title: In Vitro Mutational and Bioinformatics Analysis of the M71 Odorant Receptor and Its Superfamily.
abstract: We performed an extensive mutational analysis of the canonical mouse odorant receptor (OR) M71 to determine the properties of ORs that inhibit plasma membrane trafficking in heterologous expression systems. We employed the use of the M71::GFP fusion protein to directly assess plasma membrane localization and functionality of M71 in heterologous cells in vitro or in olfactory sensory neurons (OSNs) in vivo. OSN expression of M71::GFP show only small differences in activity compared to untagged M71. However, M71::GFP could not traffic to the plasma membrane even in the presence of proposed accessory proteins RTP1S or mβ2AR. To ask if ORs contain an internal "kill sequence", we mutated ~15 of the most highly conserved OR specific amino acids not found amongst the trafficking non-OR GPCR superfamily; none of these mutants rescued trafficking. Addition of various amino terminal signal sequences or different glycosylation motifs all failed to produce trafficking. The addition of the amino and carboxy terminal domains of mβ2AR or the mutation Y289A in the highly conserved GPCR motif NPxxY does not rescue plasma membrane trafficking. The failure of targeted mutagenesis on rescuing plasma membrane localization in heterologous cells suggests that OR trafficking deficits may not be attributable to conserved collinear motifs, but rather the overall amino acid composition of the OR family. Thus, we performed an in silico analysis comparing the OR and other amine receptor superfamilies. We find that ORs contain fewer charged residues and more hydrophobic residues distributed throughout the protein and a conserved overall amino acid composition. From our analysis, we surmise that it may be difficult to traffic ORs at high levels to the cell surface in vitro, without making significant amino acid modifications. Finally, we observed specific increases in methionine and histidine residues as well as a marked decrease in tryptophan residues, suggesting that these changes provide ORs with special characteristics needed for them to function in olfactory neurons.
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, NY, United States of America.
authors.forename: Jaclyn
authors.initials: J
authors.lastname: Bubnell
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, NY, United States of America.
authors.forename: Sophie
authors.initials: S
authors.lastname: Jamet
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, NY, United States of America.
authors.forename: Delia
authors.initials: D
authors.lastname: Tomoiaga
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, NY, United States of America.
authors.forename: Charlotte
authors.initials: C
authors.lastname: D'Hulst
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, NY, United States of America; Director of Bioinformatics, Center for Translational and Basic Research, CUNY, New York, NY, United States of America.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Krampis
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, NY, United States of America; The Graduate Center Biology Program, CUNY, New York, NY, United States of America; The Graduate Center Behavioral and Cognitive Neuroscience Program, CUNY, New York, NY, United States of America.
authors.forename: Paul
authors.initials: P
authors.lastname: Feinstein
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D056510:Position-Specific Scoring Matrices; D054730:Protein Interaction Domains and Motifs; D021382:Protein Sorting Signals; D021381:Protein Transport; D011554:Pseudopodia / Q000235:genetics / Q000378:metabolism; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment
chemical_list: C502656:Olfr151 protein, mouse; D021382:Protein Sorting Signals; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins