Abstract
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.
Highlights
The moth pheromone biosynthesis-activating neuropeptide (PBAN) and vertebrate neuromedin U (NMU) have a similar biologically essential C-terminal motif (FX1PRX2-NH2)
The potential three-dimensional spatial coordinates of the conserved residues were approximated using a homology-based molecular model of B. mori PBAN receptor (PBANR) generated from the human 2AR (PDB codes 2RH1 and 3D4S) and human A2A adenosine receptor (A2AAR) (PDB code 3EML) crystal structures
We modeled the C-terminal five residues and amide of NMU (C5NMU) with a type I -turn similar to C5PBAN, and we generated homology models of NMU receptor 1 (NMUR1) using the same structural templates (PDB codes 2Y03 and 3QAK; supplemental Data S4) as those used to construct the PBANR models
Summary
The moth pheromone biosynthesis-activating neuropeptide (PBAN) and vertebrate neuromedin U (NMU) have a similar biologically essential C-terminal motif (FX1PRX2-NH2). We used multiple sequence alignments and PBANR homology modeling to identify 27 amino acid residues in the B. mori PBANR sequence as potential ligand interaction sites. To assess their functional roles, the identified residues were Ala-substituted and expressed as PBANR-EGFP fusion proteins in cultured insect cells with their subcellular localization, ligand binding, and ligand-induced mobilization of extracellular Ca2ϩ assessed. Docking simulations using a homology-based molecular model of PBANR with the solution structure of the minimal active fragment of PBAN (i.e. FSPRL-NH2, referred to as C5PBAN) incorporating a type I -turn [42,43,44] were consistent with both mutational data and the degree and nature of sequence conservation across PBANRs and related receptors. The docking models suggest that the functionally essential amide and C-terminal residue of both PBAN and NMU interact with two glutamate residues in TM2 and TM3, both of which are highly conserved across the NMU/PBANR family of GPCRs
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