The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odors. Here we used a combination of calcium imaging and single-cell RT-PCR to identify odorant receptors (ORs) for odorants with related structures but varied odors. We found that one OR recognizes multiple odorants and that one odorant is recognized by multiple ORs, but that different odorants are recognized by different combinations of ORs. Thus, the olfactory system uses a combinatorial receptor coding scheme to encode odor identities. Our studies also indicate that slight alterations in an odorant, or a change in its concentration, can change its "code," potentially explaining how such changes can alter perceived odor quality. (+info)
A spatial map of olfactory receptor expression in the Drosophila antenna.
Insects provide an attractive system for the study of olfactory sensory perception. We have identified a novel family of seven transmembrane domain proteins, encoded by 100 to 200 genes, that is likely to represent the family of Drosophila odorant receptors. Members of this gene family are expressed in topographically defined subpopulations of olfactory sensory neurons in either the antenna or the maxillary palp. Sensory neurons express different complements of receptor genes, such that individual neurons are functionally distinct. The isolation of candidate odorant receptor genes along with a genetic analysis of olfactory-driven behavior in insects may ultimately afford a system to understand the mechanistic link between odor recognition and behavior. (+info)
Functional identification and reconstitution of an odorant receptor in single olfactory neurons.
The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand-receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription-coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest. (+info)
Conservation of sequence and structure flanking the mouse and human beta-globin loci: the beta-globin genes are embedded within an array of odorant receptor genes.
In mouse and human, the beta-globin genes reside in a linear array that is associated with a positive regulatory element located 5' to the genes known as the locus control region (LCR). The sequences of the mouse and human beta-globin LCRs are homologous, indicating conservation of an essential function in beta-globin gene regulation. We have sequenced regions flanking the beta-globin locus in both mouse and human and found that homology associated with the LCR is more extensive than previously known, making up a conserved block of approximately 40 kb. In addition, we have identified DNaseI-hypersensitive sites within the newly sequenced regions in both mouse and human, and these structural features also are conserved. Finally, we have found that both mouse and human beta-globin loci are embedded within an array of odorant receptor genes that are expressed in olfactory epithelium, and we also identify an olfactory receptor gene located 3' of the beta-globin locus in chicken. The data demonstrate an evolutionarily conserved genomic organization for the beta-globin locus and suggest a possible role for the beta-globin LCR in control of expression of these odorant receptor genes and/or the presence of mechanisms to separate regulatory signals in different tissues. (+info)
The variable and conserved interfaces of modeled olfactory receptor proteins.
The accumulation of hundreds of olfactory receptor (OR) sequences, along with the recent availability of detailed models of other G-protein-coupled receptors, allows us to analyze the OR amino acid variability patterns in a structural context. A Fourier analysis of 197 multiply aligned olfactory receptor sequences showed an alpha-helical periodicity in the variability profile. This was particularly pronounced in the more variable transmembranal segments 3, 4, and 5. Rhodopsin-based homology modeling demonstrated that the inferred variable helical faces largely point to the interior of the receptor barrel. We propose that a set of 17 hypervariable residues, which point to the barrel interior and are more extracellularly disposed, constitute the odorant complementarity determining regions. While 12 of these residues coincide with established ligand-binding contact positions in other G-protein-coupled receptors, the rest are suggested to form an olfactory-unique aspect of the binding pocket. Highly conserved olfactory receptor-specific sequence motifs, found in the second and third intracellular loops, may comprise the G-protein recognition epitope. The prediction of olfactory receptor functional sites provides concrete suggestions of site-directed mutagenesis experiments for altering ligand and G-protein specificity. (+info)
Odor response properties of rat olfactory receptor neurons.
Molecular biology studies of olfaction have identified a multigene family of molecular receptors that are likely to be involved in odor transduction mechanisms. However, because previous functional data on peripheral coding were mainly collected from inferior vertebrates, it has been difficult to document the degree of specificity of odor interaction mechanisms. As a matter of fact, studies of the functional expression of olfactory receptors have not demonstrated the low or high specificity of olfactory receptors. In this study, the selectivity of olfactory receptor neurons was investigated in the rat at the cellular level under physiological conditions by unitary extracellular recordings. Individual olfactory receptor neurons were broadly responsive to qualitatively distinct odor compounds. We conclude that peripheral coding is based on activated arrays of olfactory receptor cells with overlapping tuning profiles. (+info)
Protein kinase Cbeta and delta selectively phosphorylate odorant and metabotropic glutamate receptors.
Recombinant protein segments from a metabotropic glutamate receptor and from an odorant receptor were used as substrates in protein kinase C phosphorylation assays. Protein kinase Cbeta and delta phosphorylated an intracellular consensus phosphorylation site in the metabotropic glutamate receptor. Only protein kinase Cdelta phosphorylated a novel extracellular consensus phosphorylation site in the odorant receptor. These results suggest differential regulation of these receptors by protein kinase C isotypes. (+info)
Functional identification of a goldfish odorant receptor.
The vertebrate olfactory system utilizes odorant receptors to receive and discriminate thousands of different chemical stimuli. An understanding of how these receptors encode information about an odorant's molecular structure requires a characterization of their ligand specificities. We employed an expression cloning strategy to identify a goldfish odorant receptor that is activated by amino acids-potent odorants for fish. Structure-activity analysis indicates that the receptor is preferentially tuned to recognize basic amino acids. The receptor is a member of a multigene family of G protein-coupled receptors, sharing sequence similarities with the calcium sensing, metabotropic glutamate, and V2R class of vomeronasal receptors. The ligand tuning properties of the goldfish amino acid odorant receptor provide information for unraveling the molecular mechanisms underlying olfactory coding. (+info)