Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yea
Trang 1development of a bioassay for odorant screening
Jasmina Minic1, Marie-Annick Persuy1, Elodie Godel1, Josiane Aioun1, Ian Connerton2,
Roland Salesse1and Edith Pajot-Augy1
1 INRA, Neurobiologie de l’Olfaction et de la Prise Alimentaire, Re´cepteurs et Communication Clinique, Jouy-en-Josas, France
2 Division of Food Sciences, School of Biosciences, University of Nottingham, Loughborough, Leicestershire, UK
The olfactory receptors (ORs) are a large group of
proteins belonging to subfamily I of G protein coupled
receptors (GPCRs) that bind odorant ligands These
receptors are predicted to contain seven
transmem-brane helices that change their relative orientation
upon odorant stimulation, resulting in the
conforma-tional change of the receptor and productive
inter-action of its intracellular loops with Golf, the a subunit
of the heterotrimeric G protein [1–3] Several lines of
evidence suggest that the mechanism of OR activation
by an odorant is central to understanding odorant per-ception and coding Each OR recognizes multiple odorants and most odorants are recognized by several ORs [4–7] One OR can discriminate between odorants with different functional groups, molecular size or shape and can even be sensitive to odorant concentra-tion [8–10] In addiconcentra-tion, receptor percepconcentra-tion of an odorant can be enhanced or antagonized by the pres-ence of another odorant [8, 11,12] Despite the import-ance of OR pharmacology to olfactory detection and
Keywords
G olf ; odorant screening; olfactory receptors;
Saccharomyces cerevisiae; yeast bioassay
Correspondence
E Pajot-Augy, INRA, Neurobiologie de
l’Olfaction et de la Prise Alimentaire,
Domaine de Vilvert, 78352 Jouy-en-Josas
Cedex, France
Fax: +33 1 34 65 22 41
Tel: +33 1 34 65 25 63
E-mail: Edith.Pajot@jouy.inra.fr
(Received 13 July 2004, revised 1 October
2004, accepted 18 November 2004)
doi:10.1111/j.1742-4658.2004.04494.x
The functional expression of olfactory receptors (ORs) is a primary require-ment to examine the molecular mechanisms of odorant perception and cod-ing Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae The membrane expression of the receptor was shown by Western blotting and immunolocalization meth-ods Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter Yeasts were engineered to coexpress I7 OR and mammalian Gasubunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation
of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis The sensitivity of the bioassay was signifi-cantly enhanced using mammalian Golf compared to the Ga15 subunit, resulting in dose-dependent responses of the system The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7
OR retains its specificity and selectivity towards ligands The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional Based on these findings, the bioas-say using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms
Abbreviations
Endo H, endoglycosidase H; GPCR, G protein-coupled receptor; KLH, keyhole limpet hemocyanin; OR, olfactory receptor; PMSF,
phenylmethylsulfonyl fluoride; PNGase F, peptide N-glycosidase F; S14, somatostatin-14; SSTR2, somatostatin receptor subtype 2; PBST, 0.05% Tween, NaCl ⁄ P i
Trang 2discrimination, detailed characterization of ligand–
receptor interaction has been achieved for relatively
few ORs due to the absence of natural sources
provi-ding one receptor in sufficient amounts and to the
inherent difficulties associated with the expression of
ORs in heterologous cell systems [10]
A major hindrance to functional expression of ORs
has been the tendency of ORs to be retained in
endo-plasmic reticulum of heterologous cells due to inefficient
folding This process results in receptor sequestration
through the formation of aggregates and degradation
before they can be transported to the plasma membrane
[13,14] The failure of ORs to translocate efficiently to
the plasma membrane was also associated with the
absence of adequate accessory proteins and chaperones
in non-native cells, or with the absence of glycosylation
at the N-terminus of the OR [15] However, ORs do
not even traffic well to the plasma membrane when
expressed in a cell line derived from olfactory
epithe-lium (ODORA cells) that exhibits some olfactory
