Tài liệu Báo cáo khoa học: Olfactory receptor signaling is regulated by the post-synaptic density 95, Drosophila discs large, zona-occludens 1 (PDZ) scaffold multi-PDZ domain protein 1 pptx

Olfactory receptor signaling is regulated by the
post-synaptic density 95, Drosophila discs large,
zona-occludens 1 (PDZ) scaffold multi-PDZ domain
protein 1
Ruth Dooley
1,2,
*, Sabrina Baumgart
2,
*, Sebastian Rasche
1
, Hanns Hatt
1
and Eva M. Neuhaus
3
1 Molecular Medicine Lab RCSI, Education & Research Centre Smurfit Building, Beaumont Hospital, Dublin, Republic of Ireland
2 Department of Cell Physiology, Ruhr University Bochum, Germany
3 NeuroScience Research Center, Charite
´
, Universita
¨
tsmedizin Berlin, Germany
Keywords
MUPP1; olfactory neuron; olfactory
receptor; PDZ protein; signal transduction
Correspondence
E. M. Neuhaus, NeuroScience Research
Center, Charite
´
, Universita
¨
tsmedizin Berlin,

10117 Berlin, Germany
Fax: +49 30 450 539 970
Tel: +49 30 450 539 702
E-mail:
*These authors contributed equally to this
work
(Received 9 September 2009, revised 6
October 2009, accepted 12 October 2009)
doi:10.1111/j.1742-4658.2009.07435.x
The unique ability of mammals to detect and discriminate between thou-
sands of different odorant molecules is governed by the diverse array of
olfactory receptors expressed by olfactory sensory neurons in the nasal
epithelium. Olfactory receptors consist of seven transmembrane domain G
protein-coupled receptors and comprise the largest gene superfamily in
the mammalian genome. We found that approximately 30% of olfactory
receptors possess a classical post-synaptic density 95, Drosophila discs
large, zona-occludens 1 (PDZ) domain binding motif in their C-termini.
PDZ domains have been established as sites for protein–protein inter-
action and play a central role in organizing diverse cell signaling assem-
blies. In the present study, we show that multi-PDZ domain protein 1
(MUPP1) is expressed in the apical compartment of olfactory sensory
neurons. Furthermore, on heterologous co-expression with olfactory sen-
sory neurons, MUPP1 was shown to translocate to the plasma mem-
brane. We found direct interaction of PDZ domains 1 + 2 of MUPP1
with the C-terminus of olfactory receptors in vitro. Moreover, the odor-
ant-elicited calcium response of OR2AG1 showed a prolonged decay in
MUPP1 small interfering RNA-treated cells. We have therefore elucidated
the first building blocks of the putative ‘olfactosome’, brought together
by the scaffolding protein MUPP1, a possible central nucleator of the
olfactory response.

Structured digital abstract
l
MINT-7290305: OR2AG1 (uniprotkb:Q9H205) physically interacts (MI:0915) with MUPP1
(uniprotkb:
O75970)byanti tag coimmunoprecipitation (MI:0007)
l
MINT-7289999, MINT-7290250, MINT-7290063, MINT-7290110: OR2AG1 (uni-
protkb:
Q9H205) binds (MI:0407)toMUPP1 (uniprotkb:O75970)bypeptide array (MI:0081)
l
MINT-7290162: mOR283-1 (uniprotkb:Q9D3U9) binds (MI:0407)toMUPP1 (uni-
protkb:
O75970)bypeptide array (MI:0081)
l
MINT-7290128: mOR-EG (uniprotkb:Q920P2) binds (MI:0407)toMUPP1 (uni-
protkb:
O75970)bypeptide array (MI:0081)
Abbreviations
CamKII, calcium ⁄ calmodulin-dependent protein kinase II; GABA
B
, c-aminobutyric acid receptor B; GFP, green fluorescent protein; GST,
glutathione S-transferase; HRP, horseradish peroxidase; INAD, inactivation no after potential D; MUPP1, multi-PDZ domain protein 1; OMP,
olfactory marker protein; OR, olfactory receptor; OSN, olfactory sensory neuron; PDZ, post-synaptic density 95, Drosophila discs large,
zona-occludens 1; RNAi, RNA interference; siRNA, small interfering RNA.
FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7279
Introduction
Detection of an odorant is initiated by activation of
a fraction of many hundreds of G protein-coupled
odorant receptors (ORs) expressed in olfactory sen-
sory neurons (OSNs) of the mammalian olfactory

