A Pheromone-Binding Protein Mediates the Bombykol-Induced Activation
of a Pheromone Receptor In Vitro
Ewald Große-Wilde1, Ales ˇ Svatos ˇ2and Ju ¨rgen Krieger1
1Institute of Physiology (230), University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart,
Germany and2Research Group Mass Spectroscopy, Max-Planck-Institute for Chemical Ecology,
Hans-Kno ¨ll-Street 8, 07745 Jena, Germany
Correspondence to be sent to: Ju ¨rgen Krieger, Institute of Physiology (230), University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart,
Germany. e-mail: email@example.com
distinct sensory neurons in the antennal sensilla hairs. The hydrophobic pheromonal compounds are supposed to be ferried by
soluble pheromone-binding proteins (PBPs) through the sensillum lymph toward the receptors in the dendritic membrane. We
have generated stable cell lines expressing the candidate pheromone receptors of B. mori, BmOR-1 or BmOR-3, and assessed
activated by bombykol but also responded to bombykal, whereas cells expressing BmOR-3 responded to bombykal only. In
experiments employing the B. mori PBP, no organic solvent was necessary to mediate an activation of BmOR-1 by bombykol,
indicating that the PBP solubilizes the hydrophobic compound. Furthermore, the employed PBP selectively mediated a response
to bombykol but not to bombykal, supporting a ligand specificity of PBPs. This study provides evidence that both distinct pher-
omone receptors and PBPs play an important role in insect pheromone recognition.
Key words: Bombyx mori, expression, olfaction, pheromone detection, receptor
The recognition and discrimination of volatile chemical sig-
nals in insects supposedly involve G protein–coupled olfac-
tory receptors (ORs) residing in the dendritic membrane of
olfactory neurons housed in sensillar hair structures on the
antenna. In flies (Clyne et al., 1999; Gao and Chess, 1999;
Vosshall et al., 1999, 2000) and mosquitos (Fox et al.,
2001, 2002; Hill etal., 2002),multigene familieshave been dis-
diversity and nonspecific response spectra are considered as
the basis for a combinatorial coding of odorants (Sto ¨rtkuhl
and Kettler, 2001; Keller and Vosshall, 2003; Hallem et al.,
didate ORs for odorants and pheromones have also been
identified for the moths Heliothis virescens (Krieger et al.,
2002, 2004) and Bombyx mori (Sakurai et al., 2004; Krieger
et al., 2005; Nakagawa et al., 2005). Two of the B. mori
receptor types (BmOR-1 and BmOR-3) were shown to be
expressed in neighboring neurons within the pheromone-
sensitive hairs (long sensilla trichodea) of the male antenna
(Krieger et al., 2005; Nakagawa et al., 2005), and injection
of RNA for BmOR-1 and BmOR-3 rendered frog oocytes
responsive to components of the female sex pheromone
blend (Nakagawa et al., 2005).
of the sensory neurons which are bathing in the sensillum
lymph. Pheromones and odorants have to traverse this aque-
ous fluid before reaching the chemosensory dendritic mem-
brane. This transfer is supposed to be mediated by specific
globular proteins, the pheromone-binding proteins (PBPs)
or odorant-binding proteins (OBPs) (Steinbrecht, 1998; Leal,
evidence supporting this concept is still very sparse. In fact,
analyses of heterologously expressed receptors were all per-
formed in the absence of OBPs or PBPs. In these studies,
hydrophobic pheromonal or odorous compounds were dis-
vents, such as dimethyl sulfoxide (DMSO) (Krautwurst et al.,
a PBP (Krieger et al., 1996) and recently identified several
Chem. Senses 31: 547–555, 2006doi:10.1093/chemse/bjj059
Advance Access publication May 5, 2006
ª The Author 2006. Published by Oxford University Press. All rights reserved.
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putative pheromone receptor types (Krieger et al., 2005). In
this study, we set out to establish stable cell lines expressing
siveness by means of calcium-imaging approaches. Heterolo-
compounds to the receptors in the cell membrane.