sen-sory neuron characteristics [10,16,17]
Plasma membrane trafficking of ORs in commonly
cultured cell lines was slightly improved by appending
N-terminal protein sequences from other seven
trans-membrane domain family members Fusion proteins
have been constructed between ORs and either the
b2-adrenergic receptor [18], the N-terminal extension of
rhodopsin [19] or the membrane import sequence of the
serotonin receptor [20] Functional expression of mouse
71 OR was dramatically increased upon coexpression
with the b2-adrenergic receptor, but not that of rat I7
or human OR17-40 receptors [21] This suggested that
different ORs may require distinct GPCR partners to
drive surface expression, maybe through their persistent
physical association An alternative approach for the
functional expression of ORs utilized an adenovirus
vector to deliver OR cDNAs to the sensory neurons of
olfactory epithelia [4,8] However, this approach has
practical limitations due to the difficulty in maintaining
olfactory neurons in primary culture, the inconsistency
of viral-mediated gene transfer, and the cost if it was to
be applied to a large number of ORs
The aim of this study was to optimize the baker’s
yeast Saccharomyces cerevisiae as a host system for
properly expressing an OR at the plasma membrane,
and for its efficient coupling to a signaling pathway
that produces a measurable response to odorant
stimu-lation The yeast system was chosen for several
rea-sons Firstly, S cerevisiae has been successfully used
for functional expression of many GPCRs [22–28]
Sec-ondly, yeast constitutes an attractive system to study
membrane receptors providing a null background for
mammalian GPCRs and G proteins Finally, yeast
cells may provide a means for detailed investigation of receptor pharmacology in vivo through the use of sen-sitive reporter systems that take advantage of the func-tional homologies between yeast pheromone and mammalian GPCR signaling pathways
In genetically modified yeast strains, the reporter system is activated after receptor–ligand interaction,
Ga protein dissociation and activation of the MAP kinase pathway [29] Several GPCRs have been shown
to efficiently couple to the endogenous yeast Ga pro-tein subunit, Gpa1 Yeast Gpa1, Ste4 and Ste18 are structurally and functionally similar to mammalian Ga,
b and c subunits, respectively [30] In many cases, this functional coupling was improved by replacing Gpa1
by mammalian or chimeric Gpa1⁄ mammalian Ga sub-units that have been shown to interact with both the Ste4⁄ Ste18 complex and heterologous GPCR [31,32] Additionally, other elements of the mating signal transduction pheromone pathway were either deleted
or functionally replaced by their mammalian or mutant counterparts to optimize S cerevisiae for GPCR structure–function investigations [22,26,29,33]
In the present study we have used the rat I7 OR as
a model to investigate the OR expression in yeast since its preferred ligands (octanal, heptanal, nonanal) and their effective concentration ranges have already been determined [4,8,10,19] We recently described how
S cerevisiae can successfully be engineered as a repor-ter system for odorant detection [34] Two different yeast strains expressing an odorant receptor were only able to grow on selective media following specific odorant ligand stimulation However, this growth reporter had very limited sensitivity and was poorly adapted to the transitory nature of the response Thus, methodological improvements were drastically needed for this system to be ultimately used for pharmacologi-cal screening purposes Here, we use luciferase as a rapid reporter to study the I7 OR pharmacology We have optimized the experimental conditions for the production of the I7 OR in yeast and used biochemical and immunological methods to estimate the levels of receptor expression and its cellular localization
Results
Yeast transformations Functional expression of the I7 or OR17-40 receptor was achieved in the yeast strain MC18 modified to allow sensitive bioassay based on synthesis of lucif-erase upon odorant stimulation Strain MC18 has been reported to have an unknown mutation that prevents cell cycle arrest upon activation of the
Trang 3pheromone-mating signaling pathway and to lack GPA1 gene [35].
