Institute of Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany.
J Biol Chem. 2010 Sep 24;285(39):30355-62. doi: 10.1074/jbc.M110.114900. Epub 2010 Jul 23.
β-Arrestins are crucial regulators of G-protein coupled receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted receptor phosphorylation is required for β-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in β-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate receptor signaling and trafficking. Na(+)/H(+) exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, β-arrestin2, and the parathyroid hormone receptor type 1 (PTHR). We show that NHERF1 and β-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, β-arrestin2 binding is promoted by receptor activation. NHERF1 interacts directly with β-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and β-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing β-arrestin2 into close proximity to the PTHR, thereby facilitating β-arrestin2 recruitment after receptor activation.
β-arrestins 是 G 蛋白偶联受体 (GPCR) 信号转导、脱敏和内化的关键调节因子。尽管长期以来的范式认为激动剂促进受体磷酸化是 β-arrestin2 募集所必需的,但新出现的证据表明,磷酸化非依赖性机制在 GPCR 募集β-arrestin2 中发挥作用。几种 PDZ 蛋白已知与 GPCR 相互作用,并作为细胞溶质衔接蛋白,调节受体信号转导和转运。Na(+)/H(+) 交换调节因子 (NHERF) 在 GPCR 信号转导中发挥主要作用。通过结合成像和生化及生物物理方法,我们研究了 NHERF1、β-arrestin2 和甲状旁腺激素受体 1 (PTHR) 之间的相互作用。我们表明,NHERF1 和 β-arrestin2 可以独立地与 PTHR 结合,并在培养的人胚肾细胞和中国仓鼠卵巢细胞中形成三元复合物。尽管 NHERF1 与 PTHR 持续相互作用,但 β-arrestin2 的结合是由受体激活促进的。NHERF1 与 β-arrestin2 直接相互作用,而不使用 PTHR 作为接口。荧光共振能量转移研究表明,NHERF1 调节了 PTHR 和 β-arrestin2 相互作用的动力学。这些发现表明,NHERF1 可能作为一种衔接蛋白,将 β-arrestin2 带到 PTHR 附近,从而促进受体激活后 β-arrestin2 的募集。