Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling
Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling
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DOI：
10.1073/pnas.1608061113
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发表时间：
2016-07-12
影响因子：
11.1
通讯作者：
Stefan, Eduard
中科院分区：
文献类型：
--
作者：
Bachmann, Verena A.;Mayrhofer, Johanna E.;Stefan, Eduard
支架蛋白在空间和时间上组织从激活的 G 蛋白偶联受体 (GPCR) 到细胞内效应级联的信息流。通过这种方式，信号支架，例如 A 激酶锚定蛋白 (AKAP)，可以划分激酶活性并确保底物选择性。使用磷酸蛋白质组学方法，我们确定了蛋白激酶 A (PKA) 和 Gpr161（孤儿 GPCR）信号传导之间的物理和功能联系。我们证明 Gpr161 作为 I 型 PKA 调节亚基 (RI) 的选择性高亲和力 AKAP 发挥作用。使用基于细胞的报告基因绘制蛋白质-蛋白质相互作用图谱，我们发现 RI 直接且选择性地结合到 Gpr161 细胞质羧基末端尾部的疏水性蛋白质-蛋白质相互作用界面。此外，我们的数据表明，Gpr161 和 RI 之间的二元复合物促进 Gpr161 向质膜的区室化。此外，我们发现 Gpr161 作为 AKAP 发挥作用，将 PKA RI 募集到斑马鱼胚胎的初级纤毛中。我们还表明，Gpr161 是 PKA 磷酸化的靶标，并且 PKA 磷酸化位点的突变影响纤毛受体定位。因此，我们提出 Gpr161 本身就是 AKAP，并且 cAMP 感应 Gpr161:PKA 复合物充当纤毛区室化信号体，现在在 PKA 相关功能的分析、解释和药物靶向中需要考虑这一概念。
Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161: PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.