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

Recognition

Linda Buck and Richard AxelPublished in Cell, Volume 65, 175-187 April 5, 1991

Presented by Adam Warner on Oct. 12, 2004

Linda Buck

•Research Associate

•Assistant Investigator

1984-1991

1994-1997

Linda Buck

•Assistant Professor 1991-1996

•Associate Professor 1996-2001

•Professor 2001-2002

Linda Buck

Full Member - Division of Basic Sciences

Fred Hutchinson Cancer Research Centre, (Seattle) 2002-Current

Richard Axel

M.D. School of Medicine

Professor, Pathology and Biochemistry

1970

1978-1984

Richard Axel

Investigator 1984 - Current

Professor 1999 - Current

How Do We Smell?

•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

(Stinky Hockey Equipment)

Perception of Smell

Interaction between odourous ligands and specific olfactory receptors acknowledged

•Two major models for the interaction that results in smell being perceived

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

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

Pathway

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

Olfactory receptors are part of a large superfamily of transmembrane receptors (spanning membrane 7 times)

Hypothesis

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

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

PCR Explanation

PCR

Does molecular weight of restriction fragments = original?

No digestion

Digestion

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!

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)

Expression

Northern Blot

•hybridation only seen in olfactory epithelium preparation

•receptors are restricted to the olfactory epithelium

Screening

•Found 18 cDNA clones that encoded proteins in the same new family

•new features not seen in this new multigene family

•conserved motifs with the seven transmembrane superfamily

Protein Structure

White = homologous between clones

Black = variable amino acids

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?

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

•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

•One receptor can recognize a small number of ligands with different affinities

Overview

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

ReferencesA 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/