Hamdan Fadi F, Rochdi Moulay Driss, Breton Billy, Fessart Delphine, Michaud Douce E, Charest Pascale G, Laporte Stéphane A, Bouvier Michel
Department of Biochemistry, Groupe de Recherche Universitaire sur le Médicament, Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada.
J Biol Chem. 2007 Oct 5;282(40):29089-100. doi: 10.1074/jbc.M700577200. Epub 2007 Aug 3.
The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves beta-arrestin and clathrin-coated pits. However, both beta-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that beta-arrestin binding to the beta2 subunit of the clathrin adaptor AP-2 (beta2-adaptin) is needed for the beta-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted beta-arrestin 1 and 2 interaction with beta2-adaptin, indicating a beta-arrestin- and clathrin-dependent endocytic process. Detailed analyses of beta-arrestin interactions with both the receptor and beta2-adaptin also allowed us to demonstrate that recruitment of beta-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between beta-arrestins and beta2-adaptin. However, both receptors recruited beta-arrestins upon agonist stimulation, suggesting a beta-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based beta-arrestin/beta2-adaptin interaction assay represents a novel biosensor to assess receptor activation.
在G蛋白偶联受体（GPCR）激动剂促进的内化过程中，研究最为广泛的途径涉及β-抑制蛋白和网格蛋白包被小窝。然而，也有报道称存在不依赖β-抑制蛋白和网格蛋白的过程。传统上，内吞途径是通过药理学抑制剂和各种显性负性突变体来表征的，这有时会导致相互矛盾的结果和解释困难。在此，利用β-抑制蛋白介导GPCR靶向网格蛋白包被小窝需要β-抑制蛋白与网格蛋白衔接蛋白AP-2的β2亚基（β2-衔接蛋白）结合这一事实，我们开发了一种基于生物发光共振能量转移的方法，直接评估活细胞中GPCR内吞作用所涉及的分子步骤。在所测试的12种受体中的10种中，包括一些先前被认为通过不依赖网格蛋白的途径内化的受体，激动剂刺激促进了β-抑制蛋白1和2与β2-衔接蛋白的相互作用，表明这是一个依赖β-抑制蛋白和网格蛋白的内吞过程。对β-抑制蛋白与受体和β2-衔接蛋白相互作用的详细分析还使我们能够证明，β-抑制蛋白募集到受体以及随后的构象变化是AP-2参与和随后网格蛋白介导的内吞作用之前的主要事件。在所测试的受体中，只有内皮素A和B受体未能促进β-抑制蛋白与β2-衔接蛋白之间的相互作用。然而，这两种受体在激动剂刺激后都募集了β-抑制蛋白，表明这两种受体的内化途径是依赖β-抑制蛋白但不依赖网格蛋白的。除了提供一种剖析GPCR内吞作用所涉及分子事件的新工具外，基于生物发光共振能量转移的β-抑制蛋白/β2-衔接蛋白相互作用测定法还代表了一种评估受体激活的新型生物传感器。