Materials and methods
Odorants and pheromone components
Bombykol, (E10, Z12)-hexadeca-10,12-dien-1-ol, was pre-
pared using acetylene chemistry according to published pro-
cedures (Hoskovec et al., 2000; Kalinova et al., 2001). The
bombykol was oxidized to bombykal by pyridiniumdichro-
mate and purified on a silica gel column. The isomeric purity
of both compounds was 97% (for EZ isomer) and the chem-
ical purity 98% (determined using gas chromatography,
DB-5 phase). Using gas chromatography/mass spectroscopy,
Z-11-hexadecenal (Z11-C16:Al), Z-11-hexadecenol (Z11-
C16:OH), and ± linalool were purchased from Fluka (Buchs,
Switzerland), benzoic acid and Z-9-hexadecenol (Z9-C16:
OH) came from Sigma.
Expression of BmORs in Flp-In T-REx293 cells
Vector constructs for the generation of stable cell lines
For stable genome integration and tetracycline-regulated
heterologous expression of BmORs, the components of
the Flp-In-System (Invitrogen, Paisley, UK) and a modified
HEK 293 cell line carrying the Ga15 gene (Flp-In T-REx293/
Ga15) stably integrated in the genome were used. Ga15 has
olfactory and other G protein–coupled receptors to the
inositol trisphosphate cascade leading to intracellular Ca2+
increases (Offermanns and Simon, 1995; Krautwurst et al.,
1998; Kajiya et al., 2001). These can be monitored by cal-
cium imaging with the calcium-sensitive dye fura-2. The
Flp-In T-REx293/Ga15 cell line was kindly provided by
E. Tareilus and R. Gouka, Unilever R&D, Vlardingen. To
monitor the expression of the BmOR receptors immuno-
cytochemically, a FLAG-tag was added to the N-terminus.
First, the coding region of BmORs was polymerase chain
reaction (PCR) amplified and integrated in frame into the
pFLAG-CMV-2 vector (Kodak, New Haven, CT) using spe-
cific primers with appropriate restrictions sites (underlined).
BmOR-1–NotI: 5#-ATT TGC GGC CGC TTA TGG TTA
CTA TCC TTC AAA GA-3#; BmOR-1–XbaI: 5#-TGC TCT
AGA GCA TTA TGT TGC CAC TGT TCG GAG-3#;
BmOR-3–HindIII: 5#-ACA AGC TTA TGA TAT TCG
TCG ACG ATG CT-3#; and BmOR-3–EcoRI: 5#-TTG
AAT TCT TCA TTC GGA CAC GGT ACG AAG-3#.
PCR conditions were 1 min 40 s at 94?C, then 21 cycles with
94?C for 1 min, 50?C or 55?C for 40 s, and 72?C for 1 min,
with a decrease of the annealing temperature by 0.5?C per
cycle. Subsequently, 19 further cycles at the condition of
the last cycling step were performed followed by incubation
for 7 min at 72?C. The PCR product was gel purified, dig-
ested, and ligated into thecorrespondingsitesofthepFLAG-
CMV-2 vector. The resulting constructs pFLAG-CMV-2/
BmOR were sequenced to verify correct amplification and in-
on an ABI310 sequencing system using vector and receptor
sequence–specific primers and the BIG dye cycle sequencing
kit (Applied Biosystems, Foster City, Calif.). The FLAG-
BmOR-encoding sequences were PCR amplified from the
pFLAG-CMV-2/BmOR constructs using a combination of
FLAG–KpnI: 5#-GGG GTA CCC CAC CAT GGA CTA
CAA AGA CGA TGA CG-3# with either BmOR-1–XhoI:
5#-CCG CTC GAG CGG ATT ATG TTG CCA CTG TTC
GGA G-3# or BmOR-3–XhoI: 5#-CCG CTC GAG CGG
were gel purified, digested with KpnI/XhoI, and ligated into
the corresponding sites of the pcDNA5-FRT/TO vector
to the vector, the resulting FLAG-BmOR/FRT/TO construct
Generation of stable cell lines
Flp-In T-REx293/Ga15 cells were grown and maintained in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen,
highglucose,with L-glutamine,without Na-pyruvate)supple-
mented with 10% fetal calf serum, 100 mg/l zeocin, 10 mg/l
blasticidin, and 200 mg/l geneticin. Cells were transfected
using PerFectin (Gene Therapy Systems, San Diego, CA)
according to the supplier’s protocol. For transfection, 3 ·
105cells were seeded into 35-mm culture dishes. After 24 h,
cells were transfected with pOG44 (Invitrogen) and the
BmOR/FRT/TO construct in a 10:1 ratio. Forty-eight hour
posttransfection cells were maintained and selected for recep-
tor integration into the genome using media supplemented
with 100 mg/l hygromycin instead of zeocin. After 6 weeks,
by PCR using receptor-specific primers, genomic DNA pre-
pared from the hygromycin resistant cells and conditions as
described above. Flp-In T-Rex293/Ga15 cells positive for
BmORs were maintained and used for calcium imaging.
