In Vitro Mutational and Bioinformatics Analysis of the M71 Odorant Receptor and Its Superfamily

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

<i>Ex vivo</i> profiling of M71::GFP fusion protein.

<p>(A) Peak currents for M71 (red), M71::GFP (green) and IRES-M71 (yellow) extracellular dendritic knobs after excitation by seven odorants: five M71 specific odorants with variable affinities (mBNZ:benzaldehyde, 2aACP:2-amino acetophenone, eMLT:ethyl maltol, PIP:piperinal, 4mACP:4methyl acetophenone) and two M72 specific odorants (mSal: methyl salicylate and BTP:butryophenone). Odorants arranged by structure. Concentration for six odorants were all at 10μM except 2aACP was 1μM due to its higher affinity. The major difference observed was 2 fold higher peak current to 4mACP for M71 relative to both M71::GFP and IRES-M71. (B) Current traces for M71 and M71::GFP show similar desensitization profiles to 2aACP and 4mACP regardless of 4mACP 1 log lower EC50. Single cell dose response curves for M71 (red), M71::GFP (green) and IRES-M71 (yellow) reveal a 1 log difference lower in EC50 to 4mACP relative to M71.</p

M71::GFP does not traffic to the plasma membrane.

<p>(A) M71::GFP expression in OP6 cells in trapped perinuclearly (A’) Same cell with colabelling of plasma membrane revealed by CellMask Red. (B) mβ2AR::GFP expression in OP6 cells gives a flat morphology that has sharp focus. Expression is observed in filopodia. (B’) Same cell as (B) with colabelling of plasma membrane revealed by CellMask Red. (C, D) M71::GFP and mβ2AR::GFP expression vectors. (E) Filopodia counts by Cell Mask Red and GFP for mβ2AR::GFP and mβ2AR::GFP. (F) M71::GFP co-expression with mβ2AR::mCherry in OP6 cells maintains GFP fluorescence trapped perinuclearly whereas (F’) mβ2AR::mCherry expression is found in the filopodia.</p

Amino acid residue composition of Adrenergic, Amine, Odorant and Trace amine-associated receptors.

<p>(A) Amino acid percentages for Adrenergic, Amine, Odorant (ORs) and Trace amine-associated Receptors (TAARs). ORs and TAARs profiles differ from Adrenergic and Amine receptors. (B) Amino acid differences for ORs and TAARs expressed as percentage difference from Amine receptors. Many residues show an increase in proportion (positive percentage) and decrease in proportion (negative percentage). Three residues for ORs show a large, 60% difference (methionine-M, histidine-H and tryptophan-W). These three residues also show large differences from TAARs with methionine and histidine comprising the bulk of the statistical difference between them. Results did not change if TAARs and Adrenergic receptors were removed from the Amine receptor superfamily. Amino acids are ordered according to their hydropathy index from 4.5 to -4.5 (L to R). It appears that most of the residue differences favor ORs and TAARs to be more hydrophobic.</p

M71::GFP directed mutations that do not traffic to plasma membrane.

<p>(A) M71::GFP mutations F12D;I13H;L14D;G15V;G16T (FILGG to DHDGG), Y35A, P58V;M59T;Y60N (PMY to VTN), M98E, C169A, Y176T;F177C (FY to TC), C178A and Y217A. None of these mutations gave raise to any GFP-labeled filopodia. (B) M71::GFP mutations with N-linked glycosylation modifications: 4x NQS, 4x NSS, NQS to NGT, NQS to NAT, Nt to K4 Nt. (C) Addition of leader sequences (Kirrel2, 5HT3, Calcumenin, Rhodopsin, Endothelin, LUCY and LUCY-FLAG) to M71::GFP did not relocate GFP expression into the filopodia. (A-C) Co-expression with RTP1S for all M71::GFP mutations did not increase the presence of any GFP-labeled filopodia.</p

M71::GFP chimeras and truncations that do not traffic to plasma membrane.

