AbstractExpression of a receptor protein has, for the first time, been definitively correlated with sensitivity to a particular odorant. This receptor, expressed in the nematode Caenorhabditis elegans, appears to be distinct from the putative vertebrate odorant receptors

Firestein, Stuart

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Olfaction: Scents and sensibility
Stuart Firestein
Expression of a receptor protein has, for the first time,
been definitively correlated with sensitivity to a
particular odorant. This receptor, expressed in the
nematode Caenorhabditis elegans, appears to be
distinct from the putative vertebrate odorant receptors.
Address: Department of Biological Sciences, Columbia University,
New York, New York 10027, USA.
Current Biology 1996, Vol 6 No 6:666–667
© Current Biology Ltd ISSN 0960-9822
Here is the problem: 1000 receptors and 10 000 ligands,
match them up and find a way to make sense of the result.
This is what the vertebrate olfactory system manages to
accomplish by discriminating between at least this many
odorous molecules. When putative odorant receptor genes
were identified and cloned some five years ago, one of the
big surprises was that the gene family, with as many as
1000 members, was second in size only to that devoted to
the immune response [1]. What does this mean for under-
standing the olfactory neural code? How much of the dis-
crimination occurs at the level of the peripheral receptors,
and how much is the result of the combinatorial processing
of multiple inputs in higher brain centers?
Although it was thought that answers to these critical ques-
tions would soon be forthcoming after the cloning of
odorant receptor genes, a crucial piece of information
remained obdurately lacking: how many odors, and of what
types, do each of these receptors recognize? From physio-
logical recordings, it has been known for some time that
individual olfactory neurons typically respond to anywhere
from three to ten odors [2,3]. To do this, they must express
either multiple receptors or a single receptor that is promis-
cuous in its binding selectivity. If, as most investigators in
the field believe, the latter option is more likely, what are
the parameters that determine how many and which odor-
ants a receptor will bind? Are they related chemically, and if
so how? Nothing in the physiological literature gives a hint
about this. The difficulty is that we know precious little
about the ‘pharmacology’ of odorant receptor proteins. Yet
without this piece of the puzzle, there is little hope of
understanding the neural processing that gives rise to the
exquisite discriminations made by the olfactory system.
Why is it that we know a great deal about odorant receptor
genes, but very little about the proteins they encode? This
is primarily because odorant receptor proteins have been
frustratingly resistant to expression in a heterologous
system. In only one reported case has any of the more than
100 cloned receptor genes been functionally expressed, a
success achieved by Heinz Breer and colleagues using a
baculovirus vector in sf9 cells [4]. And even in this lone
case the response, as measured by the production of a
second messenger, to the odorants lillial and lyral was
comparatively small and saturated at a low concentration,
suggesting that receptor expression was not robust.
Adding to the difficulty of this sort of experiment are the
highly improbable odds of matching a given cloned recep-
tor with the appropriate odorant ligands. Testing 10 000
odorants on each receptor is simply not practical.
The traditional strategy for neurobiologists faced with these
sorts of hopelessly complex problems has been to search for
solutions in simpler, but no less interesting, systems.
Following this well-worn path, Sengupta et al. [5] went after
odorant receptors in the nematode Caenorhabditis elegans.
C. elegans responds to a far smaller number of odorants than
do mammals such as ourselves, and has a correspondingly
smaller number of cells devoted to the task. Nevertheless,
olfaction is still one of the primary sensory systems in this
animal, and at least 10 % of the neurons in its entire
nervous system are devoted to chemosensation [6].
By using a behavioral screen for chemosensory mutants,
Sengupta et al. [5] isolated a line of worms with a defective
ability to sense the attractant di-acetyl. From previous
experiments [7], it was known that di-acetyl signalling
requires the activity of a paired set of chemosensory
neurons known as the AWA cells. In C. elegans, perhaps
unlike vertebrates, a single olfactory cell expresses multi-
ple receptors and is able to respond to multiple odors [8].
The AWA neurons, for example, also signal the presence
of pyrazine and thiazole compounds. From the behavioral
assay, a strain defective in di-acetyl detection was deter-
mined to have a mutation in a single gene, dubbed odr-10,
which was mapped to the X chromosome. Using the
powerful genetic techniques and partial genome map
available with C. elegans, the odr-10 gene was cloned and
found to encode a protein of 339 amino acids.
