Orna Man Tsviya Olender and Doron Lancet

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Olfaction, the sense of smell, is a versatile and sensitive mechanism for detecting volatile odorous molecules [1,2]. It is mediated by hundreds of olfactory receptor (OR) proteins in the membrane of the chemosensory neurons, extended by the formation of long cilia. Odorant binding initiates a cascade of signal transduction events that involve a G-protein-dependent elevation of cAMP second messenger and opening of cAMP-gated ion channels [3]. The olfactory system utilizes a combinatorial receptor-coding scheme to discriminate different odorants [4-7]. A specific odor is recognized as a pattern of saturation values, which may be viewed as an "activity vector" generated across an array of ORs. As each sensory neuron expresses only one OR gene (in fact, a single allele thereof) [8], the pattern of receptor activation is faithfully represented in an array of cellular activities, as conveyed to the central nervous system.

In humans, a repertoire of more than 1000 OR genes has been elucidated by cloning and genomic data mining [9-12]. Genes of this "olfactory subgenome", the largest subgroup within the G-protein-coupled receptor (GPCR) hyperfamily, are disposed in dozens of clusters on most human chromosomes. This genomic disposition is accounted for by an elaborate process of gene and cluster duplication, as well as gene conversion events [13,14]. Approximately two-thirds of all human ORs are pseudogenes that have accumulated up to 27 frame-disrupting mutations [9,15]. Such an observation is consistent with the diminished importance of the sense of smell in primates, including humans. The OR subgenome consists of 17 gene families [16,17], which belong to either class I (fish-like) or class II (tetrapod-specific) receptors. Interestingly, the proportion of intact genes is greater among class I receptors, suggesting they have greater functional importance.

The genetics of ORs has only begun to be elucidated. In humans, it is commonly believed that OR polymorphisms underlie the widespread inter-individual variations in odorant threshold [18]. This is in line with genetic models developed in other species [19-21]. Providing direct evidence for this notion by genotype-phenotype correlations in humans constitutes an important future challenge that may involve the identification of pseudogenes present only in certain individuals [22].

As is the case for most integral membrane proteins, the three-dimensional structure of ORs has not yet been determined. Therefore, scientists have resorted to alternative methods of structure prediction. These include homology modeling [23,24] based on the structure of bovine rhodopsin, the only GPCR for which crystallographic structural information is available (see Chapter 22). This approach is rendered more valid because both ORs and rhodopsin belong to class A GPCRs [25]. Homology modeling was performed despite the marginal sequence similarity between ORs and rhodopsin (« 21% amino acid identity over most of the protein sequence) and was greatly aided by the occurrence of sequence motifs common to the two sets of proteins (Fig. 1). Such shared features include the overall seven-helix structure, the conserved extracellular cysteine bridge between the first and second extracellular loops, the (D/E)RY motif in the transition zone between the third transmembrane helix and second intracellular loop, the SY motif in transmembrane 5 (TM5), and the NP motif in TM7 [10,26].

Variability analysis identified 17 hypervariable residues which point to a putative odorant binding pocket formed by TM3 to TM6; these residues were proposed to form the complementarity-determining regions (CDRs). The use of enhanced variability as a criterion for functional importance is analogous to an approach originally proposed for immunoglob-ulins [26]. Additional information on potential functional residues is provided by correlated mutation analysis [27] and by comparisons of variability patterns in orthologous and paralogous sequences ([28]; Man, Gilad, and Lancet, unpublished work).

Figure 1 A three-dimensional homology model of an OR protein (OR1E1) using the structure of bovine rhodopsin ([39]; PDB code 1F88). As a template, in side view (A, extracellular at the top), and in a frontal view into the putative binding site from the extracellular side (B). Functionally important features are shaded and include the following: the conserved MAYDRYVAIC motif in the end of TM 3; exceptionally conserved residues in the second and third intracellular loops; the putative disulfide bond between the first and second extracellular loops; the N-glycosylation site near the amino terminus; the second extracellular loop that covers the binding site; the 17 putative CDRs [26].

Figure 1 A three-dimensional homology model of an OR protein (OR1E1) using the structure of bovine rhodopsin ([39]; PDB code 1F88). As a template, in side view (A, extracellular at the top), and in a frontal view into the putative binding site from the extracellular side (B). Functionally important features are shaded and include the following: the conserved MAYDRYVAIC motif in the end of TM 3; exceptionally conserved residues in the second and third intracellular loops; the putative disulfide bond between the first and second extracellular loops; the N-glycosylation site near the amino terminus; the second extracellular loop that covers the binding site; the 17 putative CDRs [26].

Functionally important residues were also highlighted using sequence conservation analysis (Fig. 1). In particular, ten sequence motifs concentrated in either the extracellular-most or intracellular-most parts of the receptors have been identified [26,29]. It was suggested that these motifs are involved in the interactions of the receptors with their signaling partners upstream (e.g., odorant-binding protein, OBP, [30]) or downstream (olfactory GTP-binding protein [31]). While many GPCRs contain conserved palmitoylation sites in their carboxy-terminal region (such as rhodopsin; see Chapter 22), which anchor them to the membrane, only 26% of intact human ORs contain such sites [10].