Calcium imaging of cells
For calcium imaging, 2 · 105BmOR/Flp-In T-Rex293/Ga15
cells were seeded onto 15-mm diameter cover slips coated
with poly-L-lysine. Cells were incubated for 36 h in DMEM
with 1 lg/ml tetracycline to induce receptor expression. For
imagingchanges intheinternal free calciumconcentrationof
washed inRingersolution(138 mMNaCl,5mM KCl,2mM
CaCl2, 1.5 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH
E. Große-Wilde et al.
room temperature with 4 lmol/l of the acetoxymethyl ester
of fura-2 in Ringer solution. Cover slips with fura-2–loaded
cells were placed into a flow chamber on the stage of an
Olympus IX70 inverted microscope equipped for epifluores-
cence. Thechamber wasrinsedwithRingersolutionataflow
rate of 1 ml/10 s. Test solutions (400 ll) were applied via
syringes connected to the system by three-way valves at
the same flow rate.
Changes in [Ca2+]iconcentration in single cells upon stim-
ulation with ligands were analyzed by monitoring the inten-
sity of fluorescent light emission at 510 nm, using excitation
at 340 and 380 nm. The ratio of fluorescence emission at 340/
380 nm excitation was used as index with an increase indi-
cating a raise in intracellular free calcium. For data analysis,
fura-2 fluorescence intensity ratios of OR-expressing cells
were determined before (F0) and after stimulation (F; peak
of the response). In a single experiment, F/F0values of at
least 30 individual cells were determined and averaged. Illu-
mination (30 ms for each wavelength) was by an IX-FLA
monochromator connected to the microscope. Cells were
viewed through a UApo/340 40· objective, and images were
detected with a MircoMax camera (Roper Scientific, Otto-
brunn, Germany). To control the illuminator, the camera,
and software was used (Visitron Systems, Puchheim,
Germany). The Ca2+response to 10 mM adenosine triphos-
phate in Ringer solution was used as an internal control of
cell viability. Washes with Ringer solution between stimula-
tions were performed for at least 5 min before applying the
next test solution. Dilutions of hydrophobic pheromone com-
ponents were prepared from stock solutions in hexane using
Ringer solution with 0.1% DMSO. Water-soluble odorants
were diluted in Ringer solution with 0.1% DMSO, adding
hexane to a final concentration of 0.1%. All dilutions were
prepared freshly and used within 3 h. To analyze the ability
of PBP to solubilize and transport bombykol or bombykal,
pheromone components were added from stock solutions
in hexane to recombinant protein in Ringer solution and in-
cubated for 1 h on ice. The solution was warmed to room
temperature and subsequently used for calcium imaging.
Expression and purification of PBPs
Recombinant B. mori PBP was expressed in E. coli and
purified from a periplasmic preparation of the bacteria as
described previously (Wojtasek and Leal, 1999; Campanacci
et al., 2001; Oldham et al., 2001). To remove hydrophobic
ligands which have been found to copurify with B. mori
levels in receptor-expressing cells after application of DMSO/Ringer solution (control) or stimulation with bombykol or bombykal solubilized with DMSO.
