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Identification in extracts from AR4-2J cells of inositol 1,4,5-trisphosphate by its susceptibility to inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.

ABSTRACT: The identity of 3H-labelled material ascribed to Ins(1,4,5)P3 in resting or bombesin-stimulated myo-[3H]inositol-labelled AR4-2J cells was investigated by determining its ability to serve as substrate for partially purified Ins(1,4,5)P3/Ins(1,3,4,5)-P4 5-phosphatase and Ins(1,4,5)P3 3-kinase. This 3H-labelled material was metabolized by these two enzymes at rates which were indistinguishable from those for an internal [32P]Ins(1,4,5)P3 standard, establishing its identity as authentic Ins(1,4,5)P3. In addition, and in contrast with findings in earlier studies utilizing substance P as an agonist, prolonged stimulation with bombesin resulted in an increase in an InsP4 which was degraded by Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. These findings serve to confirm the previous estimate of Horstman, Takemura & Putney [(1988) J. Biol. Chem. 263, 15297-15303] for the intracellular concentrations of Ins(1,4,5)P3 in resting (2 microM) and agonist-stimulated (25 microM) AR4-2J cells. The implications of these findings for the physiological regulation of intracellular Ca2+ through this intracellular messenger are discussed.

SUBMITTER: Nogimori K

PROVIDER: S-EPMC1131551 | biostudies-other | 1990 Jul

REPOSITORIES: biostudies-other

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Project description:Osteoclast activity is central to balanced bone turnover to maintain normal bone mass. A specialized osteoclast attachment to bone localizes acid secretion to remove bone mineral; in some cases, attachment is functionally impaired despite normal attachment proteins. The inositol-1,4,5-trisphosphate receptor-1 (IP3R1) is an intracellular calcium channel required for regulation of reversible osteoclast attachment by nitric oxide (NO), an important regulator of both normal and pathological bone degradation. In studies using human osteoclasts produced in vitro, we found that IP3R1 binds an endosomal isoform of the IP3R-associated cGMP-dependent kinase substrate (IRAG). IRAG is a substrate of cGMP-dependent kinase-1 (PKG1) and binds the PKG1 isoform PKG1β, which was the predominant form of PKG1 in human osteoclasts. Western blots of IRAG were consistent with NO-dependent serine phosphorylation of IRAG. An additional effect of PKG1β activity in osteoclasts was disassociation of IP3R1-IRAG complexes, as shown by analysis of IP3R1 complexes and by localization of the proteins within cells. IP3R1-IRAG complexes were stabilized by PKG or Src antagonists, Src activity being a requirement for IP3R1 calcium release downstream of PKG. IP3R1-mediated calcium release regulates cellular detachment in part through the calcium-dependent proteinase μ-calpain. In osteoclasts with IRAG suppressed by siRNA, activity of μ-calpain was increased relative to cells with normal IRAG, and regulation of μ-calpain by NO was lost. Furthermore, cells deficient in IRAG detached easily from substrate and had smaller attached diameters and randomly distributed podosomes, although IRAG knockdown did not affect cell viability. Our results indicate that IRAG is required for PKG1β-regulated cyclic calcium release during motility, and that disruption of the IP3R1-IRAG calcium regulation system is a novel cause of dysfunctional osteoclasts unrelated to defects in attachment proteins or acid secretion.

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Identification in extracts from AR4-2J cells of inositol 1,4,5-trisphosphate by its susceptibility to inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.

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