Cell-permeable non-hydrolyzable cAMP derivatives as tools for analysis of signaling pathways controlling gene regulation in Dictyostelium.
Cell-permeable non-hydrolyzable cAMP derivatives as tools for analysis of signaling pathways controlling gene regulation in Dictyostelium.
复制标题
细胞渗透性不可水解的 cAMP 衍生物作为分析盘基网柄菌中控制基因调控的信号通路的工具。
DOI：
10.1016/s0021-9258(18)53256-7
复制
发表时间：
1993
期刊：
影响因子：
--
通讯作者：
P. V. van Haastert
中科院分区：
文献类型：
--
作者：
P. Schaap;M. van Ments;R. D. Soede;R. Brandt;R. Firtel;W. Dostmann;H. Genieser;B. Jastorff;P. V. van Haastert
测试了一类新的cAMP衍生物与表面cAMP受体（CAR）、蛋白激酶A（PKA）和cAMP-磷酸二酯酶（PDE）的结合以及对盘基网柄藻中三类cAMP调节基因的诱导。这些衍生物对轴向（Sp）或赤道（Rp）环外氧原子进行硫取代，同时引入进一步的修饰以提供与CAR或PKA结合的特异性，和/或增加亲脂性并使衍生物具有膜渗透性。所有衍生物与PDE的结合都很弱，并且在与Dictyosteoblastoma细胞孵育期间几乎不降解。一种cAMP衍生物，6-硫代乙基-嘌呤核苷3 '，5'-单硫代磷酸酯，Sp-异构体（Sp-6SEtcPuMPS），满足PKA在体内选择性活化的标准。该化合物进入网骨藻细胞，并达到足以激活PKA的1 μ M的细胞内浓度，在不足以激活CAR的30 μ M的细胞外浓度。cAMP调节的前孢子和前茎基因以及聚集PDE基因的表达被CAR激动剂有效诱导，而PKA激动剂的诱导非常差。即使Sp-6SEtcPuMPS也不能有效诱导基因表达。这些数据不仅表明表面cAMP受体是cAMP诱导的基因表达的第一靶，而且反对cAMP诱导的PKA活化直接诱导这些基因的表达。
A novel class of cAMP derivatives were tested for binding to surface cAMP receptors (CAR), protein kinase A (PKA), and cAMP-phosphodiesterase (PDE) and for induction of three classes of cAMP regulated genes in Dictyostelium discoideum. These derivatives carry sulfur substitutions for either the axial (Sp) or equatorial (Rp) exocyclic oxygen atoms, while further modifications were introduced to provide specificity for binding to either CAR or PKA, and/or to increase lipophilicity and render the derivatives membrane-permeable. All derivatives bind weakly to PDE and are almost not degraded during incubation with Dictyostelium cells. One cAMP derivative, 6-thioethyl-purineriboside 3‘,5‘-monophosphorothioate, Sp-isomer (Sp-6SEtcPuMPS), fulfills the criteria for selective activation of PKA in vivo. The compound enters Dictyostelium cells and reaches an intracellular concentration of 1 microM, sufficient to activate PKA, at an extracellular concentration of 30 microM, which is insufficient to activate CAR. Expression of cAMP-regulated prespore and prestalk genes and the aggregative PDE gene are effectively induced by CAR agonists and very poorly by PKA agonists. Even Sp-6SEtcPuMPS is ineffective to induce gene expression. These data not only indicate that surface cAMP receptors are the first targets for cAMP-induced gene expression, but argue against direct induction of expression of these genes by cAMP-induced PKA activation.