Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Cell Signal. 2013 Mar;25(3):690-7. doi: 10.1016/j.cellsig.2012.11.020. Epub 2012 Nov 29.
It has been shown lately that activity of G protein-coupled receptors (GPCRs) is regulated by an array of proteins binding to carboxy (C)-terminus of GPCRs. Proteins of 4.1 family are subsets of subcortical cytoskeletal proteins and are known to stabilize cellular structures and proteins at the plasma membrane. One of the 4.1 family proteins, 4.1G has been shown to interact with the C-terminus of GPCRs and regulate intracellular distribution of the receptors, including parathyroid hormone (PTH)/PTH-related protein receptor (PTHR). PTHR is coupled to trimeric G proteins G(s) and G(q), which activate the adenylyl cyclase/cyclic AMP (cAMP) pathway and phospholipase C pathway, respectively. During the course of investigation of the role of 4.1G on adenylyl cyclase/cAMP signaling pathway, we found that 4.1G suppressed forskolin-induced cAMP production in cells. The cAMP accumulation induced by forskolin was decreased in HEK293 cells overexpressing 4.1G or increased in 4.1G-knockdown cells. Furthermore, PTH -(1-34)-stimulated cAMP production was also suppressed in the presence of exogenously expressed 4.1G despite its activity to increase the distribution of PTHR to the cell surface. In cells overexpressing FERM domain-deleted 4.1G, a mutant form of the protein deficient in plasma membrane distribution, neither forskolin-induced nor PTH -(1-34)-stimulated cAMP production was not altered. The suppression of the forskolin-induced cAMP production was observed even in membrane preparations of 4.1G-overexpressing cells. In 4.1G-knockdown HEK293 cells, plasma membrane distribution of adenylyl cyclase 6, one of the major subtypes of the enzyme in the cells, showed a slight decrease, in spite of the increased production of cAMP in those cells when stimulated by forskolin. Also, cytochalasin D treatment did not cause any influence on forskolin-induced cAMP production in HEK293 cells. These data indicate that plasma membrane-associated 4.1G regulates GPCR-mediated G(s) signaling by suppressing adenylyl cyclase-mediated cAMP production.
最近已经表明，G 蛋白偶联受体 (GPCR) 的活性受与 GPCR 羧基 (C) 末端结合的一系列蛋白质的调节。4.1 家族蛋白是皮质下细胞骨架蛋白的亚群，已知可稳定质膜上的细胞结构和蛋白质。4.1 家族蛋白之一的 4.1G 已被证明与 GPCR 的 C 末端相互作用，并调节受体的细胞内分布，包括甲状旁腺激素 (PTH)/甲状旁腺激素相关蛋白受体 (PTHR)。PTHR 与三聚体 G 蛋白 G(s) 和 G(q) 偶联，分别激活腺苷酸环化酶/cAMP (cAMP) 途径和磷脂酶 C 途径。在研究 4.1G 在腺苷酸环化酶/cAMP 信号通路中的作用过程中，我们发现 4.1G 抑制了细胞中forskolin 诱导的 cAMP 产生。在过表达 4.1G 的 HEK293 细胞中，forskolin 诱导的 cAMP 积累减少，而在 4.1G 敲低细胞中增加。此外，尽管 4.1G 增加了 PTHR 向细胞表面的分布，但 PTH-(1-34)刺激的 cAMP 产生也被抑制。在过表达 FERM 结构域缺失的 4.1G 的细胞中，该蛋白的一种缺乏质膜分布的突变形式，forskolin 诱导的或 PTH-(1-34)刺激的 cAMP 产生均未改变。即使在过表达 4.1G 的细胞的膜制剂中也观察到forskolin 诱导的 cAMP 产生的抑制。在 4.1G 敲低的 HEK293 细胞中，尽管细胞中 cAMP 的产生增加，但腺苷酸环化酶 6 的质膜分布，细胞中该酶的主要亚型之一，略有减少，当被 forskolin 刺激时。此外，细胞松弛素 D 处理对 HEK293 细胞中 forskolin 诱导的 cAMP 产生没有任何影响。这些数据表明，质膜相关的 4.1G 通过抑制腺苷酸环化酶介导的 cAMP 产生来调节 GPCR 介导的 G(s) 信号。