The second extracellular loop of ORs has attracted particular attention. It is comparatively long, and, in addition to the cysteine residue conserved in all GPCRs, it contains two conserved cysteines, perhaps forming an internal disulfide bond [10,32]. This loop also has a relatively high variability, and a correlation was found between residues within it and those in the putative binding pocket [27]. This might suggest that the loop acts as an auxiliary recognition domain, reflecting the unique specificity of the odorant-binding pocket [33]. The finding of OR mRNA in the axon terminals of olfactory receptor neurons within their target glomerular synaptic complex [34,35] led to the hypothesis [33] that the second extracellular loop participates in the guidance of olfactory neuronal axons [36].

Functional expression studies have the potential to identify the ligands that activate each receptor. Such studies have been hindered by the apparent failure of the transfected OR proteins to translocate efficiently to the plasma membrane [19]. Solutions to this problem have included in vivo infection with OR-containing viral vectors in rat olfactory epithelium [37]; expression of chimeric receptors containing rhodopsin sequences in a heterologous cell system, in conjunction with a promiscuous G protein [19]; and expression in oocytes [19]. A combination of calcium imaging and single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis has also been used to identify receptors that recognize specific odorant molecules and to elucidate a combinatorial code [5]. One study led to a relatively comprehensive elucidation of the odorant-binding characteristics of one OR protein, I7 [38]. Comparison of the highly homologous mouse and rat I7 receptors [19] resulted in the identification of a one-residue substitution, V206I, responsible for a shift in ligand binding preference from octanal in rat to heptanal in mouse. This residue resides in the extracellular region of transmembrane segment five in the vicinity of residues previously predicted to confer specificity, but points away from the homology-modeling-based proposed binding site, a point that will require additional scrutiny.

Future studies should involve a considerable augmentation of ligand-receptor relationships in the olfactory system in mammals. This should include improved protein expression methodologies, as well as genetic studies that would link olfactory sensitivity phenotypes to OR genotypes.

References

1. Shepherd, G. M. (1994). Discrimination of molecular signals by the olfactory receptor neuron. Neuron 13, 771-790.

2. Lancet, D. (1986). Vertebrate olfactory reception. Annu. Rev. Neurosci. 9, 329-355.

3. Nakamura, T. (2000). Cellular and molecular constituents of olfactory sensation in vertebrates. Compar. Biochem. Physiol. A 126, 17-32.

4. Lancet, D., Sadovsky, E., and Seidemann, E. (1993). Probability model for molecular recognition in biological receptor repertoires: significance to the olfactory system. Proc. Natl. Acad. Sci. USA 90, 3715-3719.

5. Malnic, B., Hirono, J., Sato, T., and Buck, L. B. (1999). Combinatorial receptor codes for odors. Cell. 96, 713-723.

6. Kajiya, K., Inaki, K., Tanaka, M., Haga, T., Kataoka, H., and Touhara, K. (2001). Molecular bases of odor discrimination: reconstitution of olfactory receptors that recognize overlapping sets of odorants. J. Neurosci. 21, 6018-6025.

7. Araneda, R. C., Kini, A. D., and Firestein, S. (2000). The molecular receptive range of an odorant receptor. Nat. Neurosci. 3, 1248-1254.

8. Reed, R. R. (2000). Regulating olfactory receptor expression: controlling globally, acting locally. Nat. Neurosci. 3, 638-639.

9. Glusman, G., Yanai, I., Rubin, I., and Lancet, D. (2001). The complete human olfactory subgenome. Genome Res. 11, 685-702.

10. Zozulya, S, E. F. and Nguyen, T. (2001). The human olfactory receptor repertoire. Genome Biol. 2, 1-12.

11. Human Olfactory Receptor Data Exploratorium (HORDE) (http:// bioinformatics.weizmann.ac.il/HORDE).

12. Crasto, C., Marenco, L., Skoufos, E., Healy, M. D., Singer, M. S., Nadkarni, P. M., Miller, P. L., and Shepherd, G. S. (2002). The Olfactory Receptor Database, publically available at http://ycmi.med.yale.edu/ senselab/ORDB/.

13. Sharon, D., Glusman, G., Pilpel, Y., Horn-Saban, S., and Lancet, D. (1998). Genome dynamics, evolution, and protein modeling in the olfactory receptor gene superfamily. Ann. N.Y. Acad. Sci. 855, 182-193.

14. Trask, B. J., Massa, H., Brand-Arpon, V., Chan, K., Friedman, C., Nguyen, O. T., Eichler, E., van den Engh, G., Rouquier, S., Shizuya, H., and Giorgi, D. (1998). Large multi-chromosomal duplications encompass many members of the olfactory receptor gene family in the human genome. Hum. Mol. Genet. 7, 2007-2020.