Response curves to the right represent changes of fura-2 fluorescence intensity ratios (340/380 nm) in individual cells from the same experiment. Pheromone
components were applied (arrowheads) for 4 s at 10 nM. BmOR-1–expressing cells displayed clear responses to bombykol as well as to bombykal; however,
bombykol-induced reactions showed considerably higher amplitude. Cells expressing BmOR-3 did react upon stimulation with bombykal but showed no
response to bombykol.
Pheromone-induced Ca2+responses of Flp-In T-REx293/Ga15cells expressing BmOR-1 or BmOR-3. Pseudocolor images (left panels) indicate calcium
Insect Pheromone Receptors
PBP recombinant-binding protein was dilipidated following
the protocol described earlier (Oldham et al., 2001) and fi-
was determined spectrometrically at 280 nm using absorp-
program (ExPASy molecular biology server; Swiss Institute
We have generated stable cell lines expressing BmOR-1 or
BmOR-3 to scrutinize their ligand specificity. To promote
coupling of activated receptors to an effective intracellular
reporter system, receptor cDNAs were stably integrated into
the genome of modified HEK 293 cells (Flp-In T-REx293/
Ga15cells); Ga15has been shown to facilitate the coupling
of heterologously expressed receptors to the phospholipase
C pathway leading to an increase of intracellular Ca2+con-
centration (Offermanns and Simon, 1995; Krautwurst et al.,
1998; Kajiya et al., 2001). For monitoring changes in intra-
cellular calcium levels, BmOR/Flp-In T-REx293/Ga15cells
were loaded with fura-2 and fluorescence ratios were
recorded,withan increaseof thefluorescence ratio indicating
elevated levels of calcium (Grynkiewicz et al., 1985). Due to
their hydrophobicity, the putative ligands bombykol and
bombykal were solubilized by means of DMSO. Application
of low doses of bombykol (10 nM) elicited a significant in-
crease of intracellular calcium levels in cells expressing
BmOR-1 but not in BmOR-3 cells (Figure 1). In contrast,
application of 10 nM bombykal induced a calcium response
in both BmOR-1– and BmOR-3–expressing cells. As indi-
cated by the false color pictures in Figure 1, superfusing
patches of BmOR-1 cells with 10 nM bombykol or patches
of BmOR-3 cells with 10 nM bombykal induced a strong
expressing cells were determined before (F0) and after stimulation (F; peak of the response) approaching different concentrations of bombykol (black bars) or
bombykal (gray bars). Bars represent the mean responses of cells to the pheromone concentration indicated expressed as F/F0. Each bar is based on the mean
F/F0± SD from three to eight independent experiments. BmOR-1–expressing cells (A) respond to bombykol and bombykal in a dose-dependent manner.
In contrast, cells expressing BmOR-3 (B) respond only to bombykal; no response was elicited by bombykol even at high concentration. Significant increases
in comparison to no pheromone (0) are indicated by asterisks; *P < 0.05, **P < 0.01 in a one-way ANOVA followed by Dunnett’s posttest.
Bombykol and bombykal dose-response profiles of BmOR-expressing cells. Fura-2 fluorescence intensity ratios of BmOR-1– (A) or BmOR-3 (B)–
E. Große-Wilde et al.
response in most of the cells. Monitoring the response kinet-
ics of individual BmOR-1 cells and BmOR-3 cells to bomb-
ykol or bombykal revealed in each case a transient time
course (Figure 1). Flp-In T-REx293/Ga15cells without a
et al., 2003) did not show any response to the pheromone
components (data not shown). Altogether, the calcium sig-
nals of BmOR-1 and BmOR-3 cells upon stimulation with
bombykol and bombykal, respectively, indicate that the cells
have generated functionally active receptors which render
them responsive to the pheromonal compounds.
For a more detailed analysis of the bombykol- and
bombykal-induced responses of BmOR-1 and BmOR-3
cells, recordings were performed at different concentrations.