epithelium [1]. Signal transduction begins when an
odorant molecule binds to an OR, resulting in the
activation of adenylyl cyclase type III [2] via the
olfactory G protein Ga
olf
[3]. cAMP then binds to a
cyclic nucleotide-gated channel [4–7], allowing it to
conduct cations such as Na
+
⁄ Ca
2+
. The calcium
ions then bind to a calcium-gated chloride channel
[8], further depolarizing the cell. How these diverse
signaling molecules find each other in the complex
and densely-packed environment of the cell, avoiding
cross-talk with other signaling pathways, in order to
ensure the rapidity and specificity of signaling,
remains an unanswered question. The idea of the
existence of an ‘olfactosome’, or highly ordered
multi-component protein network involving the olfac-
tory signal transducing molecules, has been previ-
ously discussed [9–11]; however, until now, no
concrete evidence has been provided. Scaffolding net-
works have been investigated in detail in the visual
system of Drosophila melanogaster, where inactivation
no after potential D (INAD), made up of five post-
synaptic density 95, Drosophila discs large, zona-
occludens 1 (PDZ) domains, has the ability to bind
to various molecules in the signal transduction cas-

cade, thereby bringing them into close proximity and
ensuring a rapid and specific signal transduction
[9,12].
PDZ domains are modular protein–protein interac-
tion domains, which are amongst the most abundant
protein interaction domains in organisms from bacteria
to mammals, and have been implicated in various pro-
cesses, including clustering, targeting and routing of
their binding partners [13–15]. PDZ target specificity is
usually dependent on the extreme carboxyl-terminal
amino acid sequence of the interacting protein; how-
ever, for some ligands, residues as far back as the )10
position may influence binding energy [16]. Peptide-
binding preferences of PDZ domains led to their
division into three discrete functional classes [16],
which may not be as strict as initially anticipated
because predictions of PDZ domain–peptide interac-
tions were recently shown to be evenly distributed
throughout selectivity space [17].
The multi-PDZ domain protein 1 (MUPP1) is com-
posed of thirteen PDZ domains, each diverse with
respect to its amino acid sequence. It was first identi-
fied through a yeast two-hybrid screening as an
interaction partner of the C-terminus of 5-hydroxy-
tryptamine receptor type 2C [18]. Subsequently, many
diverse interaction partners of MUPP1 have been
characterized, including G-protein coupled c-aminobu-
tyric acid receptor B (GABA
B
) [19] and the

calcium ⁄ calmodulin-dependent protein kinase II (Cam-
KII) [20]. In the present study, we introduce ORs as
novel interaction partners of MUPP1.
Results
MUPP1 is expressed at the sites of olfactory
signal transduction
ORs are expressed on the ciliary membranes of OSNs,
the first point of contact of the sensory cell with
incoming odorant molecules. To investigate whether a
receptor centered multi-component protein network
might exist, we tested for the expression of PDZ
scaffolding proteins in the olfactory epithelium using
RT-PCR (Fig. 1A). We detected robust expression of
MUPP1 and weak expression of ZO-1, but could not
detect Patj, Erbin or DLG-2. Because MUPP1 mRNA
was highly abundant, we examined the expression of
the protein by western blotting. Upon fractionation of
the olfactory epithelium [21], we found MUPP1 to be
present to a greater extent in the cilia-enriched fraction
compared to the remaining cell fractions (Fig. 1B).
Using olfactory marker protein (OMP)-green fluo-
rescent protein (GFP) transgenic mice, expressing GFP
in every mature olfactory sensory neuron [22], we
investigated the cellular localization of MUPP1 in the
olfactory epithelium and found MUPP1 to be
l
MINT-7290219:hOR3A1 (uniprotkb:P47881) binds (MI:0407 )toMUPP1 (uni-
protkb:
O75970)bypeptide array (MI:0081)
l

