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 .