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Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex.

ABSTRACT: Olfactory receptors (Ors) convert chemical signals--the binding of odors and pheromones--to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled receptors. The functional insect Or comprises an odor- or pheromone-specific Or subunit and the Orco co-receptor, which is highly conserved in all insect species. The insect Or-Orco complex has been proposed to function as a novel type of ligand-gated nonselective cation channel possibly modulated by G-proteins. However, the Or-Orco proteins lack homology to any known family of ion channel and lack known functional domains. Therefore, the mechanisms by which odors activate the Or-Orco complex and how ions permeate this complex remain unknown. To begin to address the relationship between Or-Orco structure and function, we performed site-directed mutagenesis of all 83 conserved Glu, Asp, or Tyr residues in the silkmoth BmOr-1-Orco pheromone receptor complex and measured functional properties of mutant channels expressed in Xenopus oocytes. 13 of 83 mutations in BmOr-1 and BmOrco altered the reversal potential and rectification index of the BmOr-1-Orco complex. Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K(+) selectivity. We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined. Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex.

SUBMITTER: Nakagawa T

PROVIDER: S-EPMC3293798 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex.

Nakagawa Tatsuro T Pellegrino Maurizio M Sato Koji K Vosshall Leslie B LB Touhara Kazushige K

PloS one 20120305 3

Olfactory receptors (Ors) convert chemical signals--the binding of odors and pheromones--to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled ...[more]

PMID: 22403649

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Project description:Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.

Project description:BackgroundInfection by jaagsiekte sheep retrovirus (JSRV) and by enzootic nasal tumor virus (ENTV) depends on cell-surface expression of the virus entry receptor, hyaluronidase 2 (Hyal2). Human Hyal2 binds the envelope (Env) proteins of these viruses and is functional as a receptor, but Hyal2 from mice does not bind Env nor does it mediate entry of either virus. Here we have explored the amino acid determinants that account for the difference in receptor function.ResultsAnalysis of human-mouse Hyal2 chimeric proteins showed that amino acid differences responsible for the difference in Hyal2 receptor activity were localized to the central third of Hyal2. Human Hyal2 mutants containing single or double amino acid replacements with the respective mouse amino acids were generated across this region and were assayed for activity. None of the single or double mutation reduced the receptor activity of human Hyal2 by more than 10-fold, whereas mouse Hyal2 activity is reduced 1,000-fold from that of human Hyal2. While the 3-dimensional structures of mammalian Hyal2 proteins are unknown, bee venom hyaluronidase shows significant amino acid similarity to human and mouse Hyal2 and its structure has been determined. Many mutations having the largest negative effects on human Hyal2 function mapped to a small region of the bee venom hyaluronidase close to but not overlapping the active site of the enzyme, suggesting that this site represents the binding site for Env. Analysis of synonymous and non-synonymous nucleotide substitutions in the coding sequences of multiple mammalian Hyal2 proteins shows that the proteins are undergoing strong selection for amino acid conservation. We found no evidence for positive selection of amino acid changes that might reflect evolution of mammalian hosts to resist JSRV or ENTV infection.ConclusionThese results show that the greatly reduced receptor activity of mouse Hyal2 in comparison to that of human Hyal2 is determined by multiple amino acid changes acting in concert. In particular, no one amino acid change blocks infection. However, the most important amino acids map to a small patch on a predicted 3-dimensional Hyal2 structure, which may represent the binding site for Env.

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Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex.

Publications

Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex.

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