Among the five senses, smell is unique in that it is dedicated to discriminating an enormous diversity of stimulating ligands. Olfactory sensory neurons achieve this detection by the expression of a multigene family of seven-transmembrane G protein–coupled receptors (GPCRs) originally identified by Linda Buck and Richard Axel (1991). These receptors, termed olfactory receptors (OR, plural ORs), are expressed in the olfactory tissue of all terrestrial vertebrates examined thus far, and have been shown to respond directly to odorant binding. The identification and subsequent study of ORs has provided great insight into the molecular and neuronal organization of the olfactory system. Indeed, in 2004, Buck and Axel were jointly awarded the Nobel Prize in Physiology for their pioneering work in odorant receptor discovery. Most of the current understanding of ORs results from experiments from the mouse model which is the focus of this review.
General Physiology and Classification of ORs
Olfactory sensory neurons reside in the main olfactory epithelium (MOE) which is located in the distal recess of the nasal cavity. The MOE covers the evaginated nasal terbinates resulting in increased surface capacity to sample ligands. The dendrite of each olfactory neuron projects toward the nasal lumen providing direct sensory contact with the odor environment. The dendrite is tufted with 20–30 sensory cilia and OR proteins localized to these cilia membranes further increase surface exposure to the ligand environment. Active sniffing draws odorant ligands into the nasal cavity enabling binding and subsequent activation of ORs. Each odorant receptor is expressed from a single coding gene. Together, individual odorant receptor genes comprise the largest GPCR family in the mammalian genome (in the human genome they account for approximately 3% of predicted exons). ORs belong to class A GPCRs (Rhodopsin family) and can be broadly divided into classes I and II (Zhang and Firestein 2002). Class I is highly conserved across evolution; related to chemosensory receptors in fish. Sequence analysis shows class II to be more divergent, suggesting that this OR class may have evolved to provide specific ecological adaptations.
Evolution of Odorant Receptors
Comparative genomics has revealed that the odorant receptor family shows rapid gene birth and death events across evolution. Tandem gene duplications have resulted in chromosomal clusters of closely related ORs with high sequence homology and similar ligand-binding profiles. In the mouse, there are 43 odorant receptor clusters found on every chromosome. Old world primates, including humans, have a significantly smaller OR repertoire (390 putatively functional genes) compared to other sequenced vertebrates such as mice (1,468 putatively functional genes), rats (1,750 putatively functional genes), and dogs (922 putatively functional genes). The evolutionary loss of functional OR coding regions has been attributed to the correlated evolution of trichromatic vision and an increased utilization of visual stimuli.
OR-Mediated Signal Transduction
Olfactory Receptor Choice: The Singularity of OR Gene Expression
A remarkable property of the olfactory system is the ability to detect and distinguish among a seemingly endless variety of odor molecules. To achieve this precise discrimination, each individual olfactory sensory neuron expresses only one of the many OR genes present in the genome. Indeed this has been referred to as the “one neuron-one receptor” rule. Expression of a single OR dedicates each sensory neuron is responsive only to the cognate ligands of the expressed receptor, and sensory neurons that express different receptors display differential response profiles to odor ligands. The mechanisms that enable a neuron to activate the expression of a single receptor, and additionally silence all other OR loci distributed across the genome, are only partially understood. Recent whole genome chromatin immunoprecipitation analysis suggests that MOE neurons initially employ a distinct type of methylation of OR loci to generate heterochromatin silencing of all ORs. In a second step, one receptor reverses this silencing. The choice of which particular OR gene will be expressed in each neuron is thought to be largely a random process. However, each OR is restricted to a stereotypic expression “zone” in the MOE which may be regulated by zone-specific transcription factors. OR genes are polyallelic, and analysis of the expression of maternal and paternal genes have revealed that only one allele is expressed per neuron. This allelic exclusion simplifies the mechanistic challenge of coordinating receptor activation between both alleles and further increases the ability of genetic variation to functionally diversify odorant detection. Activation of the chosen receptor requires physical interaction of the OR promoter with a cis-acting locus control region (LCR). For one of the OR clusters, containing mouse olfactory receptor28 (MOR28), the LCR known as the “H-region,” was identified as a 2.1 kb noncoding sequence that is evolutionary conserved between mouse and human. Deletion of the H-region abolishes the expression of MOR28 OR genes. Other LCRs that act in cis to regulate the expression of ORs within a cluster, or in trans to coordinate receptor choice across the genome, are still largely unknown. Additional experiments with transgenes have shown that if the chosen receptor cannot produce a functional protein (either through a frameshift or a deleltion) a second receptor will be activated. This suggests that the OR protein itself generates a negative feedback signaling to ultimately repress the activation of additional ORs. How functional ORs repress expression of additional ORs in olfactory sensory neurons remains unknown as mutating an ORs ability to activate G-protein mediated signaling has no effect on OR negative feedback regulation. Future experiments will be necessary to uncover the mechanisms that stabilize the expression of one receptor.
