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Regulation of inositol 1,4,5-trisphosphate receptors by cAMP independent of cAMP-dependent protein kinase.

ABSTRACT: In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP(3)R) to IP(3) that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP(3)R2. Using DT40 cells expressing single subtypes of mammalian IP(3)R, we demonstrate that high concentrations of cAMP similarly sensitize all IP(3)R isoforms to IP(3) by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP(3) binding to IP(3)R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP(3). In single channel recordings from excised nuclear patches of cells expressing IP(3)R2, cAMP alone had no effect, but it increased the open probability of IP(3)R2 activated by a submaximal concentration of IP(3) alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP(3)R subtypes to IP(3). For IP(3)R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP(3). Using stably expressed short hairpin RNA to reduce expression of the G-protein, G alpha(s), we demonstrate that attenuation of AC activity by loss of G alpha(s) more substantially reduces sensitization of IP(3)R by PTH than does comparable direct inhibition of AC. This suggests that G alpha(s) may also specifically associate with each AC x IP(3)R complex. We conclude that all three subtypes of IP(3)R are regulated by cAMP independent of PKA. In HEK cells, where IP(3)R2 selectively associates with AC6, G alpha(s) also associates with the AC x IP(3)R signaling junction.

SUBMITTER: Tovey SC

PROVIDER: S-EPMC2857138 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Regulation of inositol 1,4,5-trisphosphate receptors by cAMP independent of cAMP-dependent protein kinase.

Tovey Stephen C SC Dedos Skarlatos G SG Rahman Taufiq T Taylor Emily J A EJ Pantazaka Evangelia E Taylor Colin W CW

The Journal of biological chemistry 20100226 17

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP(3)R) to IP(3) that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP(3)R2. Using DT40 cells expressing single subtypes of mammalian IP(3)R, we demonstrate that high concentrations of cAMP similarly sensitize all IP(3)R isoforms to IP(3) by a mechanism that does not require cAMP-dependent prot ...[more]

PMID: 20189985

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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.

Dataset Information

Regulation of inositol 1,4,5-trisphosphate receptors by cAMP independent of cAMP-dependent protein kinase.

Publications

Regulation of inositol 1,4,5-trisphosphate receptors by cAMP independent of cAMP-dependent protein kinase.

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