15. Rouquier, S., Blancher, A., and Giorgi, D. (2000). The olfactory receptor gene repertoire in primates and mouse: evidence for reduction of the functional fraction in primates. Proc. Natl. Acad. Sci. USA 97, 2870-2874.

16. Fuchs, T., Glusman, G., Horn-Saban, S., Lancet, D., and Pilpel, Y. (2001). The human olfactory subgenome: from sequence to structure and evolution. Hum. Genet. 108, 1-13.

17. Glusman, G., Bahar, A., Sharon, D., Pilpel, Y., White, J., and Lancet, D. (2000). The olfactory receptor gene superfamily: data mining, classification, and nomenclature. Mamm. Genome 11, 1016-1023.

18. Amoore, J. E. (1974). Evidence for the chemical olfactory code in man. Ann. N.Y. Acad. Sci. 237, 137-143.

19. Krautwurst, D., Yau, K. W., and Reed, R. R. (1998). Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95, 917-926.

20. Griff, I. C. and Reed, R. R. (1995). The genetic basis for specific anosmia to isovaleric acid in the mouse. Cell 83, 407-414.

21. Sengupta, P., Chou, J. H., and Bargmann, C. I. (1996). odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell. 84, 899-909.

22. Menashe, I., Man, O., Lancet, D., and Gilad, Y. (2002). Population differences in haplotype structure within a human olfactory receptor gene cluster. Hum. Mol. Genet. 11(12): 1381-1390.

23. Floriano, W. B., Vaidehi, N., Goddard, 3rd, W. A., Singer, M. S., and Shepherd, G. M. (2000). Molecular mechanisms underlying differential odor responses of a mouse olfactory receptor. Proc. Natl. Acad. Sci. USA 97, 10712-10716.

24. Singer, M. S. (2000). Analysis of the molecular basis for octanal interactions in the expressed rat 17 olfactory receptor. Chem. Senses 25, 155-165.

25. Horn, F., Weare, J., Beukers, M. W., Horsch, S., Bairoch, A., Chen, W., Edvardsen, O., Campagne, F., and Vriend, G. (1998). GPCRDB: an information system for G protein-coupled receptors. Nucleic Acids Res. 26, 275-279.

26. Pilpel, Y. and Lancet, D. (1999). The variable and conserved interfaces of modeled olfactory receptor proteins. Protein Sci. 8, 969-977.

27. Singer, M. S., Oliveira, L., Vriend, G., and Shepherd, G. M. (1995). Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis. Receptors Channels 3, 89-95.

28. Lapidot, M., Pilpel, Y., Gilad, Y., Falcovitz, A., Sharon, D., Haaf, T., and Lancet, D. (2001). Mouse-human orthology relationships in an olfactory receptor gene cluster. Genomics 71, 296-306.

29. Skoufos, E. (1999). Conserved sequence motifs of olfactory receptorlike proteins may participate in upstream and downstream signal transduction. Receptors Channels 6, 401-413.

30. Tegoni, M., Pelosi, P., Vincent, F., Spinelli, S., Campanacci, V., Grolli, S., Ramoni, R., and Cambillau, C. (2000). Mammalian odorant binding proteins. Biochim. Biophys. Acta. 1482, 229-240.

31. Jones, D. T. and Reed, R. R. (1989). Golf: an olfactory neuron-specific G protein involved in odorant signal transduction. Science 244, 790-795.

32. Sosinsky, G. E. (1996). Molecular organization of gap junction membrane channels. J. Bioenerg. Biomembr. 28, 297-309.

33. Singer, M. S., Shepherd, G. M., and Greer, C. A. (1995). Olfactory receptors guide axons. Nature 377, 19-20.

34. Vassar, R., Chao, S. K., Sitcheran, R., Nunez, J. M., Vosshall, L. B., and Axel, R. (1994). Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981-991.

35. Ressler, K. J., Sullivan, S. L., and Buck, L. B. (1994). Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79, 1245-1255.

36. Mombaerts, P. (1996). Targeting olfaction. Curr. Opin. Neurobiol. 6, 481-486.

37. Zhao, H., Ivic, L., Otaki, J. M., Hashimoto, M., Mikoshiba, K., and Firestein, S. (1998). Functional expression of a mammalian odorant receptor. Science 279, 237-242.

38. Araneda, R. C., Kini, A. D., and Firestein, S. (2000). The molecular receptive range of an odorant receptor. Nat. Neurosci. 3, 1248-1255.

39. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000). Crystal structure of rhodopsin: a G protein-coupled receptor. Science. 289, 739-745.

This Page Intentionally Left Blank

Diabetes Sustenance

Diabetes Sustenance

Get All The Support And Guidance You Need To Be A Success At Dealing With Diabetes The Healthy Way. This Book Is One Of The Most Valuable Resources In The World When It Comes To Learning How Nutritional Supplements Can Control Sugar Levels.

Get My Free Ebook


Post a comment