Cerniello Flavia M, Carretero Oscar A, Longo Carbajosa Nadia A, Cerrato Bruno D, Santos Robson A, Grecco Hernán E, Gironacci Mariela M
From the Departamento de Química Biológica, IQUIFIB-CONICET, Universidad de Buenos Aires, Argentina (F.M.C., N.L.C., B.D.C., M.M.G.); Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, MI (O.A.C.); Department of Physiology, Federal University of Minas Gerais, Belo Horizonte, Brazil (R.A.S.); and Departamento de Física, Universidad de Buenos Aires and IFIBA-CONICET, Buenos Aires, Argentina (H.E.G.).
Hypertension. 2017 Nov;70(5):982-989. doi: 10.1161/HYPERTENSIONAHA.117.09789. Epub 2017 Sep 5.
The MAS1 receptor (R) exerts protective effects in the brain, heart, vessels, and kidney. R trafficking plays a critical function in signal termination and propagation and in R resensitization. We examined MAS1R internalization and trafficking on agonist stimulation and the role of β-arrestin2 in the activation of ERK1/2 (extracellular signal-regulated kinase 1/2) and Akt after MAS1R stimulation. Human embryonic kidney 293T cells were transfected with the coding sequence for MAS1R-YFP (MAS1R fused to yellow fluorescent protein). MAS1R internalization was evaluated by measuring the MAS1R present in the plasma membrane after agonist stimulation using a ligand-binding assay. MAS1R trafficking was evaluated by its colocalization with trafficking markers. MAS1R internalization was blocked in the presence of shRNAcaveolin-1 and with dominant negatives for Eps15 (a protein involved in endocytosed Rs by clathrin-coated pits) and for dynamin. After stimulation, MAS1R colocalized with Rab11-a slow recycling vesicle marker-and not with Rab4-a fast recycling vesicle marker-or LysoTracker-a lysosome marker. Cells transfected with MAS1R showed an increase in Akt and ERK1/2 activation on angiotensin-(1-7) stimulation, which was blocked when the clathrin-coated pits pathway was blocked. Suppression of β-arrestin2 by shRNA reduced the angiotensin-(1-7)-induced ERK1/2 activation, whereas Akt activation was not modified. We conclude that on agonist stimulation, MAS1R is internalized through clathrin-coated pits and caveolae in a dynamin-dependent manner and is then slowly recycled back to the plasma membrane. MAS1R induced Akt and ERK1/2 activation from early endosomes, and the activation of ERK1/2 was mediated by β-arrestin2. Thus, MAS1R activity and density may be tightly controlled by the cell.
MAS1受体（R）在脑、心脏、血管和肾脏中发挥保护作用。R的转运在信号终止与传播以及R的再敏化过程中起着关键作用。我们研究了激动剂刺激下MAS1R的内化与转运，以及β - 抑制蛋白2在MAS1R刺激后细胞外信号调节激酶1/2（ERK1/2）和Akt激活中的作用。将人胚肾293T细胞用MAS1R - YFP（与黄色荧光蛋白融合的MAS1R）的编码序列进行转染。使用配体结合测定法，通过测量激动剂刺激后质膜中存在的MAS1R来评估MAS1R的内化。通过MAS1R与转运标记物的共定位来评估MAS1R的转运。在存在小发夹RNA（shRNA）小窝蛋白 - 1以及Eps15（一种参与网格蛋白包被小窝介导的内吞性受体的蛋白）和发动蛋白的显性负性突变体的情况下，MAS1R的内化受到阻断。刺激后，MAS1R与Rab11（一种缓慢循环的囊泡标记物）共定位，而不与Rab4（一种快速循环的囊泡标记物）或溶酶体标记物溶酶体追踪染料共定位。用MAS1R转染的细胞在血管紧张素 - （1 - 7）刺激下Akt和ERK1/2的激活增加，当网格蛋白包被小窝途径被阻断时这种增加被阻断。通过shRNA抑制β - 抑制蛋白2可降低血管紧张素 - （1 - 7）诱导的ERK1/2激活，而Akt激活未改变。我们得出结论，在激动剂刺激下，MAS1R以发动蛋白依赖性方式通过网格蛋白包被小窝和小窝进行内化，然后缓慢再循环回到质膜。MAS1R从早期内体诱导Akt和ERK1/2激活，并且ERK1/2的激活由β - 抑制蛋白2介导。因此，MAS1R的活性和密度可能受到细胞的严格控制。
