A Novel Multigene Family May Encode Odorant Receptors: A Molecular Basis for Odor Recognition

1. A Novel Multigene Family May Encode Odorant Receptors: A Molecular Basis for Odor Recognition Linda Buck and Richard Axel Published in Cell, Volume 65, 175-187 April 5, 1991 Presented by Adam Warner on Oct. 12, 2004

2. Linda Buck • Research Associate • Assistant Investigator 1984-1991 1994-1997

3. Linda Buck • Assistant Professor 1991-1996 • Associate Professor 1996-2001 • Professor 2001-2002

4. Linda Buck Full Member - Division of Basic Sciences Fred Hutchinson Cancer Research Centre, (Seattle) 2002-Current

5. Richard Axel 1970 M.D. School of Medicine Professor, Pathology and Biochemistry 1978-1984

6. Richard Axel Investigator 1984 - Current Professor 1999 - Current

7. How Do We Smell? (Stinky Hockey Equipment) • How do we identify the smells around us? • Humans can distinguish between over ten thousand different odours • Even minute changes in molecular structure of an odorant can cause a different perception in its smell

8. Perception of Smell • Interaction between odourous ligands and specific olfactory receptors acknowledged • Two major models for the interaction that results in smell being perceived

9. Two Models • few receptors, each able to interact with a very large number of molecules • large number of receptors, each able to interact with only one, or very few molecules

11. Previous Findings • Bronshtein and Minor, 1977 • Removal of the cilia leads to a loss in the perception of odour • Boekhoff et al., 1990 • cilia is isolated from rat olfactory epithelium • response to odourous molecules leads to a rise in cyclic AMP • Nakamura and Gold, 1987 • rise in cyclic AMP leads to depolarization of olfactory neurons

12. Pathway

13. Similarities To Other Pathways • Similar pathway observed when neurotransmitter and hormone receptors are stimulated • superfamily of transmembrane receptor proteins • transmembrane domain spans the membrane seven times

14. Hypothesis • Olfactory receptors are part of a large superfamily of transmembrane receptors (spanning membrane 7 times) • One odorant molecule can only stimulate one or very few specific receptors • must be very diverse so part of a multigene family Olfactory receptors should be localized only to the olfactory epithelium

15. Potential Candidates? • RNA isolated from olfactory epithelium • prepare cDNA • Primer Design • 5 or 6 nucleotides in tm domains 2 and 7 • conserved in transmembrane superfamily • should amplify homologous sequences in the olfactory prep • PCR - second round of amplification • Digestion with Hinfl restriction (4 base cutter) • run on gel

16. PCR Explanation

17. PCR No digestion Digestion Does molecular weight of restriction fragments = original?

18. Part of 7-Transmembrane Superfamily? • Fragments from lane 13 were cloned into a plasmid vector sequenced • clones showed sequence similarity to transmembrane superfamily • clones also showed unique sequence motifs • new family of receptors!

19. Screening • cDNA libraries were screened to obtain full length cDNA clones • used fragments from lane 13 cDNA libraries from olfactory sensory neurons • Took hybridizing plaques and used lane 13 PCR primers, PCR 710 bp fragments were purified (original size of lane 13 band)

20. Expression • Northern Blot • hybridation only seen in olfactory epithelium preparation • receptors are restricted to the olfactory epithelium

21. Screening • Found 18 cDNA clones that encoded proteins in the same new family • conserved motifs with the seven transmembrane superfamily • new features not seen in this new multigene family

22. Protein Structure White = homologous between clones Black = variable amino acids

23. How Many Receptors? • screened rat genomic libraries • at least 200 positives per haploid chromosome • most likely several hundred genes, each with multiple subfamilies With only hundreds of positive clones, how can we distinguish between over ten thousand different odorous molecules?

24. How do we smell so well? • Several hundred genes is just the lower limit of what is actually be present • reliance on PCR and other techniques • primer design • One receptor can recognize a small number of ligands with different affinities • multiple ligands in one “smell” can be processed at once by multiple receptor types • perceived as one smell but actually is a few or many odourous molecule types

25. Overview

26. Future Directions • Better understanding of the diversity of ligand that one receptor can complex with • Number of different receptors found in olfactory epithelium • Differences between rats, humans, and other species • Evidence of DNA rearrangement? • not observed in this experiment

27. References A Novel Multigene Family May Encode Odorant Receptors: A Molecular Basis for Odor Recognition Linda Buck and Richard Axel. Cell, Volume 65, 175-187 April 5, 1991 Colorado State University Webpage http://arbl.cvmbs.colostate.edu Essential Cell Biology second edition. Garland Science. 2003 University Webpages of Harvard, Columbia, John Hopkins University Howard Hughes Medical Institute http://www.hhmi.org/ The Nobel Foundation http://nobelprize.org/