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The process of developing economic relations between Irbit and Tobolsk in the 18th century is discussed in the article. The authors come to the conclusion that the mutual trade interests realized at the Irbit Fair played a system-forming role in the process of folding and strengthening the administrative and social ties between these settlements. The process of formation of trade relations has been studied in detail. Its main content was a long way from competition to mutually beneficial cooperation between cities. An analysis of the decrees of the Tobolsk Ecclesiastical Consistory of the 18th century, as well as the generalization and systematization of the results of a study in the field of the fair trading development on the territory of the Irbit settlement and the Tobolsk province in the middle of the 17th — 18th centuries, made it possible to determine the influence of the Ural shopping center on the economy of the ancient capital of Siberia. The most common goods of the Siberian merchants sold at the Irbit Fair in the 18th century have been identified. In the course of studying the reporting and financial documentation and office materials, it was concluded that church goods and red wine were in special demand, which were later purchased for the Tobolsk clergy in large quantities on the basis of a collective order from different settlements of the Tobolsk province. It is noted that during the period under review, the Tobolsk province provided administrative assistance to the Irbitskaya Sloboda in matters of ensuring “peace” at the fair. The authors of the article show that an important result of the close mutually beneficial cooperation between the Irbitskaya Sloboda and the Tobolsk province was the development of economic ties both within the Russian Empire and beyond its borders.

Abstract Background Lung cancer is one of the most frequently diagnosed cancers characterized by high mortality, metastatic potential, and recurrence. Deregulated gene expression of lung cancer, likewise in many other solid tumors, accounts for their cell heterogeneity and plasticity. S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as Inositol triphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT), plays roles in many cellular functions, including autophagy and apoptosis but AHCYL1 role in lung cancer is largely unknown. Results Here, we analyzed the expression of AHCYL1 in Non-Small Cell Lung Cancer (NSCLC) cells from RNA-seq public data and surgical specimens, which revealed that AHCYL1 expression is downregulated in tumors and inverse correlated to proliferation marker Ki67 and the stemness signature expression. AHCYL1-silenced NSCLC cells showed enhanced stem-like properties in vitro, which correlated with higher expression levels of stem markers POU5F1 and CD133. Also, the lack of AHCYL1 enhanced tumorigenicity and angiogenesis in mouse xenograft models highlighting stemness features. Conclusions These findings indicate that AHCYL1 is a negative regulator in NSCLC tumorigenesis by modulating cell differentiation state and highlighting AHCYL1 as a potential prognostic biomarker for lung cancer.

Background: Lung cancer is one of the most frequently diagnosed cancers characterized by high mortality, metastatic potential, and recurrence. Deregulated gene expression of lung cancer, likewise in many other solid tumors, accounts for their cell heterogeneity and plasticity. S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as Inositol triphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT), plays roles in many cellular functions, including autophagy and apoptosis but AHCYL1 role in lung cancer is largely unknown. Results: Here, we analyzed the expression of AHCYL1 in Non-Small Cell Lung Cancer (NSCLC) cells from RNA-seq public data and surgical specimens, which revealed that AHCYL1 expression is downregulated in tumors and inverse correlated to proliferation marker Ki67 and the stemness signature expression. AHCYL1-silenced NSCLC cells showed enhanced stem-like properties in vitro, which correlated with higher expression levels of stem markers POU5F1 and CD133. Also, the lack of AHCYL1 enhanced tumorigenicity and angiogenesis in mouse xenograft models highlighting stemness features. Conclusions: These findings indicate that AHCYL1 is a negative regulator in NSCLC tumorigenesis by modulating cell differentiation state and highlighting AHCYL1 as a potential prognostic biomarker for lung cancer. [ABSTRACT FROM AUTHOR]

Key points: The electrogenic Na+/HCO3−cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT).The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies.Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B.We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain. The electrogenic Na+/HCO3− cotransporter NBCe1‐B is widely expressed in many tissues in the body. NBCe1‐B exhibits only basal activity due to the action of the auto‐inhibition domain (AID) in its unique amino‐terminus. However, NBCe1‐B can be activated by interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto‐inhibition of NBCe1‐B and its activation by IRBIT. The IRBIT‐binding domain (IBD) of NBCe1‐B spans residues 1−52, essentially consisting of two arms, one negatively charged (residues 1−24) and the other positively charged (residues 40−52). The AID mainly spans residues 40−85, overlapping with the IBD in the positively charged arm. The magnitude of auto‐inhibition of NBCe1‐B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1‐B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1‐B; (2) IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain. Key points: The electrogenic Na+/HCO3−cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT).The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies.Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B.We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain. [ABSTRACT FROM AUTHOR]

Inositol 1,4,5-trisphosphate (IP3) stimulates Ca2+ release from the endoplasmic reticulum (ER), and the response is potentiated by 3′,5′-cyclic AMP (cAMP). We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH) to stimulate formation of IP3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca2+ concentration, but it potentiated the Ca2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP3 receptors within a discrete Ca2+ store. We conclude that Ca2+ stores within the ER that dynamically exchange Ca2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca2+ stores adds versatility to IP3-evoked Ca2+ signals.

IP3 receptor-binding protein released with IP3 (IRBIT) interacts with various ion channels and transporters. An electroneutral type of sodium bicarbonate cotransporter, NBCn1, participates in cell migration, and its enhanced expression is related to cancer metastasis. The effect of IRBIT on NBCn1 and its relation to cancer cell migration remain obscure. We therefore aimed to determine the effect of IRBIT on NBCn1 and the regulation of cancer cell migration due to IRBIT-induced alterations in NBCn1 activity. Overexpression of IRBIT enhanced cancer cell migration and NBC activity. Knockdown of IRBIT or NBCn1 and treatment with an NBC-specific inhibitor, S0859, attenuated cell migration. Stimulation with oncogenic epidermal growth factor enhanced the expression of NBCn1 and migration of cancer cells by recruiting IRBIT. The recruited IRBIT stably maintained the expression of the NBCn1 transporter machinery in the plasma membrane. Combined inhibition of IRBIT and NBCn1 dramatically inhibited the migration of cancer cells. Combined modulation of IRBIT and NBCn1 offers an effective strategy for attenuating cancer metastasis.

The calcium ion (Ca 2+ ) is a ubiquitous intracellular signaling molecule that regulates diverse physiological and pathological processes, including cancer. Increasing evidence indicates that oncogenes and tumor suppressors regulate the Ca 2+ transport systems. Inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) are IP 3 -activated Ca 2+ release channels located on the endoplasmic reticulum (ER). They play pivotal roles in the regulation of cell death and survival by controlling Ca 2+ transfer from the ER to mitochondria through mitochondria-associated ER membranes (MAMs). Optimal levels of Ca 2+ mobilization to mitochondria are necessary for mitochondrial bioenergetics, whereas excessive Ca 2+ flux into mitochondria causes loss of mitochondrial membrane integrity and apoptotic cell death. In addition to well-known functions on outer mitochondrial membranes, B-cell lymphoma 2 (Bcl-2) family proteins are localized on the ER and regulate IP 3 Rs to control Ca 2+ transfer into mitochondria. Another regulatory protein of IP 3 R, IP 3 R-binding protein released with IP 3 (IRBIT), cooperates with or counteracts the Bcl-2 family member depending on cellular states. Furthermore, several oncogenes and tumor suppressors, including Akt, K-Ras, phosphatase and tensin homolog (PTEN), promyelocytic leukemia protein (PML), BRCA1, and BRCA1 associated protein 1 (BAP1), are localized on the ER or at MAMs and negatively or positively regulate apoptotic cell death through interactions with IP 3 Rs and regulation of Ca 2+ dynamics. The remodeling of Ca 2+ signaling by oncogenes and tumor suppressors that interact with IP 3 Rs has fundamental roles in the pathology of cancers. [ABSTRACT FROM AUTHOR]

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, 922FMDRLK927, in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922-927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO3- transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO3- secretion in several types of epithelia. [ABSTRACT FROM AUTHOR]

