Smith Thomas H, Coronel Luisa J, Li Julia G, Dores Michael R, Nieman Marvin T, Trejo JoAnn
From the Biomedical Sciences Graduate Program and Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093.
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093.
J Biol Chem. 2016 Aug 26;291(35):18453-64. doi: 10.1074/jbc.M116.729285. Epub 2016 Jul 11.
Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation.
蛋白酶激活受体-4（PAR4）是凝血酶的G蛋白偶联受体（GPCR），与典型的PAR1一样通过蛋白水解作用被激活。由于PAR1的不可逆激活，受体转运与信号调节密切相关。然而，与PAR1不同，控制PAR4转运的机制尚不清楚。在此，我们试图确定控制PAR4转运和信号传导的机制。在通过小干扰RNA（siRNA）使网格蛋白缺失的HeLa细胞中，激活的PAR4无法内化。与网格蛋白介导的内吞作用一致，发动蛋白显性负性K44A突变体的表达也阻断了激活的PAR4内化。然而，与大多数GPCR不同，PAR4内化的发生不依赖于β-抑制蛋白和受体的C末端结构域。相反，我们在PAR4的第三个细胞内环中发现了一个高度保守的基于酪氨酸的基序，并发现网格蛋白衔接蛋白复合物-2（AP-2）对内化很重要。AP-2的缺失抑制了激动剂诱导的PAR4内化。此外，基于酪氨酸的基序关键残基的突变破坏了激动剂诱导的PAR4内化。使用巨核细胞系Dami细胞，我们证实AP-2是激动剂诱导内源性PAR4内化所必需的。此外，抑制激活的PAR4内化增强了ERK1/2信号传导，而Akt信号传导则明显减弱。这些发现表明，与大多数经典GPCR不同，激活的PAR4内化需要AP-2和基于酪氨酸的基序，且其发生不依赖于β-抑制蛋白。此外，这些发现首次表明激活的PAR4内化与ERK1/2和Akt的适当激活有关。