MINT-7290191:hOR1D2 (uniprotkb:P34982) binds (MI:0407)toMUPP1 (uni-
protkb:
O75970)bypeptide array (MI:0081)
l
MINT-7289922: AC3 (uniprotkb:Q8VHH7) and MUPP1 (uniprotkb:O75970) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7289933, MINT-7289954, MINT-7289978:OR2AG1 (uniprotkb: Q9H205) binds
(
MI:0407)toMUPP1 (uniprotkb:O75970)bypull down (MI:0096)
PDZ proteins interact with olfactory receptors R. Dooley et al.
7280 FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS
expressed in the apical part of OSNs, mainly in the
cilia layer (Fig. 1C). Double immunolabeling showed
co-localization with adenylyl cyclase 3, a central mole-
cule in the olfactory signal transduction cascade in the
cilia of OSNs (Fig. 1C, D).
Interaction of PDZ domains 1 + 2 of MUPP1 with
OR2AG1 in vitro
PDZ domain interactions have been well characterized
and modes of binding have been grouped into three
main classes of PDZ binding motifs, occurring at the
C-terminus of the interacting proteins [16]. We scanned
the human olfactory receptor repertoire for putative
binding motifs and discovered them in the extreme
C-termini of approximately 30% of human ORs, with
examples from each of the three classes being outlined
to date (7% Class I, 12% Class II and 10% Class III;
Fig. 2A). Intriguingly, this suggested that a subset of

ORs could have the ability to interact with PDZ
domains of MUPP1.
We performed co-immunoprecipitation experiments
to verify the ability of ORs containing a PDZ interac-
tion motif to bind to MUPP1. Hana3A cells were
transfected with HA-OR2AG1. These cells express
members of the RTP and REEP family, which com-
prise molecular chaperones known to promote the
expression of ORs [23]. Antibodies to the HA tag were
used to co-immunoprecipitate MUPP1, as shown by a
band of 220 kDa in western blots (Fig. 2B, immuno-
precipitation: a-HA), whereas no MUPP1 immuno-
reactivity could be observed in precipitates of
nontransfected cells (Fig. 2B, control). These results
indicated an interaction between OR2AG1 and
MUPP1 in the recombinant expression system.
MUPP1 is made up entirely of thirteen different
PDZ domains, each being diverse in sequence. We set
out to determine which of these PDZ domains were
involved in the molecular interaction with ORs. We
created a glutathione S-transferase (GST) fusion pep-
tide of the C-terminus of OR2AG1 and in vitro trans-
lated the PDZ domains of MUPP1, in pairs (1 + 2,
A
C
D
B
Fig. 1. MUPP1 expression in olfactory sen-
sory neurons. (A) Expression of mRNA of
different PDZ scaffolding proteins in the

