Department of Neurological Sciences, College of Medicine, University of Vermont, Burlington, Vermont.
Department of Neurological Sciences, College of Medicine, University of Vermont, Burlington, Vermont
Am J Physiol Cell Physiol. 2014 Jun 1;306(11):C1068-79. doi: 10.1152/ajpcell.00001.2014. Epub 2014 Apr 2.
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°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.
垂体腺苷酸环化酶激活肽（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 一样，我们之前的研究表明，PAC1 受体在配体结合后会被内化，形成信号转导内体，从而招募额外的第二信使途径。使用人胚肾（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 受体刺激的质膜中环磷酸腺苷（cAMP）生成不同，PACAP 介导的 ERK 磷酸化部分依赖于受体内化，这可以通过用内吞作用的药理学抑制剂或降低温度来确定，这也抑制了受体内化。forskolin 刺激 cAMP 生成或暴露于细胞通透性 cAMP 类似物 8-溴-cAMP 和二丁酰基 cAMP 对该系统中 ERK 磷酸化的影响最小。与内吞抑制剂 Pitstop 2 和 dynasore 相比，降低温度（24°C）能够更一致地抑制 ERK 激活，这表明除了 PAC1 内化/内体激活之外，还涉及其他机制。Bisindoylmaleimide I 抑制 PAC1 受体刺激的 PLC/二酰基甘油/PKC 信号也减弱了 ERK 磷酸化，并且用佛波酯直接激活 PKC 以温度依赖性方式增加 ERK 磷酸化。抑制 PAC1 受体内吞作用和 PKC 激活完全阻断了 PACAP 刺激的 ERK 激活。PACAP 均匀地增强细胞质和核内磷酸化 ERK 的染色，并且 PKC 信号促进了核内磷酸化 ERK 的易位。总之，我们的结果表明，PACAP/PAC1 受体内吞作用和 PLC/二酰基甘油/PKC 激活代表两种互补机制，有助于 PACAP 诱导的 ERK 激活。