IRBIT [inositol 1,4,5-trisphosphate receptor (IP3R) binding protein released with inositol 1,4,5-trisphosphate (IP3)] is a multifunctional protein that regulates several target molecules such as ion channels, transporters, polyadenylation complex, and kinases. Through its interaction with multiple targets, IRBIT contributes to calcium signaling, electrolyte transport, mRNA processing, cell cycle, and neuronal function. However, the regulatory mechanism of IRBIT binding to particular targets is poorly understood. Long-IRBIT is an IRBIT homolog with high homology to IRBIT, except for a unique N-terminal appendage. Long-IRBIT splice variants have different N-terminal sequences and a common C-terminal region, which is involved in multimerization of IRBIT and Long-IRBIT. In this study, we characterized IRBIT and Long-IRBIT splice variants (IRBIT family). We determined that the IRBIT family exhibits different mRNA expression patterns in various tissues. The IRBIT family formed homo- and heteromultimers. In addition, N-terminal splicing of Long-IRBIT changed the protein stability and selectivity to target molecules. These results suggest that N-terminal diversity of the IRBIT family and various combinations of multimer formation contribute to the functional diversity of the IRBIT family. [ABSTRACT FROM AUTHOR]

Relevance of the discussed issue. Irbit diatomite deposit is one of the largest in the Russian Federation and in Trans-Urals region and it is currently used for production of low value-added goods. Despite the long history of industrial exploration, the comprehensive studies of the material composition of Irbit diatomites were never held, chemical and mineralogical features of these rocks were studied. Lack of information about the material composition of rocks does not allow planning their use in new high-tech industries. Detailed studies of diatomite material composition will allow planning their use in new high-tech industries. The main aim of the research is the comprehensive study of the material composition Irbit diatomite deposits in order to determine the prospects of expanding the scope of their use. The methods used in the research: field studies, X-ray diffraction, X-ray fluorescence analysis, infrared spectroscopy, and inductively coupled plasma mass spectrometry, differential thermal analysis, scanning electron microscopy, lithological and petrographic analysis. The results. The author has studied material composition of Irbit diatomite deposit using modern instrumental techniques. Generally, diatomite consists of silicon dioxide with signs of its partial crystallization into cristobalite and a-quartz with a small presence of cristobalite and it is characterized by a high degree of homogeneity of chemical and mineralogical composition. Fine fractions mostly consist of clay that allows planning rock cleaning due to their removal. The association of the largest metal content to less than 0,005 mm fractions can be taken into account when planning rock enrichment. The occurrence of swelling clay minerals defines the necessity to maintain the solid-liquid phase ratio within rather high range to ensure sufficient mobility of the slurry under agitation when leaching. The total content of essential microelements does not affect the use of diatomites for producing filter materials, fillers, catalysts etc. High silica content allows us to consider diatomite as a promising raw material for refractories, abrasives, as well as diatoms of high purity concentrates, raw materials for chemical, pharmaceutical and glass industry.

Summary Inositol 1,4,5-trisphosphate (IP 3 ) stimulates Ca 2+ release from the endoplasmic reticulum (ER), and the response is potentiated by 3′,5′-cyclic AMP (cAMP). We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH) to stimulate formation of IP 3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca 2+ concentration, but it potentiated the Ca 2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca 2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP 3 receptors within a discrete Ca 2+ store. We conclude that Ca 2+ stores within the ER that dynamically exchange Ca 2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca 2+ stores adds versatility to IP 3 -evoked Ca 2+ signals. [ABSTRACT FROM AUTHOR]

Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiledcoil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B. [ABSTRACT FROM AUTHOR]

IP 3 receptor (IP 3 R) was found to release Ca 2+ from non-mitochondrial store but the exact localization and the mode of action of IP 3 remained a mystery. IP 3 R was identified to be P 400 protein, a protein, which was missing in the cerebellum of ataxic mutant mice lacking Ca 2+ spikes in Pukinje cells. IP 3 R was an IP 3 binding protein and was a Ca 2+ channel localized on the endoplasmic reticulum. Full-length cDNA of IP 3 R type 1 was initially cloned and later two other isoforms of IP 3 R (IP 3 R type 2 and type 3) were cloned in vertebrates. Interestingly, the phosphorylation sites, splicing sites, associated molecules, IP 3 binding affinity and 5′ promoter sequences of each isoform were different. Thus each isoform of IP 3 receptor plays a role as a signaling hub offering a unique platform for matching various functional molecules that determines different trajectories of cell signaling. Because of this distinct role of each isoform of IP 3 R, the dysregulation of IP 3 receptor causes various kinds of diseases in human and rodents such as ataxia, vulnerability to neuronal degeneration, heart disease, exocrine secretion deficit, taste perception deficit. Moreover, IP 3 was found not only to release Ca 2+ , but also to release IRBIT ( I P 3 r eceptor b inding protein released with i nositol t risphosphate) essential for the regulation of acid-base balance, RNA synthesis and ribonucleotide reductase. [ABSTRACT FROM AUTHOR]

