Calcium-sensing receptor activation of Rho involves filamin and Rho-guanine nucleotide exchange factor
Calcium-sensing receptor activation of Rho involves filamin and Rho-guanine nucleotide exchange factor
复制标题
DOI：
10.1210/en.2002-220240
复制
发表时间：
2002-10-01
期刊：
影响因子：
4.8
通讯作者：
Quarles, LD
中科院分区：
文献类型：
--
作者：
Pi, M;Spurney, RF;Quarles, LD
我们研究了Galphaq、丝蛋白、Rho、RhoGEF Lbc和钙敏感受体（CasR） C端在CasR信号传导中的作用。我们发现Ca2+， Mg2+或N-（3-[2-氯苯基]丙基）-(R)- α -甲基-3-甲氧基苯胺（NPS-R568）的拟钙化R异构体刺激了转染CasR和SRE-荧光素酶报告结构的人胚胎肾293细胞的血清反应元件（SRE）活性。G蛋白信号（RGS）2或RGS4的调控因子Galphaq（305-359）的显性负基因的共表达抑制了CasR，刺激了SRE活性，与CasR激活Gaq一致。细胞骨架相关的Rho蛋白参与了CasR对SRE的激活，CasR介导的膜相关Rho A的增加以及肉毒杆菌C3 （C3）外酶抑制CasR和galphaqql刺激的SRE活性的能力证明了这一点。RhoGEF Lbc，缺乏dbl -同源或Pleckstrin同源结构域，以及丝状蛋白肽（1530-1875）的过表达抑制CasR介导的SRE激活。CasR羧基末端的一个小基因CasR（906-980）编码丝蛋白结合区，也阻断了CasR-和galphaqql刺激的SRE活性。通过互惠共免疫沉淀研究证实了CasR RhoGEF Lbc、Rho A、Galphaq和丝蛋白之间的潜在相互作用。我们的研究结果表明，CasR的C端可能与丝蛋白相互作用，形成一个细胞骨架支架，这对于SRE激活上游的Galphaq、RhoGEF Lbc和Rho信号通路的空间组织是必要的。
We investigated the role of Galphaq, filamin, Rho, the RhoGEF Lbc, and the C terminus of calcium-sensing receptor (CasR) in CasR signaling. We found that Ca2+, Mg2+, or the calcimimetic R isomer of N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS-R568) stimulated serum response element (SRE) activity human embryonic kidney 293 cells transfected with CasR and an SRE-luciferase reporter construct. Coexpression of either the dominant negative Galphaq(305-359) minigene, regulators of G protein signaling (RGS)2 or RGS4, inhibited CasR,stimulated SRE activity, consistent with CasR activation of Gaq. The cytoskeletal associated Rho protein is involved CasR activation of SRE, as evidenced by CasR-mediated increase in membrane-associated Rho A and by the ability of Clostridium botulinum C3 (C3) exoenzyme to inhibit both CasR and GalphaqQL-stimulated SRE activity. Overexpression of the RhoGEF Lbc, lacking either the Dbl-homology or Pleckstrin homology domain, as well as the filamin peptide (1530-1875) inhibited CasR, mediated activation of SRE. A carboxyl-terminal CasR minigene, CasR(906-980), encoding a filamin binding region, also blocked CasR- and GalphaqQL-stimulated SRE activity. Potential interactions between CasR RhoGEF Lbc, Rho A, Galphaq, and filamin were demonstrated by reciprocal coimmunoprecipitation studies. Our results suggest that the C terminus of CasR may interact with filamin to create a cytoskeletal scaffold necessary for the spatial organization of Galphaq, RhoGEF Lbc, and Rho signaling pathways upstream of SRE activation.