Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Dr., Ottawa, Ontario K1H 8M5, Canada; Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5B7, Canada.
Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Dr., Ottawa, Ontario K1H 8M5, Canada.
Cell Signal. 2018 Jul;47:109-121. doi: 10.1016/j.cellsig.2018.03.016. Epub 2018 Apr 3.
MAGI proteins are Membrane-Associated Guanylate Kinase Inverted proteins that belong to the MAGUK family. They are scaffolding proteins that were shown to mediate the trafficking and signaling of various G protein-coupled receptors (GPCRs). They contain PDZ domains in their structure and many GPCRs interact with these proteins via the PDZ motifs on the carboxyl terminal end of a receptor. In a PDZ overlay assay performed with the carboxyl terminal tail of 5-HTR, we were able to detect all three members of the MAGI subfamily, MAGI-1, MAGI-2 and MAGI-3 as interacting PDZ proteins. The PDZ motif of 5-HTR consists of three amino acids; serine (S), cysteine (C) and valine (V). In this study, we characterize these 5-HTR interactions with MAGI proteins. We first confirm the interaction using co-immunopricipitation and illustrate that the interaction is PDZ motif-dependent in human embryonic kidney (HEK 293) cells. We then assess the effects of overexpression and knockdown of the MAGI proteins on the internalization, trafficking and signaling of 5-HTR. We find that knockdown of either MAGI-1 or MAGI-3 using siRNA results in a significant reduction in the internalization of 5-HTR. As for signaling, we report here that MAGI proteins can modulate the signaling via the two transduction pathways that 5-HTR can activate. We illustrate a significant effect of modulating MAGI proteins expression on 5-HT-stimulated IP formation. We demonstrate an enhancement in 5-HTR-stimulated IP formation upon MAGI proteins overexpression. In addition, we show that knockdown of MAGI proteins with siRNA leads to a significant reduction in 5-HTR-stimulated IP formation. Furthermore, we illustrate a significant increase in 5-HT-stimulated ERK1/2 phosphorylation upon MAGI proteins knockdown. Interestingly, this effect on ERK1/2 activation is PDZ motif-independent. We also suggest two possible mechanisms of regulation for the effect of MAGI proteins on 5-HTR function. One mechanism involves the regulation of cell surface expression since we show that both MAGI-2 and MAGI-3 can enhance receptor trafficking to the plasma membrane when overexpressed in HEK 293 cells. The other mechanism points to regulation of second messengers in the signaling pathways. Specifically, we show that overexpression of any of the three MAGI proteins can enhance the recruitment of PLCβ3 to 5-HTR. In addition, we report a negative effect for knocking down MAGI-3 on β-arrestin recruitment to the receptor and this effect is PDZ motif-independent. Taken together, our findings document distinct roles for the three MAGI proteins in regulating 5-HTR trafficking and signaling and emphasize the importance of studying PDZ proteins and their interactions with GPCRs to regulate their function.
MAGI 蛋白是膜相关鸟苷酸激酶倒置蛋白，属于 MAGUK 家族。它们是支架蛋白，被证明可以介导各种 G 蛋白偶联受体 (GPCR) 的运输和信号转导。它们的结构中含有 PDZ 结构域，许多 GPCR 通过受体羧基末端的 PDZ 基序与这些蛋白相互作用。在使用 5-HTR 羧基末端尾巴进行的 PDZ 叠加测定中，我们能够检测到 MAGI 亚家族的所有三个成员，即 MAGI-1、MAGI-2 和 MAGI-3，它们是相互作用的 PDZ 蛋白。5-HTR 的 PDZ 基序由三个氨基酸组成；丝氨酸 (S)、半胱氨酸 (C) 和缬氨酸 (V)。在这项研究中，我们对 MAGI 蛋白与 5-HTR 的这些相互作用进行了表征。我们首先使用共免疫沉淀证实了相互作用，并在人胚肾 (HEK 293) 细胞中说明了相互作用依赖于 PDZ 基序。然后，我们评估了 MAGI 蛋白过表达和敲低对 5-HTR 内化、运输和信号转导的影响。我们发现，使用 siRNA 敲低 MAGI-1 或 MAGI-3 会导致 5-HTR 的内化显著减少。至于信号转导，我们在此报告 MAGI 蛋白可以调节 5-HTR 可以激活的两种转导途径的信号转导。我们说明了调节 MAGI 蛋白表达对 5-HT 刺激的 IP 形成的显著影响。我们证明了 MAGI 蛋白过表达对 5-HTR 刺激的 IP 形成有增强作用。此外，我们表明，使用 siRNA 敲低 MAGI 蛋白会导致 5-HTR 刺激的 IP 形成显著减少。此外，我们说明了在 MAGI 蛋白敲低后，5-HTR 刺激的 ERK1/2 磷酸化显著增加。有趣的是，这种对 ERK1/2 激活的影响与 PDZ 基序无关。我们还提出了 MAGI 蛋白对 5-HTR 功能的影响的两种可能的调节机制。一种机制涉及细胞表面表达的调节，因为我们表明，当在 HEK 293 细胞中过表达时，MAGI-2 和 MAGI-3 都可以增强受体向质膜的运输。另一种机制指向信号转导途径中第二信使的调节。具体来说，我们表明，三种 MAGI 蛋白中的任何一种的过表达都可以增强 PLCβ3 与 5-HTR 的募集。此外，我们报告 MAGI-3 敲低对受体 β-抑制蛋白募集的负效应，并且这种效应与 PDZ 基序无关。总之，我们的发现证明了三种 MAGI 蛋白在调节 5-HTR 运输和信号转导方面的不同作用，并强调了研究 PDZ 蛋白及其与 GPCR 的相互作用以调节其功能的重要性。