IRBIT (also called AHCYL1) was originally identified as a binding protein of the intracellular Ca 2 + channel inositol 1,4,5-trisphosphate (IP 3 ) receptor and functions as an inhibitory regulator of this receptor. Unexpectedly, many functions have subsequently been identified for IRBIT including the activation of multiple ion channels and ion transporters, such as the Na + /HCO 3 − co-transporter NBCe1-B, the Na + /H + exchanger NHE3, the Cl − channel cystic fibrosis transmembrane conductance regulator (CFTR), and the Cl − /HCO 3 − exchanger Slc26a6. The characteristic serine-rich region in IRBIT plays a critical role in the functions of this protein. In this review, we describe the evolution, domain structure, expression pattern, and physiological roles of IRBIT and discuss the potential molecular mechanisms underlying the coordinated regulation of these diverse ion channels/transporters through IRBIT. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau. [ABSTRACT FROM AUTHOR]

Abstract: The Ca2+ and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca2+ and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca2+ and cAMP signaling operate at 5–10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca2+ and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca2+ and cAMP signaling and the function of IRBIT (IP3 receptors binding protein release with IP3) as a third messenger that mediates the synergistic action of the Ca2+ and cAMP signaling. [Copyright &y& Elsevier]

Highlights: [•] AHCYL2 is a novel binding partner of the N-terminus of NBCe1-B. [•] AHCYL2 interacts with NBCe1-B in a native HCO3 −-secreting exocrine gland. [•] AHCYL2 lowers Mg2+-sensitivity of NBCe1-B in a HCO3 −-deficient cellular condition. [•] Our data may uncover a novel role of AHCYL2 in the regulation of NBCe1-B. [ABSTRACT FROM AUTHOR]

Highlights: [•] IRBIT plays a putative role in pHi regulation in HSG cells. [•] In addition to NHE1, NHE3 is also involved in pHi recovery in HSG cells. [•] IRBIT regulates pHi via its effect on membrane NHE3 translocation. [•] IRBIT-induced membrane NHE3 translocation is further regulated by SPAK. [Copyright &y& Elsevier]

Abstract: The electrogenic Na+-HCO3 − cotransporter NBCe1-B can be regulated by intracellular Mg2+ (Mg2+ i). We previously reported that under whole-cell voltage-clamp conditions, bovine NBCe1-B (bNBCe1-B) currents heterologously expressed in mammalian cells are strongly inhibited by Mg2+ i, and the inhibition is likely mediated by electrostatic interaction and relieved by truncation of the cytosolic NBCe1-B specific N-terminal region. Intriguingly, NBCe1-B-like currents natively expressed in bovine parotid acinar (BPA) cells are much less sensitive to Mg2+ i inhibition than bNBCe1-B currents. Here, we hypothesized that this apparent discrepancy may involve IRBIT, a previously identified NBCe1-B-interacting protein. RT-PCR, Western blot and immunofluorescence confocal microscopy revealed that IRBIT was not only expressed in the cytosol, but also colocalized with NBCe1-B in the region of plasma membranes of BPA cells. IRBIT was coimmunoprecipitated with NBCe1-B by an anti-NBCe1 antibody in bovine parotid cell lysate. Whole-cell patch-clamp experiments showed that coexpression of IRBIT lowered the Mg2+ i sensitivity of bNBCe1-B currents stably expressed in HEK293 cells. Collectively, these results suggest that IRBIT may reduce the apparent affinity for Mg2+ i in inhibition of NBCe1-B activity in mammalian cells. [Copyright &y& Elsevier]

Background: Huntington's Disease (HD) is a fatal hereditary neurodegenerative disease caused by the accumulation of mutant huntingtin protein (Htt) containing an expanded polyglutamine (polyQ) tract. Activation of the channel responsible for the inositol-induced Ca2+ release from ensoplasmic reticulum (ER), was found to contribute substantially to neurodegeneration in HD. Importantly, chemical and genetic inhibition of inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1) has been shown to reduce mutant Htt aggregation. Results: In this study, we propose a novel regulatory mechanism of IP3R1 activity by type III intermediate filament vimentin which sequesters the negative regulator of IP3R1, IRBIT, into perinuclear inclusions, and reduces its interaction with IP3R1 resulting in promotion of mutant Htt aggregation. Proteasome inhibitor MG132, which causes polyQ proteins accumulation and aggregation, enhanced the sequestration of IRBIT. Furthermore we found that IRBIT sequestration can be prevented by a rho kinase inhibitor, Y-27632. Conclusions: Our results suggest that vimentin represents a novel and additional target for the therapy of polyQ diseases. [ABSTRACT FROM AUTHOR]

