–23. This study completes the mitogenesis-based comparison of receptor specificity of the entire FGF family under standard conditions and should help in interpreting and predicting in vivo biological activity.

Characterization of Fyn-mediated tyrosine phosphorylation sites on GluRε2 (NR2B) subunit of the N-methyl-D-aspartate receptor

The N-methyl-d-aspartate (NMDA) receptors play critical roles in synaptic plasticity, neuronal development, and excitotoxicity. Tyrosine phosphorylation of NMDA receptors by Src-family tyrosine kinases such as Fyn is implicated in synaptic plasticity. To precisely address the roles of NMDA receptor tyrosine phosphorylation, we identified Fyn-mediated phosphorylation sites on the GluRε2 (NR2B) subunit of NMDA receptors. Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluRε2 were phosphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluRε2 were phosphorylated in human embryonic kidney fibroblasts when co-expressed with active Fyn, and Tyr-1472 was the major phosphorylation site in this system. We then generated rabbit polyclonal antibodies specific to Tyr-1472-phosphorylated GluRε2 and showed that Tyr-1472 of GluRε2 was indeed phosphorylated in murine brain using the antibodies. Importantly, Tyr-1472 phosphorylation was greatly reduced infyn mutant mice. Moreover, Tyr-1472 phosphorylation became evident when hippocampal long term potentiation started to be observed, and its magnitude became larger in murine brain. Finally, Tyr-1472 phosphorylation was significantly enhanced after induction of long term potentiation in the hippocampal CA1 region. These data suggest that Tyr-1472 phosphorylation of GluRε2 is important for synaptic plasticity.

Negative Regulation of BMP/Smad Signaling by Tob in Osteoblasts

Bone morphogenetic protein (BMP) controls osteoblast proliferation and differentiation through Smad proteins. Here we show that Tob, a member of the emerging family of antiproliferative proteins, is a negative regulator of BMP/Smad signaling in osteoblasts. Mice carrying a targeted deletion of the tob gene have a greater bone mass resulting from increased numbers of osteoblasts. Orthotopic bone formation in response to BMP2 is elevated in tob-deficient mice. Overproduction of Tob represses BMP2-induced, Smad-mediated transcriptional activation. Finally, Tob associates with receptor-regulated Smads (Smad1, 5, and 8) and colocalizes with these Smads in the nuclear bodies upon BMP2 stimulation. The results indicate that Tob negatively regulates osteoblast proliferation and differentiation by suppressing the activity of the receptor-regulated Smad proteins.

The AMPA receptor interacts with and signals through the protein tyrosine kinase Lyn

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. The ionotropic glutamate receptors are classified into two groups, NMDA (N-methyl-D-aspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors. The AMPA receptor is a ligand-gated cation channel that mediates the fast component of excitatory postsynaptic currents in the central nervous system. Here we report that AMPA receptors function not only as ion channels but also as cell-surface signal transducers by means of their interaction with the Src-family non-receptor protein tyrosine kinase Lyn. In the cerebellum, Lyn is physically associated with the AMPA receptor and is rapidly activated following stimulation of the receptor. Activation of Lyn is independent of Ca2+ and Na+ influx through AMPA receptors. As a result of activation of Lyn, the mitogen-activated protein kinase (MAPK) signalling pathway is activated, and the expression of brain-derived neurotrophic factor (BDNF) messenger RNA is increased in a Lyn-kinase-dependent manner. Thus, AMPA receptors generate intracellular signals from the cell surface to the nucleus through the Lyn–MAPK pathway, which may contribute to synaptic plasticity by regulating the expression of BDNF.

FGF22 and Its Close Relatives Are Presynaptic Organizing Molecules in the Mammalian Brain

Target-derived cues promote local differentiation of axons into nerve terminals at sites of synaptic contact. Using clustering of synaptic vesicles in cultured neurons as an assay, we purified putative target-derived presynaptic organizing molecules from mouse brain and identified FGF22 as a major active species. FGF7 and FGF10, the closest relatives of FGF22, share this activity; other FGFs have distinct effects. FGF22 is expressed by cerebellar granule cells during the period when they receive synapses. Its receptor, FGFR2, is expressed by pontine and vestibular neurons when their axons (mossy fibers) are making synapses on granule cells. Neutralization of FGF7, -10, and -22 inhibits presynaptic differentiation of mossy fibers at sites of contact with granule cells in vivo. Inactivation of FGFR2 has similar effects. These results indicate that FGF22 and its relatives are presynaptic organizing molecules in the mammalian brain and suggest new functions for this family of signaling molecules.