The yeast strains with the I7 expression vector,
pJH2-I7, or the OR17-40 expression vector, pJH2-OR17-40,
were then transformed, respectively, with either
pRGP-Golf or pRGP-Ga15 vectors to replace the lacking
GPA1 gene, or with PRGP-Golf RT–PCR analysis
conducted on RNA extracted from the strains shows
mRNA bands for the ORs (Fig 1) and the two Ga
proteins at expected sizes (data not shown) In
addi-tion, RT–PCR analysis demonstrated that mRNA of
the olfactory receptors is present in both uninduced
and induced yeast cells (Fig 1) This indicates a
leak-age of GAL1⁄ 10 promoter in glucose-containing
min-imal medium The reporter plasmid pRHF-luc was
cotransformed in the yeast strains at the same time as
the pRGP vector It places expression of luciferase
under control of the FUS1 promoter, which is
activa-ted downstream of the MAP kinase cascade (illustraactiva-ted
in Fig 2)
Biochemical characterization of the yeast I7 OR
To examine the presence of the I7 protein in
unin-duced and inunin-duced yeast cells, membrane preparations
were analyzed by immunoblotting using a polyclonal antibody raised against I7 No immunoreactivity was detected in control membrane preparations from either nontransformed MC18 yeast cells or cells transformed with the initial pJH2-somatostatin receptor subtype 2 (SSTR2) plasmid (Fig 3A) In the case of yeast cells transformed with the pJH2-I7 expression vector two immunoreactive bands were observed at approximately
40 and 51 kDa (Fig 3A) The calculated molecular weight for the I7 OR is 39 kDa, it is therefore likely that the 40 kDa band corresponds to the receptor monomer The 51 kDa band may correspond to a gly-cosylated form of the receptor since I7 OR contains two N-glycosylation sites, one at the N terminus and another in the second extracellular loop
To determine if the I7 receptor is glycosylated in
S cerevisiae, the membrane fraction from the induced yeast cells was digested with either endoglycosidase H (Endo H) or peptide N-glycosidase F (PNGase F) Figure 3B shows that the 51-kDa band is sensitive to both Endo H and PNGase F digestion, resulting in almost
Fig 1 mRNA of the rat I7 OR or human OR17-40 receptor in
trans-formed yeast strains Total RNAs from either uninduced or induced
strains transformed with pJH2-I7 or pJH2-OR17-40 expression
vec-tors were subjected to RT-PCR using specific primers to
demon-strate that mRNA synthesis of the ORs occurs even in uninduced
cells I7 cDNA in I7 plasmid and OR17-40 cDNA in
pJH2-OR17-40 plasmid were amplified in parallel as positive controls.
Fig 2 Modifications of yeast pheromone and signal transduction pathway to yield agonist-induced luciferase activity The hetero-logous OR receptor activated by its odorant ligand couples to an heterotrimetric G-protein consisting of a mammalian Ga subunit (G olf or G a15 ) and the yeast bc subunit, Ste4 ⁄ Ste18 G a dissociates from the complex and allows Ste4 ⁄ Ste18 to activate the mating MAP kinase cascade This in turn induces luciferase synthesis under the control of the FUS1 promoter, allowing quantitative read-outs of the receptor–ligand interaction.
Trang 4complete deglycosylation to yield the 40-kDa band.
The 51-kDa band thus represents the receptor
mono-mer with the exclusive addition of mannose residues
The presence of the immunoreactive bands in the
lanes relative to uninduced yeast cells (Fig 3A) is a
further sign of GAL1⁄ 10 promoter leakage, as already
seen at the mRNA level
Luciferase bioassay and functional expression
of the ORs in yeast
To address the functional integrity of the ORs
expressed in yeast, we developed a luciferase reporter
bioassay Initially, the bioassay was configured using a
control yeast strain transformed to coexpress luciferase
under control of FUS1 with the SSTR2 receptor and
Gpa1 When this strain was incubated with either
a-factor to stimulate endogenous a-factor receptor
(Ste2), or with somatostatin 14 (S14) to stimulate
SSTR2 receptor, luciferase activity was observed to
increase in a dose-dependent manner (data not shown)
Thus, the bioassay provides an effective readout of
GPCR–ligand interaction and we therefore applied the
assay to monitor the I7 or OR17-40 activity
Figure 4A summarizes differential
luciferase-medi-ated luminescence detected in the yeast strains
expres-sing I7 OR, grown at various conditions, following
their stimulation with 5 lm heptanal, octanal or non-anal In this set of experiments the effects of yeast growth temperature and GAL1⁄ 10 promoter induction were tested in order to optimize I7 OR functional expression As some GPCRs were reported to fold and traffic better to the plasma membrane when their expression level is restricted by reduced temperatures [36,37], we examined I7 OR activity in yeasts grown
at 30 or 15C Indeed, luciferase-mediated responses
to odorants were dependent on the yeast growth tem-perature The temperature shift to 15C markedly improved the functional response of the receptor (Fig 4A)
The luciferase reporter activity was compared in uninduced and galactose-induced conditions since a GAL1⁄ 10 promoter leakage had been detected in unin-duced yeasts (Figs 1 and 3A) The luciferase-mediated luminescence responses to odorant stimulations were increased in induced cells compared to uninduced cells (Fig 4A), indicating that galactose induction increases the yield of functional I7 OR relative to the leakage level
Figure 4A also shows that functional responses to odorants were several-fold higher in the strain co-expressing the Golfsubunit in comparison to the strain coexpressing the Ga15subunit when grown in the same conditions
Fig 3 Immunoblot analysis of the I7 OR in yeast membrane extracts (A) Membranes were prepared from nontransformed yeast (MC18), and transformed with either control expression vector, pJH2- SSTR2 or pJH2-I7 Each lane was loaded with 30 lg of membrane proteins Samples were analyzed by SDS ⁄ PAGE followed by Western blotting using anti-I7 IgG Yeast were grown at 15 C (B) The glycosylation sta-tus of the I7 OR was investigated on membranes from yeast transformed with pHJ2-I7 expression vector induced with 2% galactose at
15 C Membrane proteins were digested with either Endo H or PGNase F The corresponding controls were performed by using water instead of deglycosylation enzymes in respective incubation buffer Each lane was loaded with 10 lg of protein Immobilized samples were probed with anti-I7 IgG.