IRBIT (IPRs binding protein released with IP) is a protein originally identified by the Mikoshiba group as an inhibitor of IP receptors function. Subsequently it was found to have multiple functions and regulate the activity of diverse proteins, including regulation of HCO transporters to coordinate epithelial HCO secretion and to determine localization of the Fip1 subunit of the CPSF complex to regulate mRNA processing. This review highlights the remarkably divers functions of IRBIT that are likely only a fraction of all the potential functions of this protein. [ABSTRACT FROM AUTHOR]

IRBIT is an inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-binding protein that inhibits the activation of IP3R by competing with IP3 for the common binding site on IP3R. In this study, we characterize an IRBIT homologue, termed Long-IRBIT. Long-IRBIT is highly homologous to IRBIT (∼88%) and heteromerizes with IRBIT. In spite of complete conservation of critical amino acids required for the interaction with IP3R and comparable phosphorylations on critical four Ser residues for IP3R-binding, Long-IRBIT retains little ability to interact with IP3R. Deletion mutagenesis analysis revealed that this low affinity to IP3R is attributable to an inhibitory effect of the Long-IRBIT specific N-terminal appendage (LISN domain). Immunohistochemical analysis revealed the distinct distribution of Long-IRBIT and IRBIT in mouse cerebellar cortex, that is, Long-IRBIT is mainly expressed in interneurons such as basket cells, while IRBIT is mainly expressed in glial cells. Furthermore, Long-IRBIT, but not IRBIT, underwent phosphorylation during neuronal differentiation in neuroblastoma cells and this phosphorylation was dependent on the LISN domain. These results suggest that Long-IRBIT has a different function from IRBIT. [ABSTRACT FROM AUTHOR]

Abstract: Adenosylhomocysteine hydrolase (SAHase)-like protein 1 (SAH-L), also called inositol 1,4,5-triphosphate receptor-binding protein (IRBIT) is a novel protein involved in fish embryo development and calcium release in mammalian cells through protein–protein interactions. To better understand its reaction mechanism, purified protein is indispensable. Here we describe a simple purification procedure and the unique properties of SAH-L. The cDNA was isolated from mouse kidney by RT-PCR and inserted into various pET™ vectors. Escherichia coli harboring a plasmid coding for SAH-L with a C-terminal His-tag could solely produce a soluble protein. SAH-L purified through a Ni2+ column gave M rs of 59,000 and 190,000 by SDS-PAGE and gel filtration, respectively, which is suggestive of a trimer, but chemical cross-linking experiments demonstrated a dimer. The incompatible M r values implicate an irregular structure of SAH-L. In fact, SAH-L was partially purified in a form lacking the 31 N-terminal residues, and was found to be extremely susceptible to proteases in the region around residue 70. The N-terminal polypeptide (residues 1–98) was also expressed as a soluble form and was trypsin-sensitive. Circular dichroism revealed a low α-helix content but not a randomly extended structure. Interestingly, SAH-L contained tightly bound NAD+ despite showing no SAHase activity. The characterized properties of SAH-L and its N-terminal fragment present the notion that the structure of the protease-sensitive N-terminal region is relatively loose and flexible rather than compact, and which protrudes from the major SAHase-like domain. This structure is supposed to be favorable to interact with the IP3 receptor. [Copyright &y& Elsevier]

Abstract: The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can be divided in three functionally distinct regions: a ligand-binding domain, a modulatory domain and a channel domain. Numerous regulatory mechanisms including inter- and intra-molecular protein–protein interactions and phosphorylation events act via these domains to regulate the function of the IP3R. Regulation at the level of the ligand-binding domain primarily affects the affinity for IP3. The extent of IP3-induced Ca2+ release (IICR) is, however, not only determined by the affinity for IP3 but also by the effectiveness of the coupling between ligand binding and channel opening. As a result, regulation as well as malfunction of IICR may be affected by both steps in the activation mechanism. The 3D structures of the two subdomains of the ligand-binding domain have recently been determined by X-ray diffraction analysis. This allows a more detailed molecular explanation of the regulatory events situated at the ligand-binding domain of the IP3R. In this review, we will focus on recent structural and functional data on the ligand-binding domain that have extended and clarified the view on the molecular mechanisms of IP3R regulation. [Copyright &y& Elsevier]

