Joseph S K, Ryan S V
Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1993 Nov 5;268(31):23059-65.
The inositol trisphosphate receptor (IP3R) in brain has been shown to be a substrate for several different protein kinases in vitro. We have studied the phosphorylation of the IP3R in intact cells by using isolated hepatocytes and an antibody to immunoprecipitate the receptor protein from detergent extracts. Stimulation of 32P-labeled hepatocytes with glucagon or N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cAMP) markedly increased phosphorylation of the IP3R. However, no increase was observed in response to angiotensin II, vasopressin, 12-O-tetradecanoyl-phorbol-13-acetate, or epidermal growth factor. The kinetics of phosphorylation in response to glucagon was both rapid and transient. In agreement with previous studies, physiological concentrations of Ca2+ stimulated D-myo-inositol 1,4,5-trisphosphate (IP3) binding to permeabilized hepatocytes (Pietri, F., Hilly, M., and Mauger, J.-P. (1990) J. Biol. Chem. 265, 17478-17485). Pretreatment of cells with db-cAMP had no effect on binding in the absence of added Ca2+ but enhanced binding measured in the presence of basal low concentrations (0.16-0.25 microM) of Ca2+ and decreased the concentration of Ca2+ required for half-maximal stimulation. The effect of db-cAMP was associated with an increase in affinity of the IP3 binding site without a change in maximum number of binding sites. Preincubation of intact hepatocytes with okadaic acid alone produced an increase in basal phosphorylation of the IP3R, and maximal phosphorylation of the receptor was observed in the presence of both okadaic acid and db-cAMP. However, okadaic acid blocked the effect of db-cAMP and inhibited the effect of Ca2+ on IP3 binding. Detergent-solubilized binding sites were already fully activated and insensitive to modulation by Ca2+ or cAMP-dependent protein kinase. It is proposed that the receptor in native membranes is inhibited and that Ca2+ and cAMP-dependent protein kinase may act to relieve this inhibition.
脑内的肌醇三磷酸受体（IP3R）在体外已被证明是几种不同蛋白激酶的底物。我们通过使用分离的肝细胞和一种抗体从去污剂提取物中免疫沉淀受体蛋白，研究了完整细胞中IP3R的磷酸化情况。用胰高血糖素或N6,2'-O-二丁酰腺苷3',5'-环磷酸（db-cAMP）刺激32P标记的肝细胞，显著增加了IP3R的磷酸化。然而，对血管紧张素II、血管加压素、12-O-十四酰佛波醇-13-乙酸酯或表皮生长因子的反应未观察到增加。对胰高血糖素反应的磷酸化动力学既快速又短暂。与先前的研究一致，生理浓度的Ca2+刺激D-肌醇1,4,5-三磷酸（IP3）与通透化肝细胞的结合（皮耶特里，F.，希利，M.，和莫热，J.-P.（1990）《生物化学杂志》265，17478 - 17485）。在没有添加Ca2+的情况下，用db-cAMP预处理细胞对结合没有影响，但在存在基础低浓度（0.16 - 0.25 microM）Ca2+的情况下增强了结合，并降低了半最大刺激所需的Ca2+浓度。db-cAMP的作用与IP3结合位点亲和力的增加有关，而结合位点的最大数量没有变化。仅用冈田酸预孵育完整肝细胞会使IP3R的基础磷酸化增加，并且在同时存在冈田酸和db-cAMP的情况下观察到受体的最大磷酸化。然而，冈田酸阻断了db-cAMP的作用并抑制了Ca2+对IP3结合的作用。去污剂溶解的结合位点已经完全激活，并且对Ca2+或cAMP依赖性蛋白激酶的调节不敏感。有人提出天然膜中的受体受到抑制，并且Ca2+和cAMP依赖性蛋白激酶可能起到解除这种抑制的作用。