Trang 5In addition, the protein levels of I7 OR, Golf and
Ga15 in strains from Fig 4A were compared The
immunoblot analysis indicated that the levels of Golf
and Ga15 in membrane fractions are constant regard-less of the temperature and galactose induction, while the level of the I7 OR is higher in membrane fractions from yeasts induced by galactose at 15C (Fig 4B) Thus, the highest level of both membrane-associated and active I7 OR were obtained in yeast induced by galactose at 15C Therefore, we chose to perform pharmacological analysis on the yeast coexpressing I7 and Golfunder these conditions
Pharmacological characterization of the yeast I7 OR
Previous pharmacological investigations of the I7 OR expressed in mammalian cells have shown that recep-tor responses to odorants are dose dependent [10,38]
In this study, the yeast-borne I7 OR was stimulated by heptanal, octanal or nonanal over the concentration range from 5· 10)14 to 5· 10)4m All three ligands evoked luciferase reporter activity in a dose-dependent manner as presented in Fig 5A Response thresholds for heptanal, octanal and nonanal were 3 · 10)9,
7· 10)9, and 5· 10)8m, respectively The maximal amplitude was detected for 5· 10)7moctanal
Interestingly, as observed with the I7 OR expression
in mammalian cells, dose–response curves of the yeast
OR I7 were bell-shaped instead of exhibiting a plateau
at high ligand concentrations As shown in Fig 5A no significant response was detected for odorant concen-trations 5· 10)4m or higher This could be due to odorant and⁄ or its solvent toxicity to yeast cells or to receptor desensitization at the highest ligand concen-trations It was checked that a final odorant concentra-tion of 5· 10)5m corresponds to a concomitant dilution of the organic solvent that is not deleterious
to the yeast cells This was tested by cell shape exam-ination and count after incubation in the odorant dilu-tions, and also by monitoring the influence of odorant dilutions on the luciferase bioassay performed with S14 stimulation of a control yeast strain coexpressing SSTR2, Gpa1 and the luciferase reporter Only final odorant concentrations of the three aldehydes above
10)4m were toxic for yeast cells, and this was ascribed
to the presence of the organic solvent (dimethyl sulfox-ide) itself, which was deleterious for the yeast cells So the bioluminescence response as a function of odorant concentration is significant up to 5· 10)5m The nar-row bell-shaped dose–response curves in the range from 5· 10)8m to 5· 10)5mindeed define the opera-tional range of the I7 receptor
We also examined receptor specificity by testing whether a panel of nine various odorants might be recognized by the yeast I7 OR Among these, octanol,
