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 GluR⑀2 (NR2B) subunit of NMDA receptors. Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluR⑀2 were phosphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluR⑀2 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 GluR⑀2 and showed that Tyr-1472 of GluR⑀2 was indeed phosphorylated in murine brain using the antibodies. Importantly, Tyr-1472 phosphorylation was greatly reduced in fyn 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 GluR⑀2 is important for synaptic plasticity.

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Various hormonal stimuli and growth factors activate the mammalian canonical transient receptor potential (TRPC) channel through phospholipase C (PLC) activation. However, the precise mechanism of the regulation of TRPC channel activity remains unknown. Here, we provide the first evidence that direct tyrosine phosphorylation by Src family protein-tyrosine kinases (PTKs) is a novel mechanism for modulating TRPC6 channel activity. We found that TRPC6 is tyrosine-phosphorylated in COS-7 cells when coexpressed with Fyn, a member of the Src family PTKs. We also found that Fyn interacts with TRPC6 and that the interaction is mediated by the SH2 domain of Fyn and the N-terminal region of TRPC6 in a phosphorylation-independent manner. In addition, we demonstrated the physical association of TRPC6 with Fyn in the mammalian brain. Moreover, we showed that stimulation of the epidermal growth factor receptor induced rapid tyrosine phosphorylation of TRPC6 in COS-7 cells. This epidermal growth factor-induced tyrosine phosphorylation of TRPC6 was significantly blocked by PP2, a specific inhibitor of Src family PTKs, and by a dominant negative form of Fyn, suggesting that the direct phosphorylation of TRPC6 by Src family PTKs could be caused by physiological stimulation. Furthermore, using single channel recording, we showed that Fyn modulates TRPC6 channel activity via tyrosine phosphorylation. Thus, our findings demonstrated that tyrosine phosphorylation by Src family PTKs is a novel regulatory mechanism of TRPC6 channel activity.Various growth factors or hormones can induce activation of phospholipase C (PLC), 1 production of inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG), and Ca 2ϩ influx across the plasma membrane (1, 2). This PLC-dependent Ca 2ϩ influx is thought to play important roles in many physiological functions, such as cell proliferation and apoptosis, T cell activation, and the maturation and functions of B cells (3). Therefore, it is important to understand regulation of such PLC-dependent Ca 2ϩ channels, because modulation of the channel activities can profoundly affect these various physiological processes. The transient receptor potential (TRP) channel superfamily has emerged as candidates responsible for such a PLC-dependent Ca 2ϩ influx. The TRP channel superfamily can be divided into at least three subfamilies of Ca 2ϩ -permeable nonselective cation channels (TRPC, TRPV, and TRPM families), having closely related structures comprised of six transmembrane domains, a large NH 2 -terminal cytoplasmic domain, and a COOH-terminal cytoplasmic domain (4). Among the three subfamilies, TRPC channels are one of the molecules that have been extensively characterized.The TRPC channel family is composed of seven non-selective ion channels that can be divided into four subgroups (TRPC1; TRPC4 and -5; TRPC3, -6, and -7; and TRPC2) based on their amino acid sequences and functional similarities (4 -6). Recent investigations have extensively studied the regulation of TRPC channel activity. TRPC1, -4, and -5...

Intercellular cross-talk between osteoblasts and osteoclasts is important for controlling bone remolding and maintenance. However, the precise molecular mechanism by which osteoblasts regulate osteoclastogenesis is still largely unknown. Here, we show that osteoblasts can induce Ca 2+ oscillation-independent osteoclastogenesis. We found that bone marrow-derived monocyte/macrophage precursor cells (BMMs) lacking inositol 1,4,5-trisphosphate receptor type2 (IP 3 R2) did not exhibit Ca 2+ oscillation or differentiation into multinuclear osteoclasts in response to recombinant receptor activator of NF-κB ligand/macrophage colony-stimulating factor stimulation. IP 3 R2 knockout BMMs, however, underwent osteoclastogenesis when they were cocultured with osteoblasts or in vivo in the absence of Ca 2+ oscillation. Furthermore, we found that Ca 2+ oscillation-independent osteoclastogenesis was insensitive to FK506, a calcineurin inhibitor. Taken together, we conclude that both Ca 2+ oscillation/calcineurin-dependent and -independent signaling pathways contribute to NFATc1 activation, leading to efficient osteoclastogenesis in vivo .