<p>(A) M71::GFP chimeras with Nt-mβ2AR, Ct-mβ2AR, Nt-and Ct- mβ2AR. None of these chimeras led to GFP-labeled filopodia. (B) Altered NPxxY motif, Y289A M71::GFP mutants, with altered Nt and Ct: WT, ΔCt, Nt-mβ2AR, Nt-mβ2AR/ΔCt, and Nt- and Ct-mβ2AR. None of these chimeras led to GFP labeled filopodia. (A and B) Co-expression with RTP1S for all M71::GFP mutations did not increase the presence of any GFP-labeled filopodia.</p

ORs and TAARs have greater hydrophobic character than Adrenergic and Amine receptors.

<p>The percent difference in ORs and TAARs compared to Amine receptors was multiplied by the hydropathy index (HI) of each residue. Positive percent differences x positive HI values and negative percent differences x negative HI values increase the hydrophobic character to ORs. Many residues had increased the hydrophobic character or no effect. Histidine showed the most significant difference overall and in relation to TAARs. Amino acids are ordered according to their hydropathy index from 4.5 to -4.5 (L to R). (Inset) The positive and negative hydrophobic character of Adrenergic, Amine, ORs and TAARs wre calculated by adding together all residues that contribute to non-polar/neutral or polar/positive-negative hydropathy index and calculating the percentage relative to the total residues. There was a greater percentage of positive hydropathy and reduced negative hydropathy values for ORs and TAARs compared to Adrenergic and Amine receptors. This translated into higher ratios of polar/neutral to polar/positive-negative residues.</p

Distribution of Methionine, Histidine and Tryptophan in Adrenergic, Amine, OR, TAAR receptors.

<p>(A) Distribution of histidine residues; (B) Distribution of methionine residues; and (C) Distribution of tryptophan residues in Adrenergic, Amine, Odorant, and Trace amine-associted receptors. The frequency of occurrence is displayed from NxS/T to NPxxY conserved motifs, however the graphs are all anchored by the Ct NPxxY motif. Three histidine and five methionine residues are found at discrete regions in ORs compared all other receptor types (blue and black asterisks); See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141712#pone.0141712.g003" target="_blank">Fig 3</a> for exact position. Tryptophan residues are nearly abolished in ORs with only one conserved position (red asterisk), but distributions are conserved between TAARs and Adrenergic receptors.</p

Conserved OR sequences revealed by logo plot shared with M71 and mβ2AR.

<p>(A) OR logo using mouse Class I and II odorant receptor sequences. Most highly conserved OR residues are depicted with black circles. M71 and mβ2AR sequences are delineated underneath logo plot; Residues with greater than 50% bit conservation to OR logo are underlined. Conserved residues between M71 and mβ2AR are in bold and red. M71 residue swaps between M71 and mβ2AR are delineated in purple and green, respectively. M71 residues converted to alanine are described with bold blue A. Five conserved methionine residues, three conserved histidine residues and one weakly conserved tryptophan residue marked with asterisk. Two of these residues are also found in OSN expressed TAARs marked with red asterisk. (B) Schematic of OR seven transmembrane structure with conserved methionine and histidine residues depicted in linear and predicted 3 dimensional state. Methionine and histidine residues in non-transmembrane might be in close enough proximity to coordinate a metal group, like copper. Methionine residues in TM3 and TM7 may form bridge if modified.</p

MouSensor: A Versatile Genetic Platform to Create Super Sniffer Mice for Studying Human Odor Coding

Typically, ∼0.1% of the total number of olfactory sensory neurons (OSNs) in the main olfactory epithelium express the same odorant receptor (OR) in a singular fashion and their axons coalesce into homotypic glomeruli in the olfactory bulb. Here, we have dramatically increased the total number of OSNs expressing specific cloned OR coding sequences by multimerizing a 21-bp sequence encompassing the predicted homeodomain binding site sequence, TAATGA, known to be essential in OR gene choice. Singular gene choice is maintained in these “MouSensors.” In vivo synaptopHluorin imaging of odor-induced responses by known M71 ligands shows functional glomerular activation in an M71 MouSensor. Moreover, a behavioral avoidance task demonstrates that specific odor detection thresholds are significantly decreased in multiple transgenic lines, expressing mouse or human ORs. We have developed a versatile platform to study gene choice and axon identity, to create biosensors with great translational potential, and to finally decode human olfaction