A
B
Fig 4 Effects of galactose induction and temperature on functional
expression of the I7 OR in yeast (A) Luciferase bioluminescence
was measured following yeast stimulation by odorants in two
strains expressing either I7 OR and G olf , or I7 OR and G a15 Strains
were grown in minimal media with 2% glucose (uninduced) or
sub-sequently in minimal media with 2% galactose (induced) at 30 or
15 C Differential bioluminescence for each sample was calculated
with respect to controls that were prepared by replacing the odorant
by water The data were recorded as the mean ± SEM of three
sep-arate experiments (B) Western blot analysis of the I7 receptor, G olf
and Ga15expression levels in the corresponding yeast membrane
lysates as in (A) (20 lg of protein per lane) Note that the highest
level of I7 receptor expression is obtained in induced strains at
15 C while the levels of G olf and G a15 do not change significantly.
Trang 6octanon and octanoic acid were selected as they
pos-sess the same carbon chain length but have different
functional groups None of them induced any
lucif-erase activity when tested over a wide range of
concen-trations (5· 10)12to 5· 10)4m)
Similarly, the commonly used odorants
isoamyl-acet-ate, lyral, lilial, pyridine, diacetyl and
cyclohexyl-acet-ate, tested over the same concentration range, failed to
induce any luciferase activity These findings are
con-sistent with those obtained with the I7 OR expressed
in mammalian cells [8,10] and strongly suggest that
yeast expressed I7 OR retains the ligand selectivity and
specificity equivalent to its mammalian expressed
coun-terpart
The yeast-expressed OR17-40 was stimulated with
helional in the concentration range 5· 10)14 to
5· 10)4m, yielding a bioluminescence dose–response
curve shown in Fig 5B, with a threshold concentration
of 6· 10)8m, and maximal amplitude for 5· 10)6m
As in the case of I7 OR, this curve is bell shaped, and
finely tuned for helional concentrations between
5· 10)5m and 5· 10)7m In order to test OR17-40
specificity, heptanal was used as a negative control [20]
over the whole range of concentrations studied (data
not shown)
Cellular localization of the I7 OR in yeast
Immunoblot analysis showed that the I7 receptor is
associated with the yeast membrane fraction In order
to check that the I7 receptor is, at least partly,
associ-ated with yeast plasma membrane, the I7-specific anti-body was used to immunostain nonpermeabilized spheroplasts Spheroplasts of nontransformed MC18 yeast cells showed no staining with the anti-I7 IgG (Fig 6) In contrast, all the spheroplasts of I7 OR yeast strain cells that had been induced with galactose
at 15C showed an intense cortical labeling (Fig 6) indicating a clear presence of the receptor at the plasma membrane
In order to examine the ultracellular localization of the receptor, immunogold electron microscopy was performed on induced I7-transformed yeast cells grown
at 15C (Fig 7) The presence of the I7 OR was obvi-ous at the plasma membrane (two to four gold parti-cles per section) demonstrating that the I7 molecules are targeted to their functional location (long arrows)
A few gold particles were associated with vesicular structures located at the plasma membrane (double arrows), consistent with the membrane trafficking of the I7 OR molecules In the cytoplasm, gold particles were associated with endoplasmic reticulum cisternae (short arrows), thus localizing the I7 OR molecules to their secretion pathway The receptor was also present
in vacuoles (arrowheads) and sometimes associated with vacuole membranes (open arrowheads) No gold grains were observed on sections where the primary or the secondary antibody was omitted The presence of the gold particles associated with the plasma mem-brane indicates that at least some of the I7 molecules produced are inserted at the site commensurate with their ability to sense the external environment
Fig 5 Differential bioluminescence dose–response upon odorant stimulation of yeast-expressed olfactory receptors Measurements were performed on yeast transformed to coexpress the I7 OR, Golfand the luciferase reporter (A), and on yeast coexpressing the human
OR17-40, Golf, and the luciferase reporter (B) These strains were induced with 2% galactose at 15 C Dose–response curves are plotted as a dif-ference of bioluminescence response to odorants relative to controls obtained by replacing odorants with water.
Trang 7The I7 OR quantification by ELISA-type test
To quantify the level of I7 OR associated with
mem-branes, an ELISA-type test was carried out using the
specific anti-I7 IgG As purified I7 receptor is not
avail-able the calibration curve was generated by serial
dilu-tion of keyhole limpet hemocyanin (KLH)-coupled I7
antigenic N-terminal 15-amino acid peptides Fig 8A
shows this calibration curve as well as the negative
con-trol obtained by probing KLH alone Using the
KLH-coupled I7 peptide as a standard, the I7 antigen
con-centration in the range 1–100 lm could be measured
accurately (SD < 10%) Fig 8B shows the ELISA
measurements collected from the serial dilution of
membrane preparations from yeast cells expressing the
I7 OR induced at 15C Membrane proteins from
con-trol SSTR2-strain were included as a negative concon-trol
Increasing amounts of membrane proteins from the
control yeast failed to elicit an ELISA signal indicating
the specificity of the reaction (Fig 8B) In contrast, the
I7 expressing yeast produced a dose-dependent signal
that could be saturated with higher amounts of
membrane fraction bearing I7 OR The concentration
of I7 receptor produced in induced yeast was deduced
to be 327 pmolÆmg)1 of membrane protein, i.e 1.44·
105 receptor per cell This compares to 352 pmolÆmg)1
of membrane protein for recombinant expression of the a-factor receptor Ste2p itself in S cerevisiae [25], which
is the best level ever achieved for any GPCR membrane expression in yeast
Discussion
In this study we developed a novel, robust and sensi-tive yeast-based bioassay for odorant screening Yeast was engineered to functionally express an olfactory receptor in conjunction with a mammalian Ga subunit and to exhibit agonist-dependent luciferase reporter
Fig 6 Immunofluorescence confocal microscopy of the I7 OR
in yeast Optical (left panels) and confocal (right panels)
visualiza-tion of spheroplasts from nontransformed MC18 yeast and yeast
transformed with pJH2-I7 expression vector (induced at 15 C).