Distinct target-derived signals organize formation, maturation, and maintenance of motor nerve terminals

Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.

Distinct FGFs promote differentiation of excitatory and inhibitory synapses

The differential formation of excitatory (glutamate-mediated) and inhibitory (GABA-mediated) synapses is a critical step for the proper functioning of the brain. An imbalance in these synapses may lead to various neurological disorders such as autism, schizophrenia, Tourette’s syndrome and epilepsy. Synapses are formed through communication between the appropriate synaptic partners. However, the molecular mechanisms that mediate the formation of specific synaptic types are not known. Here we show that two members of the fibroblast growth factor (FGF) family, FGF22 and FGF7, promote the organization of excitatory and inhibitory presynaptic terminals, respectively, as target-derived presynaptic organizers. FGF22 and FGF7 are expressed by CA3 pyramidal neurons in the hippocampus. The differentiation of excitatory or inhibitory nerve terminals on dendrites of CA3 pyramidal neurons is specifically impaired in mutants lacking FGF22 or FGF7. These presynaptic defects are rescued by postsynaptic expression of the appropriate FGF. FGF22-deficient mice are resistant to epileptic seizures, and FGF7-deficient mice are prone to them, as expected from the alterations in excitatory/inhibitory balance. Differential effects of FGF22 and FGF7 involve both their distinct synaptic localizations and their use of different signalling pathways. These results demonstrate that specific FGFs act as target-derived presynaptic organizers and help to organize specific presynaptic terminals in the mammalian brain.

NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine‐phosphorylated in the brain, with Tyr‐1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr‐1472 site to phenylalanine (Y1472F) and show that Tyr‐1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear‐related learning and reduced amygdaloid long‐term potentiation. NMDA receptor‐mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr‐1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

Specific expressions of Fyn and Lyn, lymphocyte antigen receptor-associated tyrosine kinases, in the central nervous system.

The Src-like protein-tyrosine kinases Fyn and Lyn are expressed in lymphocytes. Fyn is expressed in T cells at elevated levels and is associated with the T cell antigen receptor complex, whereas Lyn is expressed in B cells and is associated with membrane-bound immunoglobulin. Thus, these kinases are suggested to participate in antigen-mediated signal transduction in lymphocytes. Previous report showed that fyn was also expressed in brain, but its cellular distribution was not examined. Expression of Lyn in neural tissues was not previously reported. Here we report that both fyn and lyn are expressed in discrete regions of the brain. To throw light on their functions in the brain, we investigated their expressions during brain ontogenesis in mice. In situ hybridization analysis showed that Fyn mRNA was specifically expressed in neurons of embryos and newborn mice. In adult animals, fyn mRNA was expressed in oligodendrocytes as well as neurons. In contrast, the expression of lyn mRNA was relatively low in brains of embryos and newborn mice, but in adults the transcript was specifically expressed in the granular layer of the cerebellum. Therefore, the Fyn and Lyn kinases may regulate distinct functions of specific cells during brain development. The specific expressions of Fyn and Lyn in both lymphatic and neural tissues could suggest common signalling mechanisms in the immune system and central nervous system.

ANA, a novel member of Tob/BTG1 family, is expressed in the ventricular zone of the developing central nervous system

Using a polymerase chain reaction-mediated cloning procedure, we have identified a novel member, termed ANA (from Abundant in Nueroepithelium Area), of Tob/BTG1 family of antiproliferative genes. Molecular cloning and analysis of cDNAs revealed that the human and mouse ANA encoded a protein of 252 amino acids. The amino-terminal half of ANA was homologous to the previously characterized antiproliferative gene products, BTG1, PC3/TIS21/BTG2, and Tob. The human ANA gene was localized at chromosome 21q11.2-q21.1. ANA was expressed in a variety of tissues and cell lines, its expression being high in the ovary, testis, prostate, thymus, and lung. Further analysis revealed that ANA expression was high in the ventricular zone of the developing central nervous system. Finally, overexpression of ANA impaired serum-induced cell cycle progression from the G0/G1 to S phase. In conclusion, ANA is a fourth member of the Tob/BTG1 family that might play roles in neurogenesis in the central nervous system.