Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated Ca 2؉ channels that are located on intracellular Ca 2؉ stores. We previously identified an IP 3R binding protein, termed IP3R binding protein released with IP3 (IRBIT). Because IRBIT is released from IP3R by physiological concentrations of IP3, we hypothesized that IRBIT is a signaling molecule that is released from IP3R and regulates downstream target molecules in response to the production of IP3. Therefore, in this study, we attempted to identify the target molecules of IRBIT, and we succeeded in identifying Na ؉ ͞HCO3 ؊ cotransporter 1 (NBC1) as an IRBIT binding protein. Of the two major splicing variants of NBC1, pancreas-type NBC1 (pNBC1) and kidney-type NBC1 (kNBC1), IRBIT was found to bind specifically to pNBC1 and not to bind to kNBC1. IRBIT binds to the N-terminal pNBC1-specific domain, and its binding depends on the phosphorylation of multiple serine residues of IRBIT. Also, an electrophysiological analysis in Xenopus oocytes revealed that pNBC1 requires coexpression of IRBIT to manifest substantial activity comparable with that of kNBC1, which displays substantial activity independently of IRBIT. These results strongly suggest that pNBC1 is the target molecule of IRBIT and that IRBIT has an important role in pH regulation through pNBC1. Also, our findings raise the possibility that the regulation through IRBIT enables NBC1 variants to have different physiological roles.pH ͉ acidosis ͉ phosphorylation I nositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) are intracellular Ca 2ϩ -release channels that are located on intracellular Ca 2ϩ -storage organelles, mainly the endoplasmic reticulum (ER) (1). IP 3 Rs release Ca 2ϩ from the ER into the cytoplasm and increase the cytoplasmic concentration of Ca 2ϩ in response to the binding of a second messenger, IP 3 . This IP 3 -Ca 2ϩ pathway regulates many biological processes, including cell growth, cell differentiation, apoptosis, synaptic plasticity, secretion, and fertilization (1).We identified (2) an IP 3 R binding protein, termed IP 3 R binding protein released with IP 3 (IRBIT). IRBIT consists of an N-terminal domain (residues 1-104), which contains a serine͞threonine-rich region, and a C-terminal domain (residues 105-530), which has homology with the methylation pathway enzyme S-adenosylhomocysteine hydrolase. We found (2) that the N-terminal amino acids 1-277 of IRBIT are sufficient for the interaction with the IP 3 R and that the interaction between IRBIT and the IP 3 R is inhibited by physiological concentrations of IP 3 , indicating that IRBIT interacts with the IP 3 R in the resting state and dissociates from the IP 3 R when IP 3 production is induced by extracellular stimuli. Therefore, we speculated that IRBIT acts as a signaling molecule that dissociates from the IP 3 R and regulates target proteins in response to IP 3 production, raising the possibility of the existence of an unidentified pathway, the IP 3 -IRBIT pathway.The Na ϩ ͞HCO 3 Ϫ cotransporter 1 (NBC1) is a membrane...

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated intracellular Ca2+ channels. We previously identified an IP3R binding protein, IRBIT, which binds to the IP3 binding domain of IP3R and is dissociated from IP3R in the presence of IP3. In the present study, we showed that IRBIT suppresses the activation of IP3R by competing with IP3 by [3H]IP3 binding assays, in vitro Ca2+ release assays, and Ca2+ imaging of intact cells. Multiserine phosphorylation of IRBIT was essential for the binding, and 10 of the 12 key amino acids in IP3R for IP3 recognition participated in binding to IRBIT. We propose a unique mode of IP3R regulation in which IP3 sensitivity is regulated by IRBIT acting as an endogenous "pseudoligand" whose inhibitory activity can be modulated by its phosphorylation status.

50 of ϳ0.5 M, i.e. it was 50 times more potent than other inositol polyphosphates. Moreover, alkaline phosphatase treatment abolished the interaction, suggesting that the interaction was dualistically regulated by IP 3 and phosphorylation. Immunohistochemical studies and co-immunoprecipitation assays showed the relevance of the interaction in a physiological context. These results suggest that IRBIT is released from activated IP 3 R, raising the possibility that IRBIT acts as a signaling molecule downstream from IP 3 R.The hydrolysis of phosphatidylinositol 4,5-bisphosphate in response to cell surface receptor activation leads to the production of an intracellular second messenger, inositol 1,4,5-trisphosphate (IP 3 ).1 IP 3 mediates the release of Ca 2ϩ from intracellular Ca 2ϩ storage organelles, mainly the endoplasmic reticulum, by binding to its receptor (IP 3 R). In these IP 3 /Ca 2ϩ signaling cascades, IP 3 R works as a signal converter from IP 3 to Ca 2ϩ (1-3). IP 3 R is a tetrameric intracellular IP 3 -gated Ca 2ϩ release channel (3, 4). There are three distinct types of IP 3 R in mammals (5-7). Type 1 IP 3 R (IP 3 R1) is highly expressed in the central nervous system, particularly in the cerebellum (8, 9). Mouse IP 3 R1 is composed of 2749 amino acids (5), and is divided into three functionally distinct regions: the IP 3 -binding domain near the N terminus, the channel-forming domain with six membrane-spanning regions close to the C terminus, and the regulatory domain separating the two regions (10, 11). Deletion mutagenesis analysis of the IP 3 -binding domain has shown that residues 226 -578 of IP 3 R1 are close to the minimum for specific and high affinity ligand binding, thus assigned to the IP 3 binding core (12). The precise gating mechanism of IP 3 R triggered by IP 3 remains unclear, but IP 3 binding induces a substantial but as yet undefined conformational change, which may cause channel opening (10). Besides this channel opening, such IP 3 -induced conformational change has been assumed to be responsible for degradation of IP 3 R (13, 14).The increase in the cytoplasmic Ca 2ϩ concentration resulting from IP 3 R activation regulates the activities of thousands of downstream targets that play key roles in many aspects of cellular processes, including fertilization, development, proliferation, secretion, and synaptic plasticity (1, 2, 15). To control such a vast array of cell functions, Ca 2ϩ signals need to be precisely regulated in terms of space, time and amplitude (2, 15). Such a complex regulation of Ca 2ϩ signals has been partly attributed to the diversity of IP 3 R isoform expression, assembly of heterotetrameric complexes of IP 3 R isoforms, subcellular distributions of IP 3 R, and regulation of IP 3 R by Ca 2ϩ itself, ATP, and phosphorylation (3,4,16). IP 3 R channels are also regulated by their interacting proteins (4, 17), including calmodulin (18, 19), FKBP12 (Refs. 20 -22, but also see Refs. 23 and 24), calcineurin (Refs. 21 and 25, but also see Refs. 23 and 24), ankyrin (26 -28), si...

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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...