MAGI proteins can differentially regulate the signaling pathways of 5-HT2AR by enhancing receptor trafficking and PLC recruitment |
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| Institution: | 1. Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Dr., Ottawa, Ontario K1H 8M5, Canada;2. Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5B7, Canada;1. Department of Medicine, Hofstra North-Shore LIJ Medical School, Feinstein Institute for Medical Research, NY, United States;2. Department of Chemistry, Bar-Ilan University, Ramat Gan, Israel;3. Jamia Millia Islamia, New Delhi, India;1. J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute, and the Department of Physiology and Pharmacology, University of Western Ontario, 100 Perth Dr. London, Ontario N6A 5K8, Canada;2. Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 3K7, Canada;3. Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Dr. Ottawa, Ontario K1H 8M5, Canada |
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| Abstract: | 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. |
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