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-HT2AR, 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-HT2AR consists of three amino acids; serine (S), cysteine (C) and valine (V). In this study, we characterize these 5-HT2AR 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-HT2AR. We find that knockdown of either MAGI-1 or MAGI-3 using siRNA results in a significant reduction in the internalization of 5-HT2AR. As for signaling, we report here that MAGI proteins can modulate the signaling via the two transduction pathways that 5-HT2AR can activate. We illustrate a significant effect of modulating MAGI proteins expression on 5-HT-stimulated IP formation. We demonstrate an enhancement in 5-HT2AR-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-HT2AR-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-HT2AR 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-HT2AR. 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-HT2AR trafficking and signaling and emphasize the importance of studying PDZ proteins and their interactions with GPCRs to regulate their function.

中文翻译：

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MAGI蛋白可以通过增强受体运输和PLC募集来差异调节5-HT2AR的信号传导途径。

MAGI蛋白是膜相关的鸟苷酸激酶反转蛋白，属于MAGUK家族。它们是脚手架蛋白，被证明可介导各种G蛋白偶联受体（GPCR）的运输和信号传导。它们在其结构中包含PDZ结构域，许多GPCR通过受体羧基末端的PDZ基序与这些蛋白质相互作用。在用5-HT2AR的羧基末端尾巴进行的PDZ重叠分析中，我们能够检测到MAGI亚家族的所有三个成员，MAGI-1，MAGI-2和MAGI-3作为相互作用的PDZ蛋白。5-HT2AR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，我们表征了这些5-HT2AR与MAGI蛋白的相互作用。我们首先确认使用共免疫沉淀的相互作用，并说明该相互作用是人类胚胎肾脏（HEK 293）细胞中的PDZ基序依赖性。然后，我们评估了MAGI蛋白的过表达和敲低对5-HT2AR内在化，运输和信号传导的影响。我们发现使用siRNA敲低MAGI-1或MAGI-3会导致5-HT2AR内在化的显着降低。至于信号传导，我们在这里报告说，MAGI蛋白可以通过5-HT2AR可以激活的两个转导途径调节信号传导。我们说明了调节MAGI蛋白表达对5-HT刺激的IP形成的重大影响。我们证明了MAGI蛋白过表达后5-HT2AR刺激的IP形成的增强。此外，我们表明，用siRNA敲低MAGI蛋白可导致5-HT2AR刺激的IP形成显着减少。此外，我们举例说明了敲除MAGI蛋白后5-HT刺激的ERK1 / 2磷酸化的显着增加。有趣的是，这种对ERK1 / 2激活的作用是与PDZ基序无关的。我们还建议调节MAGI蛋白对5-HT2AR功能的影响的两种可能的机制。一种机制涉及细胞表面表达的调节，因为我们证明当在HEK 293细胞中过表达时，MAGI-2和MAGI-3均可增强受体向质膜的转运。其他机制指向信号通路中第二信使的调节。具体而言，我们显示了三种MAGI蛋白中任何一种的过表达都可以增强PLCβ3向5-HT2AR的募集。此外，我们报道了敲低MAGI-3对β-arrestin募集到受体的负面影响，并且这种作用是PDZ基序独立的。综上所述，我们的发现证明了三种MAGI蛋白在调节5-HT2AR转运和信号传导中的独特作用，并强调了研究PDZ蛋白及其与GPCR相互作用以调节其功能的重要性。