Odorant Receptors

The receptors responsible for odorant discrimination were first cloned in 1991 by Linda Buck and Richard Axel (Buck and Axel 1991). A series of physiological and biochemical experiments performed during the mid-1980s indicated that odorant activation of olfactory sensory neurons was mediated by a G-protein-dependent pathway, which led to activation of adenylyl cyclase, increases in intracellular concentrations of cyclic adenosine monophosphate (cAMP), activation of cyclic nucleotide-gated channels, and neuron depolarization (Firestein et al. 1991; Lowe et al. 1989; Nakamura and Gold 1987; Pace et al. 1985; Sklar et al. 1986; see also Chapter 8). The subsequent cloning of olfactory-specific genes coding for a Gα protein (Gαolf) (Jones and Reed 1989) and for a cAMP-gated channel (Dhallan et al. 1990) further strengthened the involvement of cAMP in odorant signal transduction. These experiments strongly indicated that the odorant receptors (ORs) should be G-protein-coupled receptors (GPCRs). About the same time, the polymerase chain reaction (PCR) technique was developed (Saiki et al. 1988), and the first GPCRs had been identified. Comparison between the sequences of rhodopsin and (β-adrenergic receptors indicated that receptors that couple to G-proteins showed related structures, with seven membrane-spanning regions (Dixon et al. 1986). Comparison of the sequences of a higher number of GPCRs (around 20 G-protein-compled receptor (GPCR) sequences were known by 1989) revealed that they all shared a related seven-transmembrane structure and they also shared limited sequence motifs. In 1989, it was shown for the first time that degenerate primers could be used in PCR reactions to identify new members of the GPCR family (Libert et al. 1989). The approach used by Buck and Axel to isolate the Odorant receptor (OR) genes was based on the assumptions that the receptors should belong to a large family of GPCRs and their expression should be restricted to the olfactory epithelium (Buck and Axel 1991). Eleven degenerate primers that would allow amplification of all known GPCRs at the time were designed and all possible combinations were used in PCR reactions with rat olfactory epithelium cDNA. As a result, 64 bands of appropriate sizes were obtained in agarose gels. The next step was to screen these bands for the ones containing the OR genes. It was reasoned that if one of the bands contained cDNAs corresponding to multiple OR genes, four-cutter restriction enzymes would cleave the DNA into smaller fragments showing sizes that, when summed up, would produce a size greater than that of the original band. The 64 PCR bands were treated with the I or III restriction enzymes, and for most of the bands, restriction digestion generated fragments with sizes that summed up to the original band size. Therefore, these PCR products contained a single DNA species. However, restriction digestion of one of the bands produced fragments with sizes that summed to a value far greater than that of the original PCR product. This PCR band contained a mixture of different DNA species, each of which was amplified by the same pair of degenerate primers. The band was cloned into plasmid, and individual recombinant plasmids were sequenced. All sequences were different, but they all showed a GPCR-like structure. Using Northern blot analysis, it was also demonstrated that these receptors are expressed in the olfactory epithelium, but not in a further eight tissues analyzed, including the brain, retina, and liver (Buck and Axel 1991). In addition, in order to estimate the approximate size of the OR gene family, rat genomic libraries were screened for OR genes using a mixture of the OR cDNAs as probes. It was estimated then that the rat haploid genome should contain at least 500–1000 OR genes (Buck and Axel 1991; Buck 1992). Comparison of different rat OR amino acid sequences revealed that, even though they are extremely diverse, they share conserved motifs that are characteristic of the OR family, such as GN in transmembrane domain I, PMYF/LFL in transmembrane domain II, MAYDRYVAIC in transmembrane domain III, KAFSTCA/GSHLSVV in transmembrane domain 6, and PMLNPFIYSLRN in transmembrane domain VII (Buck and Axel 1991) (Figure 7.1). Additional members of the OR family were identified by using degenerate primers matching these OR motifs in PCR reactions with olfactory epithelium cDNA or genomic DNA (since the OR coding region is contained in one single exon). Degenerate primers matching to the highly conserved motifs in transmembrane III and VI were very efficient in amplifying a large fraction of the mouse OR genes (Malnic et al. 1999; Michaloski et al. 2006; Ressler et al. 1993).