Inositol 1,4,5-trisphosphate (IP3) is a second messenger that induces the release of Ca2+ from the endoplasmic reticulum (ER). The IP3 receptor (IP3R) was discovered as a developmentally regulated glyco-phosphoprotein, P400, that was missing in strains of mutant mice. IP3R can allosterically and dynamically change its form in a reversible manner. The crystal structures of the IP3-binding core and N-terminal suppressor sequence of IP3R have been identified. An IP3 indicator (known as IP3R-based IP3 sensor) was developed from the IP3-binding core. The IP3-binding core’s affinity to IP3 is very similar among the three isoforms of IP3R; instead, the N-terminal IP3 binding suppressor region is responsible for isoform-specific IP3-binding affinity tuning. Various pathways for the trafficking of IP3R have been identified; for example, the ER forms a meshwork upon which IP3R moves by lateral diffusion, and vesicular ER subcompartments containing IP3R move rapidly along microtubles using a kinesin motor. Furthermore, IP3R mRNA within mRNA granules also moves along microtubules. IP3Rs are involved in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP3R1 activity. IP3 has been found to release Ca2+, but it also releases IRBIT (IP3R-binding protein released with IP3). IRBIT is a pseudo-ligand for IP3 that regulates the frequency and amplitude of Ca2+ oscillations through IP3R. IRBIT binds to pancreas-type Na, bicarbonate co-transporter 1, which is important for acid-base balance. The presence of many kinds of binding partners, like homer, protein 4.1N, huntingtin-associated protein-1A, protein phosphatases (PPI and PP2A), RACK1, ankyrin, chromogranin, carbonic anhydrase-related protein, IRBIT, Na,K-ATPase, and ERp44, suggest that IP3Rs form a macro signal complex and function as a center for signaling cascades. The structure of IP3R1, as revealed by cryoelectron microscopy, fits closely with these molecules. [ABSTRACT FROM AUTHOR]

Abstract: IRBIT has previously been shown to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in an IP3-sensitive way. So far it remained to be elucidated whether this interaction was direct or indirect, and whether it was functionally relevant. We now show that IRBIT can directly interact with the IP3R, and that both the suppressor domain and the IP3-binding core of the IP3R are essential for a strong interaction. Moreover, we identified a PEST motif and a PDZ-ligand on IRBIT which were critical for the interaction with the IP3R. Furthermore, we identified Asp-73 as a critical residue for this interaction. Finally, we demonstrated that this interaction functionally affects the IP3R: IRBIT inhibits both IP3 binding and IP3-induced Ca2+ release. [Copyright &y& Elsevier]

IP3 receptor-binding protein released with IP3 (IRBIT) interacts with various ion channels and transporters. An electroneutral type of sodium bicarbonate cotransporter, NBCn1, participates in cell migration, and its enhanced expression is related to cancer metastasis. The effect of IRBIT on NBCn1 and its relation to cancer cell migration remain obscure. We therefore aimed to determine the effect of IRBIT on NBCn1 and the regulation of cancer cell migration due to IRBIT-induced alterations in NBCn1 activity. Overexpression of IRBIT enhanced cancer cell migration and NBC activity. Knockdown of IRBIT or NBCn1 and treatment with an NBC-specific inhibitor, S0859, attenuated cell migration. Stimulation with oncogenic epidermal growth factor enhanced the expression of NBCn1 and migration of cancer cells by recruiting IRBIT. The recruited IRBIT stably maintained the expression of the NBCn1 transporter machinery in the plasma membrane. Combined inhibition of IRBIT and NBCn1 dramatically inhibited the migration of cancer cells. Combined modulation of IRBIT and NBCn1 offers an effective strategy for attenuating cancer metastasis. [ABSTRACT FROM AUTHOR]