Immunolabeling was performed with the anti-I7 IgG and an
Alexa488-coupled secondary antibody on nonpermeabilized
sphero-plasts Scale bar, 5 lm.
Fig 7 Ultrastructural localization of the I7 OR in yeast Yeast strain transformed with pJH2-I7 expression vector and induced at 15 C was immuno-labeled with the primary anti-I7 IgG and 10 nm gold-conjugated secondary antibody Gold grains are present on the plasma membrane (long arrows) and vesicles near the plasma membrane (double arrows) They are also associated with endo-plasmic reticulum cisternae (short arrows), the vacuole (arrow-heads) and sometimes with vacuolar membrane (open arrows) Bar, 0.25 lm.
Trang 8activity By taking advantage of structural and
func-tional similarities between yeast and mammalian
GPCR signaling pathways, this assay enables the
quantitative measurement of receptor activity, or
alter-nately the detection of its ligands Using known
lig-ands of I7 OR (heptanal, octanal and nonanal), we
successfully demonstrated that they act as agonists as
already experienced in mammalian cells Odorants of
the same carbon chain length but with different
func-tional groups failed to induce any luciferase activity
demonstrating that the yeast borne receptor retains its
ligand specificity and selectivity In addition, validation
of the system was completed by demonstrating that six
commonly used odorants do not stimulate luciferase
activity OR17-40 also exhibited an intense and specific
bioluminescence in response to helional stimulation This corroborates the adequacy of the bioassay for a totally different olfactory receptor
By fusing the pheromone inducible FUS1 promoter sequence to the coding sequence of luc, the expression
of luciferase was regulated through activation of the MAP kinase signaling pathway upon OR–odorant interaction, to allow quantification of the dose– response to odorants The luciferase reporter was cho-sen for its cho-sensitivity, rapidity and easy to perform enzymatic reaction In a previous study we used the FUS1–His1 or FUS1–Hph reporters to provide odor-ant-dependent yeast growth on histidine-deficient or hygromycin-containing medium, respectively [34] Such assays are commonly used for GPCRs but have low sensitivity and are time consuming as they include a delay of 24–48 h in response [24,34] Therefore, they are not best adapted to study ORs regarding the trans-itory nature of their response to odorants, their desen-sitization and recycling observed in mammalian cells and possible degradation of odorant molecules at yeast growth temperatures b-galactosidase assay for GPCR agonist screening is more rapid but requires relatively expensive fluorogenic substrates for sensitive readout [31] We believe that our luciferase sensor can be at the basis of efficient, rapid and low cost screening of a large range of odorants
The signal can be significantly enhanced through Ga subunit engineering The intense reporter activity regis-tered demonstrates that the receptor naturally coupling
Ga protein, Golf, is able to interact efficiently with both the heterologous OR and the endogenous Gbc complex, Ste4⁄ Ste18 The efficient coupling of Golf to the pheromone response pathway was previously dem-onstrated when it complemented a Gpa1 null mutation
in S cerevisiae [35] This is in contrast to a chimeric Gpa1–Golf, which showed poor coupling efficiency with either the OR and⁄ or Ste4 ⁄ Ste18 [34] We also observed higher sensor sensitivity with Golf than with the promiscuous Ga15commonly used for pharmacolo-gical studies of recombinant ORs This probably arises from the poor affinity of Ga15for yeast Gbc, as such a lack of affinity has already been reported for Gpa1⁄ Ga15,16chimeras in S cerevisiae [31,32]
Another aspect of this study was the optimization of
OR functional expression in S cerevisiae During the last decade only a handful of mammalian ORs have been functionally expressed in heterologous systems due to inefficient receptor insertion into the plasma membrane [10,13,14,17] Here, we found that I7 OR functional responses to odorants were notably enhanced when yeasts were induced in galactose-containing medium at 15C The achievement of high
Fig 8 ELISA-type test for quantification of I7 expressed in yeast
cells The ELISA test was performed by using an anti-I7 primary
antibody and a biotin-conjugated antibody plus horseradish
strept-avidine peroxidase to quantify the presence of I7 OR (A)
Calibra-tion curve obtained by probing KLH-coupled I7 antigenic N-terminal
15-amino acid peptides Uncoupled KLH was probed as a control.