In a single stimulation experiment, fura-2 fluorescence inten-
sity ratios of at least 30 OR-expressing cells were measured
before (F0) and after stimulation (F; peak of the response)
to calculate a mean F/F0ratio. Mean F/F0values from single
independent experiments were averaged to determine the re-
sponsiveness of cells. Upon application of control stimuli
(e.g., application of Ringer/DMSO/hexane), this value was
determined to be slightly above 1.0 due to slight fluctuations
intrinsic to calcium-imaging measurements. Application of
increasing concentrations of bombykol to BmOR-1 cells
led to an increase in the F/F0values in a dose-dependent
manner (Figure 2A) resulting from a rise in the number of
reacting cells as well as higher amplitudes of the bombykol-
induced calcium signals. Half-maximal effects were ob-
served at ;100 pM. Threshold concentration for calcium
responses of individual cells was at about 10 pM bombykol.
BmOR-1 cells also respond to bombykal; however, com-
pared to bombykol, signals were lower at higher concentra-
tions. BmOR-3 cells selectively respond to bombykal;
bombykol elicited no response in BmOR-3 cells even at con-
centrations as high as 10 nM (Figure 2B). To further assess
the specificity of the receptors, BmOR-expressing cells were
challenged with pheromone components from other moth
species as well as with general odorants which are recognized
by female B. mori (linalool, benzoic acid) (Heinbockel and
Kaissling, 1996). It was found that neither the BmOR-1– nor
the BmOR-3–expressing cells responded to 10 nM Z9-
C16:OH (Figure 3A,B). Also weak reactions observed after
applying the same high concentration of the pheromone
components Z11-C16:Al and Z11-C16:OH or the odorants
linalool and benzoic acid were not significantly different
from control. These results indicate a selective interaction
of BmOR-1 and BmOR-3 with components of the B. mori
In the aqueous sensillum lymph of the sensory hairs, the
ubilized by means of PBPs. To scrutinize this long-standing
hypothesis, we have examined if the PBP of B. mori may ful-
fillthis role in vitro. RecombinantB. moriPBP was employed
to solubilize bombykol or bombykal in the buffer medium
substituting the organic solvent DMSO. Activation of recep-
tors upon interaction with pheromone components was
monitored by calcium imaging of the cells. Superfusion of
BmOR-1 cells with Ringer solution containing B. mori
PBP (1 lM) preincubated with bombykol (100 pM) led to
a clear calcium response in the cells (Figure 4A), with a tran-
sient time course on the single-cell level. The mean calcium
signal intensity in BmOR-1 cells elicited by application of
B. mori PBP/bombykol was comparable to the response
obtained withthesame concentrationof bombykol dissolved
of solvent in Ringer solution did not induce any response;
also PBP alone did not have any effect. Applying PBP
(1 lM) together with bombykal (100 pM) also elicited no re-
sponse in BmOR-1 cells (Figure 4C,D), suggesting that
B. mori PBP may selectively bind bombykol. This notion
was further supported by experiments demonstrating that
also BmOR-3–expressing cells were not activated by 1 lM
B. mori PBP preincubated with 100 pM bombykal (Figure
4E). The response of BmOR-1 cells to bombykol mediated
of a solution with the same concentration of PBP (1 lM)
pounds and general odorants. Bars represent the responses of cells express-
ing BmOR-1 (A) and BmOR-3 (B) upon stimulation with 10 nM solutions of
pheromone components from other moth species or general odorants rele-
vant to Bombyx mori. At this high ligand concentrations, structurally related
no significant response. Also no significant reaction of BmOR-1 or BmOR-3
solubilized in 0.1% DMSO (control). F/F0ratios represent the mean F/F0± SD
ANOVA followed by Dunnett’s posttest revealed no significant increases of
F/F0values compared to control.
Stimulation of BmOR-expressing cells with pheromonal com-
Insect Pheromone Receptors
loaded with increasing concentrations of bombykol (1 pM to
1 nM) led to dose-dependent calcium signals (Figure 5A).