olfactory epithelium by RT-PCR. *Weak
band for ZO-1. (B) Fractional preparation of
whole olfactory epithelium shows MUPP1,
at 220 kDa, enriched in the cilia fraction (1)
compared to the remaining cell fractions
(2–4); a Coomassie-stained gel is shown as
a loading control. (C) MUPP1 is co-localized
with adenylyl cyclase 3 in the apical layer of
the olfactory epithelium. Immunohistochemi-
cal staining of 14 lm cryosections of
OMP-GFP mouse olfactory epithelium using
specific antibodies against MUPP1 (green)
and adenylyl cyclase 3 (red). Overlay shows
mature OSNs in blue. White arrow denotes
the apical layer. Scale bars = 50 lm. (D).
Higher magnification image of MUPP1 ⁄ ade-
nylyl cyclase 3 stained olfactory epithelium.
The arrow shows MUPP1 expression in cilia
and in dendritic knobs. Scale bar = 5 lm.
R. Dooley et al. PDZ proteins interact with olfactory receptors
FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7281
3 + 4, etc.) (Fig. 2C). Interaction assays were then
carried out by incubating different pairs of PDZ
domains as in vitro translation products with OR2AG1
C-terminus GST fusion peptides. A specific binding of
PDZ domains 1 + 2 was determined via western blot-
ting, whereas, for example, in vitro translated PDZ
domains 3 + 4 did not have the ability to bind to the
C-terminus of OR2AG1 (Fig. 2D). None of the PDZ
domains could bind to GST alone. We then tested

binding of single PDZ domains 1 + 2, and found that
both could bind to the OR C-terminus (Fig. 2D).
Next, we investigated the ability of PDZ domains
1 + 2 to bind to receptor C-termini of 15 amino
acids in length, which were spotted on microarrays.
The arrays were probed four times with PDZ
domains 1 + 2 fused to the HA tag for subsequent
analysis of binding by antibody incubation and
chemiluminescence detection (Fig. 2E). Interactions
were analyzed in every experiment with positive (HA
tag spotted directly) and negative (FLAG tag spotted
directly and A1 ⁄ A2) control spots. Interactions that
yielded robust interactions of PDZ domains 1 + 2
with the olfactory receptors hOR2AG1 (S-T-L),
mOR283-1 (A-T-V) and hOR3A1 (S-L-A), which all
contain PDZ domain binding motifs in their C-ter-
mini, were scored as array positives (Fig. 2E).
hOR1D2 and mOR-EG, which are olfactory receptors
that do not contain classical PDZ interaction motifs
in their C-termini, also showed positive interactions
but, in the case of hOR1D2, only in two out of four
experiments. Other olfactory receptors, such as
mOR167-4, mOR199-1, M71, M72 and mOR241-1
A
C
DE
B
Fig. 2. The C-terminus of OR2AG1 interacts with MUPP1 in vitro. (A) Pie chart illustrating the abundance of classical PDZ motifs in human
OR C-termini. (B) MUPP1 was immunoprecipitated in HA-OR2AG1 expressing Hana3A cells using a-HA antibodies, detection was performed
with a-MUPP1 (*MUPP1) and a control was performed with identical amounts of nontransfected cell lysates from Hana3A cells. The blot

shown is representative of three independent immunoprecipitation experiments. (C) Western blot using HA-specific antibodies showing the
in vitro translation products of PDZ domain pairwise constructs. Three nonspecific bands appear at 170, 70 and 30 kDa. Specific bands at
the correct molecular weights are outlined (white asterisk). (D) PDZ domains 1 + 2 both interact with OR2AG1_GST in vitro. Interaction
assay using in vitro translated PDZ domains 1 + 2, PDZ domains 3 + 4, PDZ domain 1 and PDZ domain 2 with GST alone or C-terminus
OR2AG1_GST. The blots shown are representative of four independent experiments for each interaction assay described. (E) Peptide micro-
array with C-termini of different receptors incubated with PDZ domains 1 + 2 fused to HA; chemiluminescence detection on film after incu-
bation with a-HA antibodies and HRP-coupled secondary antibodies. The array shown is representative of four independent experiments;
peptide sequences for spots A1–A12 (row 1), A13–A24 (row 2) and B1 (FLAG tag) and B2 (HA tag, positive control) are listed in Table S1.
A1 and A2 serve as negative controls.
PDZ proteins interact with olfactory receptors R. Dooley et al.
7282 FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS
and mGluR2, as well as the olfactory cyclic nucleo-
tide-gated ion channel subunit A2, did not show any
interaction with the PDZ domains investigated.
We furthermore investigated the binding determi-
nants in the C-terminus of hOR2AG1 by spotting pep-
tides that correspond to mutated or shortened receptor
C-termini. Truncation of the last amino acids abol-
ished binding of the C-terminus of hOR2AG1 to PDZ
domains 1 + 2. hOR2AG1 constructs where the last
four amino acids H-S-T-L were mutated to H-A-T-A
[OR2AG1_deltaPDZ(A)] still bound to PDZ domains
1 + 2, whereas mutation to H-W-T-W [OR2AG1_del-
taPDZ(W)] completely abolished binding (Fig. 2E).
MUPP1 shows plasma membrane localization
upon co-expression of ORs
MUPP1 is a cytosolic protein, and MUPP1-GFP, simi-
lar to endogenous MUPP1, shows a homogenous,
predominantly cytosolic distribution when expressed
in Hana3A cells (Fig. 3A). Interestingly, when

