Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor |
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Authors: | Nobuaki Hirota Daisuke Yasuda Tomomi Hashidate Teruyasu Yamamoto Satoshi Yamaguchi Teruyuki Nagamune Takahide Nagase Takao Shimizu and Motonao Nakamura |
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Institution: | From the Departments of ‡Biochemistry and Molecular Biology and ;§Respiratory Medicine, Faculty of Medicine, ;¶Chemistry and Biotechnology, Graduate School of Engineering, and ;‖Center for NanoBio Integration, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan |
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Abstract: | Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro247, in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR. |
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Keywords: | Cell/Trafficking Chaperones G Proteins/Coupled Receptors (GPCR) Membrane/Proteins Methods/Fluorescence Protein/Cell Surface Protein/Export Receptors/Recycling |
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