PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling
PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling
复制标题
DOI：
10.1152/ajpcell.00001.2014
复制
发表时间：
2014-06-01
影响因子：
5.5
通讯作者：
Parsons, Rodney L.
中科院分区：
文献类型：
--
作者：
May, Victor;Buttolph, Thomas R.;Parsons, Rodney L.
垂体腺苷酸环化酶激活多肽（PACAP）选择性PAC1受体（Adcyap1r1）是一种G蛋白偶联受体（GPCR），可激活腺苷酸环化酶和PLC。与许多其他gpcr类似，我们之前的研究表明，PAC1受体在配体结合后被内化，形成信号内体，并招募额外的第二信使途径。利用人胚胎肾（HEK 293） PAC1Hop1-EGFP受体细胞系，我们研究了不同的PAC1受体信号机制如何促进MEK/ERK激活。与PAC1受体刺激的质膜腺苷酸环化酶/cAMP产生不同，pacap介导的ERK磷酸化部分依赖于受体内化，这是通过内吞作用或温度降低的药物抑制剂治疗确定的，这也抑制了受体内化。福斯克林刺激cAMP生成或暴露于细胞渗透性cAMP类似物8-溴-cAMP和二丁基cAMP对该系统中ERK磷酸化的影响很小。与内吞抑制剂Pitstop 2和dynasore相比，降低温度（24℃）持续抑制ERK激活的能力更大，这表明除了PAC1内化/内核体激活外，还涉及其他机制。双吲哚酰马来酰亚胺I抑制PAC1受体刺激的PLC/二酰基甘油/ PKC信号传导也减弱了ERK的磷酸化，而磷酸酯直接激活PKC以温度依赖的方式增加了ERK的磷酸化。抑制PAC1受体内吞作用和PKC激活完全阻断pacap刺激的ERK激活。PACAP增强了磷酸化ERK在细胞质和细胞核上的均匀染色，PKC信号传导促进了核磷酸化ERK的易位。总之，我们的研究结果表明，PACAP/PAC1受体内吞作用和PLC/二酰基甘油/ PKC激活是PACAP诱导的ERK激活的两种互补机制。
The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (Adcyap1r1) is a G protein-coupled receptor (GPCR) that activates adenylyl cyclase and PLC. Similar to many other GPCRs, our previous studies showed that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a human embryonic kidney (HEK 293) PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization, as determined by treatment with pharmacological inhibitors of endocytosis or temperature reduction, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell-permeable cAMP analogs 8-bromo-cAMP and dibutyryl cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24 degrees C) to consistently suppress ERK activation to a greater extent than the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms, in addition to PAC1 internalization/ endosome activation, were involved. Inhibition of PAC1 receptor-stimulated PLC/diacylglycerol/ PKC signaling by bisindoylmaleimide I also attenuated ERK phosphorylation, and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. Inhibition of PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/diacylglycerol/ PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation.