IRBIT, an inositol 1,4,5-trisphosphate receptor-binding protein, specifically binds to and activates pancreas-type Na ⁺ /HCO ₃ ⁻ cotransporter 1 (pNBC1)

Abstract

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...

IRBIT, Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3, Binds Na+/H+ Exchanger NHE3 and Activates NHE3 Activity in Response to Calcium

“…pNBC1 differs from kNBC1 in that it has a unique NH 2 -terminal domain, which is enriched with positively charged residues clustered together between aa 40 and 60. It was proposed that the NH 2 -terminal domain of pNBC1 interacts with the serine-rich PEST domain of IRBIT (see the next paragraph) (6). In the present study, we showed that the region between aa 591 and 696 of NHE3 was essential for its interaction with IRBIT as shown by yeast two-hybrid and co-immunoprecipitation in PS120 cells.…”

“…We first examined whether the interaction between IRBIT and NHE3 was regulated by thapsigargin or ionomycin treatment, because a previous study demonstrated that binding of IRBIT to pNBC1 was increased by the presence of Ca 2ϩ in lysate buffer (6). On the contrary, we found that the IRBIT-NHE3 interaction was not regulated by elevation of Ca 2ϩ by either thapsigargin or ionomycin treatments (Fig.…”

“…Therefore, we conclude that the Ca 2ϩ /CaM/CaMKII signaling axis regulates IRBIT-dependent trafficking of NHE3. This mechanism of NHE3 regulation differs from that of pNBC1 in which co-expression of IRBIT in Xenopus oocytes did not affect the surface expression of pNBC1 despite the activation of the transport activity (6).…”

“…IRBIT inhibits activation of IP 3 R and Ca 2ϩ release by competing with IP 3 (5). Interestingly, IRBIT binds to the pancreastype electrogenic Na ϩ /HCO 3 Ϫ cotransporter 1 (pNBC1) and co-expression of IRBIT and pNBC1 in Xenopus oocytes activated Na ϩ /HCO 3 Ϫ cotransporter activity (6). Na ϩ /H ϩ exchange is a process present in all organisms from prokaryotes to human (7).…”

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) stimulates the electrogenic Na/HCO 3 cotransporter NBCe1-A expressed in Xenopus oocytes

“…Such automodulation may involve additional binding proteins and signaling molecules. Indeed, Shirakabe et al (9) reported that the IP 3 receptor binding protein released with IP 3 (IRBIT) can stimulate NBCe1-B co-expressed in oocytes by binding to the transporter's amino terminus and masking an autoinhibitory domain.…”