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Aim: To understand how the olfactory transduction system is organized• Are there several receptor protein “species” each of which detect a class

of odorant molecules, or, like the retina, are there relatively few species that respond to a relatively broad panel of odorants?• Humans can discriminate ~10,000 monomolecular odorants

Approach: 1. Receptors should be related to similar proteins found elsewhere, based on

their functional properties.2. The broad sensitivity of olfactory systems suggests a multigene family of

receptor proteins (unlike retina) that should have 7 transmembrane (7TM) spanning regions.

3. Odor receptor proteins should be unique to the olfactory epithelium (based on comparison of cDNA from different tissues).

Overview of the olfactory epithelium 3 major cell types:• Olfactory sensory neurons• Supporting cells• Basal (stem) cells

Molecular basis of sensory transduction:1. G-protein coupled receptors activate2. Adenylate cyclase production of cAMP3. cAMP activates ion channels

Activation of this pathway results in depolarization of the cell membrane potential.

Hypothesis: Olfactory receptors are related to other G-protein coupled receptors.

“Digestion of one of these, PCR 13, yielded a large number of fragments whose sizes summed to a value much greater than that of the undigested PCR 13 DNA, indicating that PCR 13 might contain multiple species of DNA that are representatives of a multigene family.”

“cDNA prepared from olfactory epithelium RNA was subjected to PCR amplification with a series of different primer oligonucleotides [designed based on other 7TMPs] ; the DNA products of appropriate size [based on other 7TMPs (600-1300bp)] were isolated, further amplified by PCR, and size fractionated on agarose gels.”

Northern blot analysis indicates that isolated RNAs are specific to olfactory epithelium. Hence these 7TMPs are specific to the olfactory epithelium.

Analysis of DNA nucleotide sequences reveal several distinct 7-TMPsThese sequences are also novel among other known 7-TMPs

external cytoplasmic

Analysis of TM5 region + flanking loops suggests that these 7TMP receptors can be grouped into subfamilies based on sequence homology

external cytoplasmic

Analysis of DNA sequences reveal several distinct 7-transmembrane proteins Prototypical 7-TMP highlighting regions of variability (black circles).• Remember the primers originally used were for TM regions 2 and 6 hence

they should be the least variable• The high overall variability of the TM regions suggests that these are

binding sites for unique classes of odor

external

cytoplasmic

70 from 7: Olfactory receptors represent a gene super family!Using 7 primers designed from 7 different sequences (Fig 4) a total of 70 bands emerge on a Southern blot suggesting 70 unique DNA sequences (aka. 70 receptor proteins).