Similarly, the response of BmOR-1 cells to a PBP/bombykol
combination was also dependent on the concentration of
PBP in the solution (Figure 5B). The same concentration
of bombykol (100 pM) elicited a stronger response with in-
creasing concentrations of PBP (10 nM to 10 lM). Together,
these results indicate that B. mori PBP in a ligand-specific
The present study demonstrates a specific interaction of the
two candidate pheromone receptors BmOR-1 and BmOR-3
with pheromone components of the silkmoth B. mori, bomb-
ykol and bombykal in vitro. This result is in line with the pre-
vious observation that BmOR-1 and BmOR-3 are expressed
in neighboring neurons of long trichoid hairs on male silk-
moth antenna (Krieger et al., 2005) as well as with earlier
electrophysiological recordings from sensilla trichodea, indi-
cating that these long olfactory hairs contain two sensory
neurons, one responsive to bombykol and the second to
bombykal (Kaissling et al., 1978). The functional character-
ization of the BmOR-3 receptor, heterologously expressed in
modified HEK 293 cells, has demonstrated its highly sensi-
tive and selective responsiveness to bombykal but not to
bombykol. A similar result has recently been reported after
injecting a combination of RNAs for BmOR-3 and BmOR-2
images (left panels) indicate calcium levels in BmOR-1–expressing cells after application of DMSO/Ringer solution (control) or stimulation with BmorPBP/bomb-
ykol (BOL) in Ringer. The response curve to the right represent changes of fura-2 fluorescence intensity ratios (340/380 nm) of an individual cell from the same
experiment. (B) BmOR-1 cells were stimulated with bombykol, BmorPBP and DMSO alone, or in combinations. Bombyx mori PBP with bombykol elicited
a significant response; the signal intensity was similar to bombykol solubilized with DMSO (data from Figure 2). Bombykol, BmorPBP, or DMSO (control) alone
respond to a combination of BmorPBP/bombykal. Only stimulation of BmOR-1 cells with bombykal solubilized by DMSO (values from Figure 2) revealed a clear
response. (E) BmOR-3–expressing cells did not respond to stimulation with BmorPBP/bombykal. Responses of cells in B and D are displayed as mean F/F0± SD
ratios determined from a minimum of three independent measurements. Significant increases in comparison to stimulation with bombykol or bombykal alone
are indicated by asterisks; **P < 0.01 in a one-way ANOVA followedby Dunnett’s posttest. Concentrations used in stimulation experiments shownin A–E were
100 pM for bombykol or bombykal, 1 lM for B. mori PBP, or 0.1% for DMSO.
Bombyx mori pheromone-binding protein (BmorPBP) mediated responses of BmOR-1 cells to bombykol but not to bombykal. (A) Pseudocolor
E. Große-Wilde et al.
in frog oocytes; expression of BmOR-3 receptor made the
oocytes responsive to bombykal but only very weakly to
bombykol (Nakagawa et al., 2005). In the same study, it
was shown that oocytes expressing BmOR-1 led to a strong
response to bombykol but elicited only a weak response
to bombykal. BmOR-2 improved functional expression of
BmOR-1 and BmOR-3 in oocytes (Nakagawa et al., 2005),
and its Drosophila homologue (Or83B) was found necessary
for membrane targeting and function of other odorant recep-
tor types (Larsson et al., 2004; Neuhaus et al., 2005; Benton
et al., 2006). Functional expression of BmOR-1 and BmOR-3
in modified HEK 293 cells was possible without BmOR-2,
probably due to the ability of HEK 293 cells to correctly
process G protein–coupled receptors (Couve et al., 2002;
Ivic et al., 2002; Thomas and Smart, 2005). We have found
that BmOR-1 expressed in modified HEK 293 cells did re-
spond to both bombykol and bombykal, although at higher
doses (1–10 nM), the response to bombykol was stronger. If
this difference in ligand specificity of BmOR-1 in HEK 293
cells compared to oocytes is due to the different host cells
Analyzing the responsiveness of heterologously expressed
receptors for pheromones and odorants in cultured cells is
always hampered by the strong hydrophobicity of the appro-
priate ligands. Usually, organic solvents are employed to
minimize this problem. In fact, the solvent DMSO has been
Sakurai et al., 2004; Nakagawa et al., 2005). Under these cir-
cumstances, the solubilized pheromones or odorants were
capable to activate the receptor (see also Figures 1 and 2).
