Kuramoto Ritsuki, Ikuta Tatsuya, Carino Carlo Marion C, Kawakami Kouki, Kushiro Miisha, Watanabe Chihiro, Uchida Yasunori, Abe Mitsuhiro, Sako Yasushi, Taguchi Tomohiko, Yanagawa Masataka, Inoue Asuka
Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
Nat Chem Biol. 2025 Aug 6. doi: 10.1038/s41589-025-01967-4.
Upon ligand-induced activation, G-protein-coupled receptors (GPCRs) recruit β-arrestins (βarrs) to the plasma membrane, where phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P) stabilizes the GPCR-βarr complex. Although PtdIns(4,5)P is reported to form nanoscale clusters, the spatiotemporal dynamics of how the GPCR-βarr-PtdIns(4,5)P complex assembles and organizes in living cells remain unexplored. Here we demonstrate that multiple PtdIns(4,5)P-binding sites on βarrs cooperatively promote GPCR-βarr assembly in membrane domains. Using molecular dynamics simulations, we identify a noncanonical (NC) PtdIns(4,5)P-binding site, distinct from the known canonical site. Biochemical assays confirm that both sites are essential for βarr binding to PtdIns(4,5)P-containing liposomes, while NanoBiT assays reveal synergistic contributions of both sites for βarr recruitment in living cells. Notably, single-molecule imaging demonstrates that the NC site is required for the rapid accumulation of the GPCR-βarr-PtdIns(4,5)P complex into immobile membrane domains upon ligand stimulation. Collectively, our findings highlight how multivalent βarr-PtdIns(4,5)P interactions drive GPCR-βarr compartmentalization, adding complexity to GPCR signaling dynamics.
在配体诱导激活后，G蛋白偶联受体（GPCRs）会将β抑制蛋白（βarrs）招募至质膜，在那里磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P）可稳定GPCR-βarr复合物。尽管据报道PtdIns(4,5)P会形成纳米级簇，但GPCR-βarr-PtdIns(4,5)P复合物在活细胞中组装和组织的时空动态仍未得到探索。在此，我们证明βarrs上的多个PtdIns(4,5)P结合位点协同促进膜结构域中的GPCR-βarr组装。通过分子动力学模拟，我们鉴定出一个与已知经典位点不同的非经典（NC）PtdIns(4,5)P结合位点。生化分析证实，这两个位点对于βarr与含PtdIns(4,5)P的脂质体结合均至关重要，而纳米BiT分析揭示了这两个位点对活细胞中βarr招募的协同作用。值得注意的是，单分子成像表明，在配体刺激后，GPCR-βarr-PtdIns(4,5)P复合物快速积累到固定膜结构域中需要NC位点。总的来说，我们的研究结果突出了多价βarr-PtdIns(4,5)P相互作用如何驱动GPCR-βarr的区室化，为GPCR信号动力学增添了复杂性。
