Hawes B E, van Biesen T, Koch W J, Luttrell L M, Lefkowitz R J
Howard Hughes Medical Institute, Department of Medicine (Cardiology), Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1995 Jul 21;270(29):17148-53. doi: 10.1074/jbc.270.29.17148.
Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.
与异源三聚体G蛋白Gi或Gq偶联的受体，可刺激磷酸肌醇（PI）水解和丝裂原活化蛋白激酶（MAPK）激活。PI水解产生肌醇1,4,5 - 三磷酸和二酰基甘油，导致蛋白激酶C（PKC）激活，进而刺激MAPK活性增加。然而，Gi和Gq信号通路中PI水解与MAPK激活之间的关系尚未明确界定，这也是本研究的主题。评估了几种信号抑制剂的作用，包括源自β肾上腺素能受体激酶1（βARKct）羧基末端的肽的表达，该肽可特异性阻断由G蛋白的βγ亚基（Gβγ）介导的信号传导；p21ras（RasN17）和p74raf - 1（NδRaf）的显性负突变体的表达；蛋白酪氨酸激酶（PTK）抑制剂以及PKC的细胞内耗竭。与Gi偶联的α2A肾上腺素能受体（AR）刺激MAPK激活，该激活可被βARKct、RasN17或NδRaf的表达，或PTK抑制剂阻断，但不受PKC细胞内耗竭的影响。相比之下，由与Gq偶联的α1B AR或M1毒蕈碱胆碱能受体刺激的MAPK激活，不受βARKct或RasN17表达或PTK抑制剂的影响，但可被NδRaf的表达或PKC耗竭阻断。这些数据表明，与Gi和Gq偶联的受体通过不同的信号通路刺激MAPK激活。Gβγ通过一种利用p21ras和p74raf且不依赖PKC的机制，介导与Gi偶联的受体刺激的MAPK激活。相反，Gα通过一种不依赖p21ras的机制，利用PKC和p74raf介导与Gq偶联的受体刺激的MAPK激活。为了确定Gβγ在与Gi偶联的受体介导的PI水解和MAPK激活中的作用，使用了Gβγ的直接刺激。Gβγ的表达导致MAPK激活，该激活对βARKct、RasN17或NδRaf的表达或PTK抑制剂的抑制敏感，但对PKC耗竭不敏感。相比之下，Gβγ介导的PI水解不受βARKct、RasN17或NδRaf表达或PTK抑制剂的影响。总之，这些结果表明，Gβγ通过独立的信号通路介导MAPK激活和PI水解。