Analysis of the distribution of hydrophobic residues in the
sequence of the ODR-10 protein revealed seven putative
transmembrane regions, suggestive of a G-protein-coupled
receptor. A search of the Genbank database, however,
revealed little similarity to any known protein, except for a
low (12 %) level of sequence identity to one of the verte-
brate olfactory receptors. Indeed, ODR-10 appears to lack
almost all of the sequences that characterize the mammalian
odorant receptor family. Furthermore, the putative third
intracellular loop, notably short in the vertebrate odorant
receptors (17 residues), is twice as long in ODR-10. This
region has been implicated in G-protein coupling inter-
actions, so this difference may be useful in determining the
second messenger system used by the C. elegans receptor
[9]. The presence of introns in odr-10 is also unusual;
although a few G-protein-coupled receptor genes do have
introns, most, including the vertebrate odorant receptor
genes, do not [9]. What this may mean for the control of
gene expression and the possibility that alternative splicing
generates transcripts encoding additional receptors will
undoubtedly be the subject of intense investigation. This is
particularly interesting in view of the fact that multiple
receptors are expressed by individual cells in C. elegans.
The novel sequence of the C. elegans receptor provides a
possible explanation for the curious failure to identify
candidate odorant receptor genes, using the known
sequences of the vertebrate receptors, in invertebrates.
This failure has been especially galling in the insect world,
where olfaction is known to be a primary sense and where
so much behavioral and neurophysiological data regarding
the olfactory system have been compiled. In spite of the
best efforts of a number of laboratories working in animals
from Drosophila to honeybee to moth, invertebrate odorant
receptors have previously gone undetected. Indeed, one of
the most important consequences of the Sengupta et al. [5]
work is likely to be the provision of potential primer
sequences to search for insect odorant receptor genes
using the  polymerase chain reaction (PCR).
But the major immediate contribution of this work is the
correlation it provides between a cloned odorant receptor
and a specific odorant ligand sensitivity. ODR-10 appears
to have a high affinity for di-acetyl, and the mutation that
enabled the gene to be identified — a substitution of a
tyrosine for a histidine in the third transmembrane domain
— leaves the animal with virtually no di-acetyl sensitivity.
The possible discovery of other, equally subtle mutations
in this gene, and the characterization of their effects on
ligand sensitivity, could provide invaluable insight into
the key residues and structural features of these receptors.
The evidence that ODR-10 is indeed an odorant receptor
is comprehensive: odr-10 mutants are specifically defect-
ive in responses to di-acetyl and not to pyrazine or thiaz-
oles; the odr-10 phenotype can be rescued by injection of
the mutant animals with a cosmid containing the normal
gene; deletion of a significant portion of the odr-10 gene
results in a di-acetyl-insensitive animal; the odr-10 gene is
specifically expressed in AWA cells; and the protein is
localized to the sensory cilia of those cells.
In a particularly elegant experiment, wild-type odr-10 was
placed under the control of the odr-3 promoter, which
drives expression not only in the AWA neuron, but also in
AWC neurons that are not normally sensitive to di-acetyl.
Animals expressing the odr-3::odr-10 construct were able to
detect di-acetyl, indicating that ODR-10 can confer di-
acetyl sensitivity on either the AWA or the AWC neurons,
or both. An intriguing extension of this experiment, at least
theoretically possible in C. elegans, might be to use an alter-
native promoter to express wild-type odr-10 in another
class of chemosensitive cell, perhaps the ASE neurons that
sense non-volatiles such as cAMP and salts. This would
amount to a heterologous expression system within the
animal itself, and could provide a method for exploring
receptor structure–function relationships through in vitro
mutagenesis, expression and testing.
What are the implications for the strategy used by the
vertebrate olfactory system to encode a chemical environ-
ment rich in stimuli? First there is the issue of similarity,
or rather the lack of it, to the vertebrate family of recep-
tors. Many of the differences suggest different strategies
are used to control gene expression: for example, the fact
that chemosensitive neurons in C. elegans clearly express
more than one receptor suggests that specific transcrip-
tional regulatory mechanisms, distinct from those in verte-
brates, are employed. ODR-10 itself clearly differs from
the vertebrate receptors, but is nonetheless a member of
the G-protein- coupled receptor family. This new receptor
gives us a further tool to explore the critical molecular
features of ligand binding and receptor activation. There
is, however, still a profound need to develop a system in
which many individual odorant receptors can be function-
ally expressed and assayed for ligand binding, and that
could be used in a kind of pharmacological analysis of
these receptors. The enormous odorant receptor family is
a kind of gift of nature in which all the mutations that an
experimenter might want to assay for different ligand
sensitivities have already been produced by evolution. We
just have to figure out how to unwrap the present.
Acknowledgements
Supported by the NIDCD and the Human Frontiers Science Program.
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