Allosteric modulation of a human odorant receptor.

Odor perception is first determined by how the myriad of environmental volatiles are detected at the periphery of the olfactory system. The combinatorial activation of dedicated odorant receptors generates enough encoding power for the discrimination of tens of thousands of odorants. Recent studies have revealed that odorant receptors undergo widespread inhibitory modulation of their activity when presented with mixtures of odorants, a property likely required to maintain discrimination and ensure sparsity of the code for complex mixtures. Here, we establish the role of human OR5AN1 in the detection of musks and identify distinct odorants capable of enhancing its activity in binary mixtures. Chemical and pharmacological characterization indicate that specific α-ß unsaturated aliphatic aldehydes act as positive allosteric modulators. Sensory experiments show decreased odor detection threshold in humans, suggesting that allosteric modulation of odorant receptors is perceptually relevant and likely adds another layer of complexity to how odors are encoded in the peripheral olfactory system.

Odorant Receptor Inhibition Is Fundamental to Odor Encoding.

Most natural odors are complex mixtures of volatile components, competing to bind odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) of the nose. To date, surprisingly little is known about how OR antagonism shapes neuronal representations in the detection layer of the olfactory system. Here, we investigated its prevalence, the degree to which it disrupts OR ensemble activity, and its conservation across phylogenetically related ORs. Calcium imaging microscopy of dissociated OSNs revealed significant inhibition, often complete attenuation, of responses to indole-a commonly occurring volatile associated with both floral and fecal odors-by a set of 36 tested odorants. To confirm an OR mechanism for the observed inhibition, we performed single-cell transcriptomics on OSNs exhibiting specific response profiles to a diagnostic panel of odorants and identified three paralogous receptors-Olfr740, Olfr741, and Olfr743-which, when tested in vitro, recapitulated OSN responses. We screened ten ORs from the Olfr740 gene family with ∼800 perfumery-related odorants spanning a range of chemical scaffolds and functional groups. Over half of these compounds (430) antagonized at least one of the ten ORs. OR activity fitted a mathematical model of competitive receptor binding and suggests normalization of OSN ensemble responses to odorant mixtures is the rule rather than the exception. In summary, we observed OR antagonism occurred frequently and in a combinatorial manner. Thus, extensive receptor-mediated computation of mixture information appears to occur in the olfactory epithelium prior to transmission of odor information to the olfactory bulb.

In Vitro Mutational Analysis of the ß2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor.

Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse ß2-adrenergic-receptor (mß2AR), robustly traffics to the plasma membrane. We set out to characterize mß2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mß2AR using a Green Fluorescent Protein-tagged mß2AR (mß2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mß2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mß2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mß2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mß2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mß2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.

ß2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the ß2 adrenergic receptor (ß2AR). We have characterized these ß2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound ß2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All ß2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality.

Divergent evolution among teleost V1r receptor genes.

The survival of vertebrate species is dependent on the ability of individuals to adequately interact with each other, a function often mediated by the olfactory system. Diverse olfactory receptor repertoires are used by this system to recognize chemicals. Among these receptors, the V1rs, encoded by a very large gene family in most mammals, are able to detect pheromones. Teleosts, which also express V1r receptors, possess a very limited V1r repertoire. Here, taking advantage of the possibility to unequivocally identify V1r orthologs in teleosts, we analyzed the olfactory expression and evolutionary constraints of a pair of clustered fish V1r receptor genes, V1r1 and V1r2. Orthologs of the two genes were found in zebrafish, medaka, and threespine stickleback, but a single representative was observed in tetraodontidae species. Analysis of V1r1 and V1r2 sequences from 12 different euteleost species indicate different evolutionary rates between the two paralogous genes, leading to a highly conserved V1r2 gene and a V1r1 gene under more relaxed selective constraint. Moreover, positively-selected sites were detected in specific branches of the V1r1 clade. Our results suggest a conserved agonist specificity of the V1R2 receptor between euteleost species, its loss in the tetraodontidae lineage, and the acquisition of different chemosensory characteristics for the V1R1 receptor.

Olfactory expression of a single and highly variable V1r pheromone receptor-like gene in fish species.

Sensory neurons expressing members of the seven-transmembrane V1r receptor superfamily allow mice to perceive pheromones. These receptors, which exhibit no sequence homology to any known protein except a weak similarity to taste receptors, have only been found in mammals. In the mouse, the V1r repertoire contains >150 members, which are expressed by neurons of the vomeronasal organ, a structure present exclusively in some tetrapod species. Here, we report the existence of a single V1r gene in multiple species of a non-terrestrial, vomeronasal organ-lacking taxon, the teleosts. In zebrafish, this V1r gene is expressed in chemosensory neurons of the olfactory rosette with a punctate distribution, strongly suggesting a role in chemodetection. This unique receptor gene exhibits a remarkably high degree of sequence variability between fish species. It likely corresponds to the original V1r present in the common ancestor of vertebrates, which led to the large and very diverse expansion of vertebrate pheromone receptor repertoires, and suggests the presence of V1rs in multiple nonmammalian phyla.