Lecat Sandra, Belemnaba Lazare, Galzi Jean-Luc, Bucher Bernard
GPCRs, Pain and Inflammation Team, UMR7242, CNRS-University of Strasbourg, LabEx Medalis 300 Bvd Sébastien Brant, CS 10413, 67412 Illkirch, France.
UMR 7213, CNRS-University of Strasbourg, Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, 74 route du Rhin, CS 60024, 67401 Illkirch, France.
Cell Signal. 2015 Jul;27(7):1297-304. doi: 10.1016/j.cellsig.2015.03.016. Epub 2015 Mar 25.
Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit β-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gβ/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gβ/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that β-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.
神经肽Y与G蛋白偶联受体结合，其作用导致腺苷酸环化酶活性受到抑制。利用稳定表达天然神经肽Y Y1受体的HEK293细胞，我们发现神经肽Y激动剂可引发细胞外信号调节激酶（ERK1/2）的瞬时磷酸化。我们首先表明，Y1受体刺激后ERK1/2的激活依赖于异源三聚体Gi/o，因为用百日咳毒素预处理可完全抑制该激活。此外，ERK1/2的激活不依赖于内化，因为无法募集β - 抑制蛋白的突变型Y1受体仍能与野生型受体一样程度地激活ERK信号。接下来我们表明，MAPK途径的这种激活受到MEK抑制剂U0126的抑制，不依赖于Y1受体处的钙信号传导（抑制磷脂酶C、蛋白激酶C或蛋白激酶D无影响），而是依赖于激活PI3激酶的Gβ/γ及相关信号通路。虽然抑制表皮生长因子受体酪氨酸激酶不影响神经肽Y诱导的ERK1/2激活，但我们表明AG1024抑制胰岛素生长因子受体IGFR可完全阻断Y1受体对ERK1/2的激活。这种对Gβ/γ - PI3K - AG1024敏感的途径不涉及通过金属蛋白酶释放可溶性配体来激活IGFR，因为它不受金属蛋白酶抑制剂马立马司他的影响。最后，我们发现一条对渥曼青霉素 - AG1024敏感但对马立马司他不敏感的类似途径，参与内源性表达的与Gq蛋白偶联（毒蕈碱M3受体）或与Gs蛋白偶联（内皮素ETB受体）的GPCR在HEK293细胞中激活ERK信号。我们的分析首次表明，β - 抑制蛋白募集到神经肽Y Y1受体并非该受体激活MAPK所必需，但IGFR受体的反式激活是必需的。