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Astrocytes regulate hippocampal synaptic plasticity by the Ca dependent release of the N-methyl d-aspartate receptor (NMDAR) co-agonist d-serine. Previous evidence indicated that d-serine release would be regulated by the intracellular Ca release channel IP receptor (IP R), however, genetic deletion of IP R2, the putative astrocytic IP R subtype, had no impact on synaptic plasticity or transmission. Although IP R2 is widely believed to be the only functional IP R in astrocytes, three IP R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP R and the contribution of the three IP R subtypes to Ca signalling. As a proxy for gliotransmission, we found that long-term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP R blocker heparin, and rescued by exogenous d-serine, indicating that astrocytic IP Rs regulate d-serine release. To explore which IP R subtypes are functional in astrocytes, we used pharmacology and two-photon Ca imaging of hippocampal slices from transgenic mice (IP R2 and IP R2 ;3 ). This approach revealed that underneath IP R2-mediated global Ca events are an overlooked class of IP R-mediated local events, occurring in astroglial processes. Notably, multiple IP Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca release by astrocytic IP Rs. GLIA 2017;65:502-513.

Inositol 1,4,5-trisphosphate receptor (IP 3 R) is one of the important calcium channels expressed in the endoplasmic reticulum and has been shown to play crucial roles in various physiological phenomena. Type 3 IP 3 R is expressed in taste cells, but the physiological relevance of this receptor in taste perception in vivo is still unknown. Here, we show that mice lacking IP 3 R3 show abnormal behavioral and electrophysiological responses to sweet, umami, and bitter substances that trigger G-proteincoupled receptor activation. In contrast, responses to salty and acid tastes are largely normal in the mutant mice. We conclude that IP 3 R3 is a principal mediator of sweet, bitter, and umami taste perception and would be a missing molecule linking phospholipase C ␤2 to TRPM5 activation.Taste perception is a pivotal and primitive sensory system for survival in animals. By sensing taste, animals are provided with valuable information about foods (e.g. qualities and nature) and can choose the nutrient-rich foods necessary for living or avoid harmful and toxic substances. There are five taste categories (sweet, bitter, umami, sour, and salty), and recent studies have furthered our understanding of the molecular mechanisms of taste perception, especially for sweet, bitter, and umami tastes (1, 2).For perception of sweet, bitter, and umami taste, phospholipase C ␤2 (PLC␤2) 3 activation through G-protein-coupled receptor (sweet, T1R2 ϩ T1R3; umami, T1R1 ϩ T1R3; bitter, T2Rs) (1, 3-8) and the subsequent activation of PLC␤2 and transient-receptor potential receptor M5 (TRPM5) are necessary (8, 9), but the molecular mechanism by which PLC␤2 activation leads to TRPM5 in vivo is still unclear (2). Several reports have suggested the possible involvement of Ca 2ϩ , probably released from the intracellular stores, in the activation of TRPM5 in heterologously expressed cells (10 -14) and in taste cells (15); however this remains controversial (9). Because PLC␤2 activation actually leads to production of both IP 3 and diacylglycerol, it is an important issue to definitely determine, which is a major player for gustatory systems. To clarify whether IP 3 R is necessary for taste perception in vivo, we analyzed the taste signaling of IP 3 R-deficient mice in this study (16). We found that mice lacking IP 3 R3 showed altered taste recognition for sweet, bitter, and umami, whereas they were indistinguishable from wild-type (WT) mice in their recognition for salty and sour stimuli. However, they showed residual responses to high concentrations of sweets and bitter. Our data present the direct validation that IP 3 R3 is a key molecule in taste perception for sweet, bitter, and umami and also suggest the existence of IP 3 R3-independent taste signal transduction for recognition of high dose of these tastants. EXPERIMENTAL PROCEDURESMice-IP 3 R3-and IP 3 R2-deficient mice were generated as described previously (16), and the mice intercrossed with C57BL/6 mice at least twelve times were used. WT C57BL/6 mice were littermates or purchased from ...

Deranged Ca(2+) signaling and an accumulation of aberrant proteins cause endoplasmic reticulum (ER) stress, which is a hallmark of cell death implicated in many neurodegenerative diseases. However, the underlying mechanisms are elusive. Here, we report that dysfunction of an ER-resident Ca(2+) channel, inositol 1,4,5-trisphosphate receptor (IP(3)R), promotes cell death during ER stress. Heterozygous knockout of brain-dominant type1 IP(3)R (IP(3)R1) resulted in neuronal vulnerability to ER stress in vivo, and IP(3)R1 knockdown enhanced ER stress-induced apoptosis via mitochondria in cultured cells. The IP(3)R1 tetrameric assembly was positively regulated by the ER chaperone GRP78 in an energy-dependent manner. ER stress induced IP(3)R1 dysfunction through an impaired IP(3)R1-GRP78 interaction, which has also been observed in the brain of Huntington's disease model mice. These results suggest that IP(3)R1 senses ER stress through GRP78 to alter the Ca(2+) signal to promote neuronal cell death implicated in neurodegenerative diseases.