Regulation of inositol 1,4,5-trisphosphate receptors by cAMP independent of cAMP-dependent protein kinase.
Regulation of inositol 1,4,5-trisphosphate receptors by cAMP independent of cAMP-dependent protein kinase.
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DOI：
10.1074/jbc.m109.096016
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发表时间：
2010-04-23
期刊：
影响因子：
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通讯作者：
Taylor CW
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文献类型：
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作者：
Tovey SC;Dedos SG;Rahman T;Taylor EJ;Pantazaka E;Taylor CW
在稳定表达甲状旁腺激素（PTH）的1型受体的HEK细胞中，PTH引起肌醇1，4，5-三磷酸受体（IP 3R）对IP 3的敏化，其完全由cAMP介导，并且需要cAMP直接从6型腺苷酸环化酶（AC 6）传递到IP 3R 2。使用DT 40细胞表达单一亚型的哺乳动物IP 3R，我们表明，高浓度的cAMP同样敏感的所有IP 3R亚型IP 3的机制，不需要cAMP依赖性蛋白激酶（PKA）。IP 3与IP 3R 2的结合不受cAMP的影响，并且致敏作用不受ATP增强对IP 3反应的位点介导。在单通道记录从切除的细胞核补丁表达IP 3R 2，cAMP单独没有影响，但它增加了开放的概率IP 3R 2激活的亚最大浓度的IP 3单独或与最大有效浓度的ATP。这些结果证实cAMP本身增加了所有IP 3R亚型对IP 3的敏感性。对于IP 3R 2，这种致敏作用是由cAMP结合到增加IP 3功效的新位点引起的。使用稳定表达的短发夹RNA来减少G蛋白Gαs的表达，我们证明，与AC的直接抑制相比，通过Gαs的缺失来减弱AC活性更显著地降低了PTH对IP 3R的致敏作用。这表明Gαs也可能与每个AC·IP 3R复合物特异性结合。我们的结论是，所有三种亚型的IP 3R调节cAMP的PKA的独立。在HEK细胞中，IP 3R 2选择性地与AC 6结合，Gαs也与AC·IP 3R信号连接结合。
In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP3R) to IP3 that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP3R2. Using DT40 cells expressing single subtypes of mammalian IP3R, we demonstrate that high concentrations of cAMP similarly sensitize all IP3R isoforms to IP3 by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP3 binding to IP3R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP3. In single channel recordings from excised nuclear patches of cells expressing IP3R2, cAMP alone had no effect, but it increased the open probability of IP3R2 activated by a submaximal concentration of IP3 alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP3R subtypes to IP3. For IP3R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP3. Using stably expressed short hairpin RNA to reduce expression of the G-protein, Gαs, we demonstrate that attenuation of AC activity by loss of Gαs more substantially reduces sensitization of IP3R by PTH than does comparable direct inhibition of AC. This suggests that Gαs may also specifically associate with each AC·IP3R complex. We conclude that all three subtypes of IP3R are regulated by cAMP independent of PKA. In HEK cells, where IP3R2 selectively associates with AC6, Gαs also associates with the AC·IP3R signaling junction.