Dept. of Cancer Studies and Molecular Medicine, Leicester Royal Infirmary, Leicester, UK.
Am J Physiol Cell Physiol. 2012 Mar 1;302(5):C723-34. doi: 10.1152/ajpcell.00202.2011. Epub 2011 Dec 7.
Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.
内皮素类型 A（ET(A)）和核苷酸（P2Y）Gα(q)-偶联受体的过度刺激会导致血管平滑肌收缩、高血压，最终导致肥大和血管闭塞。G 蛋白偶联受体激酶（GRK）和阻滞蛋白被激动剂占据的 Gα(q)-偶联受体依次募集，以终止磷脂酶 C 信号转导，防止延长/不适当的收缩信号转导。然而，这些蛋白也在调节几种丝裂原激活的蛋白激酶（MAPK）信号通路中发挥作用，这些信号通路对血管重塑至关重要。在这里，我们研究了不同的阻滞蛋白异构体是否调节大鼠主动脉平滑肌细胞（ASMC）中的内皮素和核苷酸受体 MAPK 信号转导。当用 Ca(2+)敏感染料负载分离的 ASMC 来评估细胞内 Ca(2+)水平时，P2Y(2)和 ET(A)受体脱敏被选择性小干扰 (si)RNA 介导的 G 蛋白偶联受体激酶 2（GRK2）耗竭所减弱。使用类似的 siRNA 技术，阻滞蛋白 2 的敲低可防止 P2Y(2)受体脱敏，并增强和延长 p38 和 ERK MAPK 信号，而阻滞蛋白 3 的敲低则无效。相反，阻滞蛋白 3 的敲低可防止 ET(A)受体脱敏，并减弱 ET1 刺激的 p38 和 ERK 信号，而阻滞蛋白 2 的敲低则没有影响。使用 Transwell 测定法来评估激动剂刺激的 ASMC 迁移，我们发现，在阻滞蛋白 2 的敲低后，UTP 刺激的迁移明显减弱，而在敲低任何一种阻滞蛋白后，ET1 刺激的迁移都减弱。这些数据突出了 ET(A)和 P2Y(2)受体刺激的 MAPK 信号转导的差异依赖于阻滞蛋白的调节。GRK2 和阻滞蛋白的表达对激动剂刺激的 ASMC 迁移是必不可少的，而 ASMC 迁移是血管重塑的关键过程，这突出了 GRK2 和阻滞蛋白在血管疾病进展中的潜在作用。
