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...

Enlargeosomes are small cytoplasmic vesicles that undergo rapid, Ca2+‐dependent exo/endocytosis. The role of the cytoskeleton in these processes was unknown. In PC12‐27 cells, microtubule disassembly had little effect on enlargeosomes, whereas microfilament disassembly increased markedly both their resting and stimulated exocytosis, and inhibited their endocytosis. Even at rest enlargeosomes are coated at their cytosolic surface by an actin‐associated protein, annexin2, bound by a dual, Ca2+‐dependent and Ca2+‐independent mechanism. In contrast, the other enlargeosome marker, desmoyokin/Ahnak, is transported across the organelle membrane, apparently by an ABC transporter, and binds to its lumenal face. Annexin2‐GFP expression revealed that, upon stimulation, the slow and random enlargeosome movement increases markedly and becomes oriented toward the plasma membrane. After annexin2 downregulation enlargeosome exocytosis induced by both [Ca2+]i rise and cytoskeleton disruption is inhibited, and the NGF‐induced differentiation is blocked. Binding of annexin2 to the enlargeosome membrane, the most extensive ever reported (>50% annexin2 bound to ∼3% of total membrane area), seems therefore to participate in the regulation of their exocytosis.

No abstract

Glycogen storage disease (GSD) 1b is the deficiency of endoplasmic reticulum glucose-6-phosphate (G6P) transport. We here report the structure of the gene encoding a protein likely to be responsible for G6P transport, and its mapping to human chromosome 11q23.3. The gene is composed of nine exons spanning a genomic region of approximately 4 kb. Primers based on the genomic sequence were used in single strand conformation polymorphism (SSCP) analysis and mutations were found in six out of seven GSD 1b patients analysed.z 1998 Federation of European Biochemical Societies.

SUMMARYWe demonstrate that human 2Ј-deoxycytidine kinase (dCK) is a nonenantioselective enzyme because it phosphorylates ␤-D-2Ј-deoxycytidine (D-dCyd), the natural substrate, and ␤-L-2Ј-deoxycytidine (L-dCyd), its enantiomer, with the same efficiency. Kinetic studies showed that L-dCyd is a competitive inhibitor of the phosphorylation of D-dCyd with a K i value of 0.12 M, which is lower than the K m value for D-dCyd (1.2 M). Chemical modifications of either the base or the pentose ring strongly decrease the inhibitory potency of L-dCyd. L-dCyd is resistant to cytidine deaminase and competes in cell cultures with the natural D-dCyd as substrate for dCK, thus reducing the incorporation of exogenous [ 3 H]dCyd into DNA. L-dCyd had no effect on the pool of dTTP deriving from the salvage or from the de novo synthesis, does not inhibit short term RNA and protein syntheses, and shows little or no cytotoxicity. Our results indicate a catalytic similarity between human dCK and herpetic thymidine kinases, enzymes that also lack stereospecificity. This functional analogy underlines the potential role of dCK as activator of L-deoxycytidine analogs as antiviral and antineoplastic agents and lends support to the hypothesis that herpesvirus thymidine kinase might have evolved from a captured cellular dCK gene, developing the ability to phosphorylate thymidine and retaining that to phosphorylate deoxycytidine.