title: Amino acid residues critical for endoplasmic reticulum export and trafficking of platelet-activating factor receptor.
abstract: Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Hirota
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yasuda
authors.forename: Tomomi
authors.initials: T
authors.lastname: Hashidate
authors.forename: Teruyasu
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
authors.forename: Takahide
authors.initials: T
authors.lastname: Nagase
authors.forename: Takao
authors.initials: T
authors.lastname: Shimizu
authors.forename: Motonao
authors.initials: M
authors.lastname: Nakamura
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D010449:Peptide Mapping / Q000379:methods; D010742:Phospholipid Ethers; D010980:Platelet Membrane Glycoproteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014230:Triazoles / Q000494:pharmacology
chemical_list: D001381:Azepines; D010742:Phospholipid Ethers; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D014230:Triazoles; C039351:platelet activating factor receptor; C053479:1-O-hexadecyl-2-N-methylcarbamylphosphatidylcholine; C068035:4-(2-chlorophenyl)-2-(2-(4-isobutylphenyl)ethyl)-6,9-dimethyl-6H-thieno(3,2-f)(1,2,4)triazolo(4,3-a)(1,4)diazepine; D011392:Proline