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of age 1+ juvenile spring Chinook salmon reared as part of a
hatchery program in the Yakima River, Washington. One-year-old
juveniles were sampled at their central rearing hatchery and then
again just prior to release as smolts from three different sites where
they were transferred for acclimation and release. Using
quantitative PCR, we assessed mRNA expression levels of several
ORs representing distinct subfamilies of ORs expressed in the
olfactory epithelium. After several weeks of rearing at different
sites, smolts reared in different waters displayed distinct patterns of
OR expression. In a similar experiment, sockeye salmon siblings
reared in two different water sources also displayed distinct
patterns of OR mRNA expression. These results are consistent with
our hypothesis that distinct odors present in the waters of different
tributaries may influence mRNA expression in the olfactory
epithelium. These findings may ultimately facilitate development of
molecular tools for assessing imprinting and identifying critical
chemical components in natural waters that are important for
imprinting. Acknowledgements: Funding provided by NWFSC
#P211
POSTER SESSION V:
TRIGEMINAL SYSTEM; BEHAVIOR
AND PSYCHOPHYSICS; ODORANT
RECEPTORS & OLFACTION PERIPHERY
Functional Analysis of Nematode GPCRS in Yeast
Muhammad Tehseen, Alisha Anderson, Mira Dumancic,
Stephen Trowell
CSIRO Food Futures Flagship and Division of Ecosystem Sciences,
Black Mountain Laboratories Canberra, Australia
Given its ease of genetic manipulation, rapid growth, and
eukaryotic secretory pathway, yeast is an attractive host system for
the development of robust heterologous expression systems. Yeast
has been successfully used for the heterologous expression of
mammalian membrane proteins, specifically G-protein coupled
receptors (GPCRs). Although the
Caenorhabditis elegans
genome
was sequenced 13 years ago and encodes over 1,000 GPCRs, of
which several hundred are believed to respond to volatile organic
ligands, only one of these receptors, ODR-10, has been linked to a
specific ligand, 2,3-butanedione. Here we report the tailoring of a
Saccharomyces cerevisiae
strain for the analysis of
C. elegans
olfactory receptor function. In this study, a yeast
gpa1D ste2D
double mutant was used to develop a strain that efficiently couples
a nematode olfactory receptor with the yeast signalling pathway.
We used three different reporter genes: green fluorescent protein
(GFP), histidine (HIS
3
) and MEL1 to verify activation of the signal
transduction pathway by ligand-GPCR interactions. With this
heterologously engineered yeast system, we will be able to
accelerate the de-orphaning of
C. elegans
GPCR proteins.
Acknowledgements: Australian Department of Defence for
CTD and Department of Prime Minister and Cabinet and
Attorney General’s.
#P212
POSTER SESSION V:
TRIGEMINAL SYSTEM; BEHAVIOR
AND PSYCHOPHYSICS; ODORANT
RECEPTORS & OLFACTION PERIPHERY
M3 Muscarinic Acetylcholine Receptor Potentiates
Mammalian Odorant Receptors through Inhibiting
ß-Arrestin2 Recruitment
Yue Jiang
1
, Hiroaki Matsunami
1,2
1
Department of Molecular Genetics and Microbiology, Duke
University Medical Center Durham, NC, USA,
2
Department of
Neurobiology, Duke University Medical Center Durham, NC, USA
In mammals, activation of odorant receptors (ORs) expressed in the
olfactory epithelium mediates the perception of smell. These ORs
form the largest class of G-protein coupled receptors (GPCRs).
The M3 muscarinic acetylcholine receptor (M3-R), which is
co-expressed with ORs in the olfactory epithelium, couples to and
enhances the G protein based cAMP response of ORs. This
enhancement is further increased by M3-R agonists and reduced
by M3-R antagonists. As the olfactory epithelium contains a
population of cholinergic cells and is innervated by
parasympathetic nerves that release acetylcholine, the potentiation
of OR mediated cAMP response by M3-R suggests the layer of
olfactory signaling regulation by the neurotransmitter. However,
the mechanism of OR potentiation by M3-R is not well understood.
In accordance with classic GPCR signaling, OR desensitization
involves ß-arrestin2 recruited to the activated receptor.
We developed a split luciferase based assay system to measure
ß-arrestin2 recruitment by activated ORs in live heterologous cells.
In this system, dose-dependent ß-arrestin2 recruitment is observed
for various ORs when stimulated by their cognate ligands. Using
this assay, we show that M3-R reduces ß-arrestin2 recruitment for
various ORs, raising the possibility that M3-R potentiates OR
mediated G protein activation by inhibiting ß-arrestin2 recruitment
and receptor desensitization. Activation of M3-R with agonist
further inhibits ß-arrestin2 recruitment to the OR, while M3-R
antagonist reduces the inhibition. When ß-arrestin2 is knocked
down using siRNA, the potentiation of OR cAMP response by
M3-R is attenuated. These results suggest that M3-R potentiates
cAMP response of ORs, at least in part, through attenuating
ß-arrestin2 recruitment by activated ORs. Acknowledgements:
This work is supported by NIH.
#P213
POSTER SESSION V:
TRIGEMINAL SYSTEM; BEHAVIOR
AND PSYCHOPHYSICS; ODORANT
RECEPTORS & OLFACTION PERIPHERY
Chemosensory processing of odorants in the skin
Daniela Busse, Anna Christina Sondersorg, Heike Benecke,
Hanns Hatt
Ruhr-University Bochum/ Cellphysiology Bochum, Germany
In its essential role as a protective barrier the skin is exposed to
multiple environmental factors. Keratinocytes represent the major
cell type of the epidermal layer of the skin. They express a variety
of different sensory receptors that enable to react to various
thermal, mechanical and chemical stimuli and thus contribute to
information processing in the skin. This study is dedicated to
unravel the molecular and cellular mechanisms underlying odorant
perception in the human skin. For this purpose, the physiological
impact of odorant exposure was analyzed in cultured human
keratinocytes using the calcium imaging technique. Several
fragrances have been identified that induce transient calcium
98 | AChemS Abstracts 2012
Abstracts are printed as submitted by the author(s)
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