Identification of functionally important residues of the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate neuromedin U receptor.
The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBAN(R2K)) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca(2+) mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca(2+) mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide.
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Takeshi
T
Kawai
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Yukie
Y
Katayama
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Linjun
L
Guo
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Desheng
D
Liu
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Tatsuya
T
Suzuki
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Kou
K
Hayakawa
the Molecular Entomology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan, and.
Jae Min
JM
Lee
the Molecular Entomology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan, and.
Toshihiro
T
Nagamine
the United States Department of Agriculture-Arid Land Agricultural Research Center, Maricopa, Arizona 85138.
J Joe
JJ
Hull
the Molecular Entomology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan, and.
Shogo
S
Matsumoto
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Hiromichi
H
Nagasawa
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, amtanok@mail.ecc.u-tokyo.ac.jp.
Masaru
M
Tanokura
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, aknagata@mail.ecc.u-tokyo.ac.jp.
Koji
K
Nagata
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D012831:Bombyx; D002118:Calcium / Q000378:metabolism; D019295:Computational Biology; D017124:Conserved Sequence; D018698:Glutamic Acid; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008024:Ligands; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D021381:Protein Transport; D018013:Receptors, Neuropeptide / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D008024:Ligands; D009479:Neuropeptides; D018013:Receptors, Neuropeptide; D018698:Glutamic Acid; D002118:Calcium
FXPRX-amide Motif; G-protein-coupled Receptor (GPCR); Ligand-Receptor Interaction; Molecular Docking; Molecular Modeling; Neuromedin U; Peptide Hormone; Receptor Structure-Function; Sex Pheromone Biosynthesis; Silkworm