(B) ELISA signal plotted as a function of increasing amount of total
proteins in membrane fractions from I7 yeast strain induced at
15 C, and from yeast expressing SSTR2 as a control.
Trang 9I7 response to ligand stimulation was correlated to its
improved expression, since on Western blots a
signifi-cant increase in receptor level was observed in the
membrane fraction Under these conditions, neither
aggregation of possibly misfolded receptors within the
yeast, nor yeast vacuole overloading with species
inten-ded for degradation were observed by immunogold
labeling Thus, it appears that galactose induction at
15C provides adequate conditions for functional
receptor expression It remains unclear how S
cerevisi-ae responds to mild low temperatures and at which
stage of the folding⁄ trafficking process the reduced
temperatures have an effect Recently it was reported
that in S cerevisiae a temperature downshift to 10–
18C leads to an induction of specific ‘cold shock
pro-teins’, some of which are able to serve as molecular
chaperones [39] Such proteins could be involved in the
upregulation of I7 OR functional expression observed
at 15C However, other mechanisms that arise upon
lowering the temperature must also be considered For
instance, lower temperature may positively affect the
yield of properly folded proteins [40–42] Also, it is
interesting to note that reduced temperatures increase
the content in higher sterols within yeast cell
mem-branes [43] This may not only improve receptor
inser-tion into the plasma membrane [44], but also allow
correct receptor activity [45]
The achievement of receptor plasma membrane
insertion was demonstrated by confocal
immunofluo-rescence microscopy of nonpermeabilized spheroplasts
and by ultrastructural immunogold analysis In
addi-tion, immunological analysis of raw and deglycosylated
samples showed that the predominant receptor form in
the membrane fraction is the mannose-glycosylated
monomer Indeed, only high mannose elongation of
core sugars can occur in S cerevisiae, contrary to
mammalian cells [46] However, considering the yeast
I7 receptor discrimination between closely related
lig-ands strongly suggests the authenticity of its ligand
binding and the maintenance of the coding ability at
the receptor level Consequently this suggests that
gly-cosylation of I7 OR is not a major determinant of
receptor pharmacology
Odorant concentrations giving rise to responses in
yeast cells are several orders of magnitude higher than
those observed in COS or ODORA cells [10] This
dis-crepancy in the behavior of I7 and OR17-40 receptors
expressed in yeast vs mammalian cells could be due to
differences in the lipid membrane composition and
organization between the two heterologous systems
[45] Nevertheless, by comparing the threshold
concen-trations for I7 OR response to odorant stimulation, we
find that heptanal ranks first as in COS cells, where as
octanal and nonanal are less potent ligands Thus, although higher odorant concentrations are necessary
to activate the receptor in this nonmammalian cellular background, the receptor affinity ranking and selectiv-ity are close to those in mammalian cells
The yeast system was optimized for functional expression and sensitive characterization of the olfac-tory receptors It could be amenable to a rapid, inex-pensive screening assay with an extended dynamic range, in which the many orphan ORs could be inves-tigated against the extraordinary large number of nat-urally occurring odorants Although optimization is certainly required for transfer to a high throughput format, this method demonstrates a potential for con-veniently screening a large number of organic mole-cules as novel GPCR ligands which could serve as leads for drug discovery
Experimental procedures
Odorants and other reagents
Odorant solutions were prepared just before use as des-cribed previously [10,34] Octanal, nonanal, heptanal, diace-tyl, cyclohexyl-acetate, octanol, octanon, octanoic acid, isoamyl-acetate, pyridine were from Sigma-Aldrich (Saint Quentin, Fallavier, France) Helional was a generous gift from Givaudan-Roure (Du¨bendorf, Switzerland), courtesy
of B Schilling Lyral and lilial were kindly provided by Roche (Meylan, France)
Complete protease inhibitor cocktail, Endo H and PNG-ase F were from Roche Diagnostics GmbH (Mannheim,