co-expressed with hOR2AG1, MUPP1 exhibited a lar-
gely plasma membrane expression in a subset of cells,
forming clusters at the cell surface (Fig. 2A, B).
Approximately 5% of transfected cells exhibited this
translocation effect of MUPP1-GFP. This apparently
low proportion of cells reflects the notoriously low
expression rate of olfactory receptors in heterologous
systems and the relatively high amount of cells express-
ing MUPP1-GFP. However, the results obtained in the
present study correlate with various studies showing a
similar proportion of transiently transfected cells
responding to odorant in ratiometric calcium imaging
experiments [24–26]. This alteration in the subcellular
distribution of MUPP1 upon co-expression with ORs
supported the finding of a physical association between
MUPP1 and ORs in the heterologous expression system.
We hypothesized that, by deleting the components
of the PDZ binding motif, we could disrupt MUPP1
translocation. Truncation of the receptor from the final
eight amino acids (amino acids 309–316) did result in
a clear abrogation of the association, as outlined by
the predominantly cytosolic localization of MUPP1-
GFP (Fig. 3A). We then investigated the in vitro bind-
ing properties of the truncated C-terminal mutant of
OR2AG1 by creating a GST fusion construct. The
A
BC D
Fig. 3. MUPP1 plasma membrane translocation on co-expression of odorant receptors. (A) MUPP1-GFP expressed alone in Hana3A cells
exhibits a diffuse cytosolic expression. Co-transfection of OR2AG1 with MUPP1-GFP leads to a predominant plasma membrane expression
of MUPP1-GFP with clustering apparent. Truncation of OR2AG1 from the final eight amino acids leads to a cytosolic expression of MUPP1-

GFP; at least five independent experiments were performed for each condition. (B) Higher magnification of the plasma membrane of the
cells shown in (A). (C) In vitro interaction properties of truncated hOR2AG1 C-terminus. Western blot showing HA-PDZ1 + 2 probed with
2AG1-GST and trunc8-GST, at 55 kDa, using a-HA antibodies. The experiment was repeated three times with similar results being obtained.
(D) Co-expression of hOR1D2 and hOR3A1 also resulted in translocation of MUPP1-GFP to the plasma membrane; at least five independent
experiments were performed for each receptor. Scale bars = 20 lm.
R. Dooley et al. PDZ proteins interact with olfactory receptors
FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7283
C-terminal mutant peptide was incubated with PDZ
domains 1 + 2 of MUPP1 and failed to interact with
truncated mutant (Fig. 3C).
Consistent with this observation is the fact that
other olfactory receptors showing interactions with
PDZ domains 1 + 2 (hOR1D2, hOR3A1) also caused
A
C
DE
FG
HI
B
Fig. 4. Functional role of MUPP1 in OR signaling. (A) Western blot showing MUPP1 expression in Hana3A cells (control) compared to 48
and 72 h after siRNA (exon5) transfection. (B) Representative ratiometric calcium imaging responses of transiently transfected Hana3A cells
[siRNA(1)]; the arrow represents the beginning of application of amylbutyrate, lasting for 10 s. (C) Western blot showing MUPP1 expression
in Hana3A cells (control) compared to scrambled siRNA, siRNA against exon 5 of MUPP1 and siRNA against exon 45 of MUPP1, 72 h after
transfection. (D) Bar chart showing the rise time (10–90%) of Hana3A cells responding to amylbutyrate, transfected with OR2AG1 (ctrl)
(n = 15) or siRNA(exon5)-treated Hana3A cells transfected with OR2AG1 (RNAi) (n = 15). (E) Time of decay from 90% of peak amplitude to
10% of average baseline (n = 15) for each condition and the percentage of cell responses decaying to basal levels within the time-frames
outlined. Cell responses not decaying within the time-frame of experiment were included in the > 20 s section; n = 15 for control, n =27
for RNAi(exon5). (F) Bar chart showing the rise time (10–90%) for siRNA(exon45) transfected Hana3A cells; n = 12 for control, n = 12 for
RNAi(exon45). (G) Bar chart showing the time of decay (90–10%) and percentages of cell responses for siRNA(exon45) transfected Hana3A
cells; n = 12 for control, n = 12 for RNAi(exon45). (H) Bar chart showing the rise time (10–90%) for Hana3A cells transfected with scrambled