Role of ORs in Axon Pathfinding
Olfactory sensory neurons that express the same OR converge in specific neuropil, called glomeruli, in the olfactory bulb in the brain. Sensory neurons use both dorsal/ventral and anterior/posterior coordinates to direct their axons to the correct glomeruli. While the general process of receptor choice is stochastic, each OR is expressed in a stereotypic “zone” within the olfactory epithelium. The mechanisms that regulate this zonally restricted anatomic organization have not been identified; however, the positional organization of ORs correlates with the expression of complementary gradients of guidance molecules including Robo-2, Neuropilin-2 (Nrp2), and Sema-3f in the sensory neurons. Corresponding gradients of Slit-1 and Slit-3 are present in the olfactory bulb thereby creating a topographic organization of glomeruli along the dorsal/ventral axis that mirrors the general spatial organization of ORs in the MOE (Cho et al. 2007).
In contrast to these genetically determined mechanisms, an elegant series of experiments has shown involvement of an activity driven mechanism, signaling from the OR itself, to direct anterior/posterior axon guidance. The first indication of this arose from generation of mice in which pairs of genomic OR coding regions were swapped. These mutant mice displayed OR-driven mis-localization of their glomeruli. Moreover, OR protein was found to be localized to the axon terminus; a pivotal position to direct axon guidance. How do ORs guide axons? Key insights came from the observation that ORs mutant in Gs signaling failed to form proper glomeruli. Complementary studies of constitutively active Gs and PKA mutants indicate that high levels of OR-mediated cAMP directs axons to the posterior, while low cAMP levels directs axons to the anterior. cAMP levels directly correspond to the transcription of neuropilin-1 (Nrp1) an axon guidance molecule. Nrp1 and Sema3A expression levels form complementary anterior/posterior gradients in olfactory neurons. Axon–axon interactions are thought to sort and organize neurons as they develop toward their targets (Imai et al. 2009). While these mechanisms account for general axon targeting, it is thought that OR-mediated cAMP signaling additionally further refines glomerular patterning. Levels of neuronal activity regulate additional sets of axon guidance and adhesion molecules including Kirrel2, Kirrel3, EphA5, and ephrin-A. The repulsive and adhesive effects of these molecules are thought to promote homotypic formation of individual glomeruli (Serizawa et al. 2006). The mechanism of how individual ORs generate differential levels of cAMP activity that can distinguish the position of neighboring glomeruli remains to be understood.
The Combinatorial Code of ORs to Encode Odor Identity
Other Olfactory Receptors
In addition to the ORs, the main olfactory epithelium also expresses a family of trace amine receptors (TAARs) which have been shown to detect volatile amines (Liberles and Buck 2006). Additionally most vertebrates have an accessory olfactory system, the vomeronasal organ (VNO), which is physiologically and morphologically distinct from the main olfactory epithelium. The VNO has been shown to express at least three classes of chemosensory receptors that are evolutionarily distinct both from the odorant receptors and from each other: vomeronasal receptors class I (V1Rs, Vmn1Rs) (Dulac and Axel 1995), vomeronasal receptors class II (V2Rs, Vmn2Rs) (Herrada and Dulac 1997; Matsunami and Buck 1997; Ryba and Tirindelli 1997), and formyl peptide receptors (FPRs) (Liberles et al. 2009; Riviere et al. 2009). GPCRs expressed in the VNO are thought to largely detect volatile and peptide pheromonal ligands that mediate social behaviors like courtship, territorial aggression, gender and individual recognition, maternal aggression, and interspecies defense (Bean and Wysocki 1989; Chamero et al. 2007; Del Punta et al. 2002; Haga et al. 2010; Kimoto et al. 2005; Leinders-Zufall et al. 2000; Papes et al. 2010; Wysocki and Lepri 1991).
Environmental odor cues are detected by hundreds of seven-membrane-spanning odorant receptors, expressed in the olfactory system. These olfactory receptors can be broadly classified into type I and type II, type I being evolutionarily more ancient and conserved. On binding odors, ORs can signal through a canonical pathway involving cAMP second messenger that regulates the activity of the CNG channel, depolarizing the neurons. Additionally there are other noncanonical signal transductions involving TrpM5 or cGMP. Each olfactory neuron expresses a specific receptor and activation of different combinations of receptors by an odor or mixture of odors helps an organism to distinguish an endless repertoire of chemicals.