meta-perio-date and Tween 20 were from Sigma-Aldrich RQ1 RNAse-free DNAse was from Promega (Charbonnieres-les-Bains, France) Enzymes used for molecular cloning were from Promega and New England Biolabs (Beverly, MA, USA) The DNA size marker was DRIgest III from Amersham Pharmacia Biotech Europe (Orsay, France) Protein size
Stand-ards from Bio-Rad (Marnes la Coquette, France)
Expression vectors
All plasmid manipulations were performed in E coli strain DH5a from Gibco BRL (Invitrogen, Cergy Pontoise, France) After selection, a single colony was used to isolate the plasmid for yeast transformation The multicopy plas-mid construct, pJH2-I7, for I7 receptor expression, was obtained by homologous recombination in the pJH2-SSTR2 expression vector (kindly provided by MH Pausch, Cyanamid Agricultural Research Center, Princeton, NJ,
Trang 10USA) as described previously [34] OR17-40 full-length
sequence was cloned into a pGEM-T vector, then inserted
in the pCMV-Tag3 expression vector for N-terminal c-myc
tagging using sites BamHI and XhoI of the multiple cloning
site as described previously [10] pJH2-OR17-40 expression
vector was obtained from pJH2-SSTR2 by homologous
sequence, using primers (5¢-CGTCAAGGAGAAAAAAC
CCCGGATCTAAAAAATGGAGCAGAAACTCATCTC
TGAAGAGGATCTG-3¢) and (5¢-GCATGCCTGCAGG
TCGACTCTAGAGGATCTCAAGCCAGTGACCGCCT
CCC-3¢), and checked for the presence and sequence of the
new insert, as in the case of pJH2-I7
Plasmids pJH2-I7 and pJH2-OR17-40 carry a galactose
contains a GAL4 gene under the control of the GAL10
pro-moter The induction of the yeast, by galactose containing
media, results in overexpression of GAL4, in turn inducing
an increase of the expression of the OR gene under control
of GAL1 The pJH2 vector contains the URA3-selectable
marker
coding sequence The pRGP vector contains
HIS3-selecta-ble marker To endow the yeast strain with a reporter
capa-city, a pRHF-luc plasmid was constructed by replacing the
the luciferase coding sequence In this vector the Photinus
promoter The pRHF-luc vector contains TRP1-selectable
marker
Yeast transformation, growth and galactose
induction
The S cerevisiae strain MC18 (MATa gpa1::lacZ [LEU2]
ade2-1 his3-11, 15 leu2-3112 trp1-1 ura3-1 can1–100) [35]
was transformed with either pJH2-I7 or pJH2-OR17-40,
pRHF-luc expression vectors using the lithium acetate
method [47] Transformed cells were plated on 2% agar in
media A: yeast nitrogen base (Difco, Detroit, MI, USA),
synthetic drop-out CSM media without HIS, LEU, TRP,
complemented with 2% glucose The colonies were grown
in liquid media A complemented with 2% glucose at either
pres-ence of plasmids in transformed cells was verified by PCR
on nucleic acid extracts Induction of I7 expression was
per-formed as reported for the SSTR2 induction [24] with the
exception of the temperature In brief, the cells were washed
to remove glucose and cultured for 4–6 h in the selection media containing 3% lactate, then pelleted and diluted to a
60 h, respectively All subsequent experiments with either uninduced or induced yeasts were carried out with cells in
RNA extraction and RT-PCR
RNA was extracted from yeast cells following the hot aci-dic phenol procedure RT-PCR was performed on DNAse-treated RNA extracts Primers used for RT-PCR were: for the I7 OR (5¢-CGTCAAGGAGAAAAAACCCCGGATCT AAAAAATGGAGCGAAGGAACCACAG-3¢) and (5¢-AG CTGCCTGCAGGTCGACTCTAGAGGATCCTAACCAA
AAAAAACCCCGGATCTAAAAAATGGAGCAGAAAC
CCTGCAGGTCGACTCTAGAGGATCTCAAGCCAGT
TGGGGTGTTTGGGCAAC-3¢) and (5¢-GCGGCCGCCT
GGCCCGGTCCCTGACTTGG-3¢) and (5¢-TCACAGCA GGTTGATCTCGTCC-3¢) Negative controls for the pres-ence of remaining DNA were provided by RT-PCR with the same primers performed on nonreverse transcribed mRNA
Isolation of yeast membranes
Membranes were prepared from yeast cells washed twice with ice-cold water, harvested by centrifugation and resus-pended in an equal volume of ice-cold lysis buffer (50 mm
sorbitol) and the Complete protease inhibitor cocktail Glass beads (425–600 lm, Sigma) were added and cells were disrupted by seven cycles of 1 min of vigorous
cells and cell walls The supernatant was further centrifuged
in membranes, was resuspended in the lysis buffer with a
The protein concentration of the membrane preparation was determined using the BCA reagent (Pierce, Brebieres, France) with BSA as a standard
Immunoblot analysis
Proteins of the membrane fraction were separated by elec-trophoresis on 12% SDS polyacrylamide gels and electro-transferred onto Hybond-C Extra membrane (Amersham Pharmacia Biotech Europe) The membrane was blocked