The I P3 r eceptor b inding protein released with i nositol 1,4,5- t risphosphate (IRBIT) plays important roles in the regulation of intracellular Ca2+ signaling and intracellular pH. The mammals express two IRBIT paralogs, i.e., IRBIT1 (encoded by AHCYL1) and IRBIT2 (encoded by AHCYL2). The clawed frog Xenopus laevis oocyte is widely used for biophysical studies on ion channels and transporters. It remains unknown whether endogenous IRBIT is expressed in Xenopus oocytes. Here, we cloned from frog oocyte irbit2.L and irbit2.S, orthologs of mammalian IRBIT2. When over-expressed, the frog IRBITs powerfully stimulate the electrogenic Na+/HCO 3 – cotransporter NBCe1-B as mouse IRBIT2-V2 does. Expression of an isolated Nt fragment of NBCe1-B containing the IRBIT-binding domain greatly decreases NBCe1-B activity in oocytes, suggesting that the basal activity of NBCe1-B contains a large component derived from the stimulation by endogenous frog IRBIT. The frog IRBITs are highly homologous to the mammalian ones in the carboxyl-terminal region, but varies greatly in the amino-terminal (Nt) appendage. Interestingly, truncation study showed that the Nt appendage of IRBIT1 and the long Nt appendage of IRBIT2-V2 modestly enhance, whereas the short Nt appendage of IRBIT2-V4 greatly inhibits the functional interaction between IRBIT and NBCe1-B. Finally, Ala-substitution of Ser68, a key phosphorylation site in the PEST domain of IRBIT, causes distinct functional consequences depending on the structural context of the Nt appendage in different IRBIT isoforms. We conclude that the Nt appendage of IRBITs is not necessary for, but plays an important regulatory role in the functional interaction between IRBIT and NBCe1-B. Unlabelled Image • By protein interaction, the mammalian IRBIT can modulate the function of a series of membrane channels and transporters. • Xenopus laevis oocyte expresses two endogenous IRBITs that are highly homologous to the mammalian ones. • When over-expressed, the Xenopus IRBITs can fully activate mouse NBCe1-B. • Nt appendage of IRBITs affect the effect of mutating the phosphorylation site in the PEST domain. [ABSTRACT FROM AUTHOR]

Background/aim: We investigated whether the expression of inositol 1, 4, 5-trisphosphate receptor-binding protein released with inositol 1, 4, 5-trisphosphate (IRBIT) in clinical gastric cancer (GC) patients could predict the therapeutic response to postoperative adjuvant chemotherapy., Materials and Methods: Immunohistochemistry was used to investigate IRBIT expression in 115 GC patients. To clarify whether IRBIT had a relationship with the therapeutic effects of chemotherapy, we compared two groups - 62 patients treated with postoperative adjuvant chemotherapy and 53 patients treated with postoperative adjuvant chemotherapy., Results: Regarding the postoperative adjuvant chemotherapy-free group, we did not find any statistically significant correlation between clinicopathological features and recurrence regardless of the expression of IRBIT. In contrast, in the group receiving postoperative adjuvant chemotherapy, a significant association was found between IRBIT expression and both overall and disease-free survival., Conclusion: IRBIT may be used as a useful predictive marker for chemotherapy., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Anion exchanger 2 (AE2) has a critical role in epithelial cells and is involved in the ionic homeostasis such as Cl(-) uptake and HCO3(-) secretion. However, little is known about the regulatory mechanism of AE2. The main goal of the present study was to investigate potential regulators, such as spinophilin (SPL), inositol-1,4,5-trisphosphate [IP3] receptors binding protein released with IP3 (IRBIT), STE20/SPS1-related proline/alanine-rich kinase (SPAK) kinase, and carbonic anhydrase XII (CA XII). We found that SPL binds to AE2 and markedly increased the Cl(-)/HCO3(-) exchange activity of AE2. Especially SPL 1-480 domain is required for enhancing AE2 activity. For other regulatory components that affect the fidelity of fluid and HCO3(-) secretion, IRBIT and SPAK had no effect on the activity of AE2 and no protein-protein interaction with AE2. It has been proposed that CA activity is closely associated with AE activity. In this study, we provide evidence that the basolateral membrane-associated CA isoform CA XII significantly increased the activity of AE2 and co-localized with AE2 to the plasma membrane. Collectively, SPL and CA XII enhanced the Cl(-)/HCO3(-) exchange activity of AE2. The modulating action of these regulatory proteins could serve as potential therapeutic targets for secretory diseases mediated by AE2.