title: The atypical N-glycosylation motif, Asn-Cys-Cys, in human GPR109A is required for normal cell surface expression and intracellular signaling.
abstract: Asparagine-linked glycosylation (N-glycosylation) is necessary for the proper folding of secreted and membrane proteins, including GPCRs. Thus, many GPCRs possess the N-glycosylation motif Asn-X-Ser/Thr at their N-termini and/or extracellular loops. We found that human GPR109A (hGPR109A) has an N-glycosylation site at Asn(17) in the N-terminal atypical motif, Asn(17)-Cys(18)-Cys(19). Why does hGPR109A require the atypical motif, rather than the typical sequence? Here we show that Asn(17)-Cys(18)-Cys(19) sequence of hGPR109A possesses 2 biologic roles. First, Asn(17)-X-Cys(19) contributed to hGPR109A N-glycosylation by acting as an atypical motif. This modification is required for the normal surface expression of hGPR109A, as evidenced by the reduced surface expression of the nonglycosylated mutants, hGPR109A/N17A, and the finding that hGPR109A/C19S and hGPR109A/C19T, which are N-glycosylated at Asn(17), exhibited expression similar to the wild-type receptor. Second, the X-Cys(18)-Cys(19) dicysteine is indispensable for hGPR109A function. Substitution of Cys(18) or Cys(19) residue to Ala impaired Gi-mediated signaling via hGPR109A. We propose the disulfide bond formations of these residues with other Cys existed in the extracellular loops for the proper folding. Together, these results suggest that the atypical motif Asn(17)-Cys(18)-Cys(19) is crucial for the normal surface trafficking and function of hGPR109A.
authors.affiliation: *Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Department of Immunology, Faculty of Medicine, Akita University, Akita, Japan; Department of Lipid Signaling, National Center for Global Health and Medicine, Tokyo, Japan; and Department of Life Science, Faculty of Science, Okayama University of Science, Okayama, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yasuda
authors.affiliation: *Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Department of Immunology, Faculty of Medicine, Akita University, Akita, Japan; Department of Lipid Signaling, National Center for Global Health and Medicine, Tokyo, Japan; and Department of Life Science, Faculty of Science, Okayama University of Science, Okayama, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Imura
authors.affiliation: *Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Department of Immunology, Faculty of Medicine, Akita University, Akita, Japan; Department of Lipid Signaling, National Center for Global Health and Medicine, Tokyo, Japan; and Department of Life Science, Faculty of Science, Okayama University of Science, Okayama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ishii
authors.affiliation: *Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Department of Immunology, Faculty of Medicine, Akita University, Akita, Japan; Department of Lipid Signaling, National Center for Global Health and Medicine, Tokyo, Japan; and Department of Life Science, Faculty of Science, Okayama University of Science, Okayama, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: *Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Department of Immunology, Faculty of Medicine, Akita University, Akita, Japan; Department of Lipid Signaling, National Center for Global Health and Medicine, Tokyo, Japan; and Department of Life Science, Faculty of Science, Okayama University of Science, Okayama, Japan moto-nakamura@dls.ous.ac.jp.
authors.forename: Motonao
authors.initials: M
authors.lastname: Nakamura
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004151:Dipeptides / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D057809:HEK293 Cells; D006367:HeLa Cells; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D034622:RNA Interference; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D004151:Dipeptides; C530636:HCAR2 protein, human; D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled; D011978:Receptors, Nicotinic; C046557:cysteinylcysteine; D001216:Asparagine; D006602:Hexosyltransferases; C099573:STT3A protein, human; C551978:STT3B protein, human
keywords: Asn-X-Cys motif; N-linked glycosylation; Nicotinic acid receptor; STT3B; receptor activation