For the insect antennae, it has been proposed that globular
proteins in the sensillum lymph, PBPs and OBPs, act as sol-
ubilizer for the hydrophobic ligands (Vogt and Riddiford,
1981; Leal, 2003; Vogt, 2003). In view of the fact that exper-
imentalevidence forthis conceptisstillveryweak, theresults
of this study demonstrate that in the presence of PBP, bomb-
ykol elicited a response of BmOR-1–expressing cells compa-
rable to bombykol dissolved by DMSO, indicating that the
PBPcancompletely substitutetheorganicsolvent (Figure4).
This result suggests that indeed one of the functions of PBPs
in the sensillum lymph may be solubilization of the phero-
The observation that stimulation of BmOR-expressing
cells by bombykal only occurs when it was solubilized by
means of DMSO but not in the presence of the B. mori
PBP (Figure 4) strongly suggests that the B. mori PBP spe-
cifically interacts with bombykol but not with bombykal.
This finding indicates that the binding proteins are not just
pounds,thusconfirmingresultsobtainedin previous binding
assays. It has been shown that different PBPs of a moth dis-
pounds (Du and Prestwich, 1995; Feixas et al., 1995; Plettner
et al., 2000; Maida et al., 2003); moreover, a binding protein
can interact differentially with different pheromone compo-
nents (Bette et al., 2002; Mohl et al., 2002). In addition, elec-
trophysiological studies have demonstrated that the B. mori
PBP applied in combination with bombykol to receptor neu-
rons via tip-opened sensillar hairs led to the activation of the
bombykol-sensitive neuron, whereas application of bomby-
kal with PBP failed to have any effect (Pophof, 2004). Sim-
ilarly, the OBP LUSH was shown to be absolutely necessary
for activation of pheromone-sensitive neurons in Drosophila
by 11-cis vaccenyl acetate (Xu et al., 2005).
The selective role of the PBP for bombykol rather than
bombykal implies that another binding protein of B. mori may
exist. The presence of multiple PBPs have been demonstrated
trations. (A) Response profile of cells upon stimulation with 1 lM BmorPBP
preincubated with different bombykol concentrations (1 pM to 1 nM). At
constant BmorPBP concentration, the response of BmOR-1 cells was depen-
dent on the bombykol dose. (B) Stimulation of BmOR-1 cells with varying
amounts of BmorPBP (10 nM to 10 lM) loaded with 100 pM bombykol each.
Applying a fixed concentration of the pheromone component but varying the
amount of BmorPBP, the response of BmOR-1 cells was dependent on the PBP
concentration. Data represent the mean reponses of cells expressed as F/F0±
SD ratios determined from at least three independent experiments. Significant
increases in comparison to nopheromone or no PBP are indicatedbyasterisks;
**P < 0.01 in a one-way ANOVA followed by Dunnett’s post test.
Responses of BmOR-1 cells to varying BmorPBP/bombykol concen-
Insect Pheromone Receptors
for several other moths species (Krieger et al., 1991; Merritt
etal., 1998;Robertsonetal., 1999;Maida etal., 2000). Forth-
coming studies have to show if indeed a further PBP exists
in B. mori which could be specialized for bombykal.
and generous support. Wethank Erwin Tareilus and Robin Gouka,
Unilever R&D, Vlardingen for expert help with the heterologous
expression system and Gesa Dreesman for excellent technical assis-
tance. This work was supported by the Deutsche Forschungsge-
meinschaft (grant KR1786/3-2).
Benton, R., Sachse, S., Michnick, S.W. and Vosshall, L.B. (2006) Atypical
membrane topology and heteromeric function of Drosophila odorant
receptors in vivo. PLoS Biol., 4, e20.
Bette, S., Breer, H. and Krieger, J. (2002) Probing a pheromone binding
protein of the silkmoth Antheraea polyphemus by endogenous trypto-
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Accepted April 13, 2006
Insect Pheromone Receptors