siRNA. (I) Bar chart showing the time of decay (90–10%) and percentages of cell responses for scrambled siRNA transfected Hana3A cells.
Error bars show the SEM. **P < 0.01, ***P < 0.001.
PDZ proteins interact with olfactory receptors R. Dooley et al.
7284 FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS
MUPP1-GFP translocation to the plasma membrane
in the Hana3A cells (Fig. 3D).
MUPP1 controls the duration of Ca
2+
signaling
mediated by recombinant ORs
To determine whether MUPP1 has the ability to
regulate OR function, we investigated the role of this
scaffolding protein in hOR2AG1-mediated Ca
2+
mobilization by performing ratiometric Ca
2+
imaging
in Hana3A cells. These cells express MUPP1 endoge-
nously and, by reducing the amount of MUPP1 by
RNA interference, we aimed to investigate the func-
tionality of the interaction between MUPP1 and
hOR2AG1. Transfection of small interfering RNA
(siRNA) against Mupp1 led to an almost complete
knockdown of the MUPP1 protein, as shown by wes-
tern blotting (Fig. 4A). Next, we monitored the
response of transiently transfected OR2AG1 to its spe-
cific odorant ligand, amylbutyrate [24] via ratiometric
calcium imaging in siRNA-treated cells (Fig. 4B).
When MUPP1 was largely absent, the OR-elicited
response exhibited a similar rise time to that of the

control cells, 5.54 ± 0.87 s for RNA interference
(RNAi) compared to 4.27 ± 0.65 s for the control
(Fig. 4B, C). However, the response failed to decay
within the normal average time-frame in siRNA-trea-
ted cells (19.3 ± 2.9 s) compared to the control cells
(7.2 ± 2.1 s) (Fig. 4B, D). Using another siRNA
directed against an alternative exon of Mupp1, similar
results were obtained. The rise time of the OR-elicited
response was similar to that of the control cells
AB
CD
EF
Fig. 5. Interaction of MUPP1 with OR2AG1 is important for controlled signal decay. (A) Immunocytochemistry (a-HA antibody) showing sta-
ble expression of MUPP1-PDZ1 + 2-HA in Hana3A cells. Scale bar = 20 lm. (B) Representative ratiometric calcium imaging traces for
Hana3A cells (control) and MUPP1-PDZ1 + 2-HA cells (1 + 2) transiently transfected with OR2AG1. Arrows denote amylbutyrate application.
(C) Bar chart showing the rise time (10–90%) for Hana3A cells stably expressing MUPP1-PDZ1 + 2-HA, transiently transfected with
OR2AG1; n = 13 for control, n = 24 for PDZ domains 1 + 2. (D) Decay of response (90–10%) (n = 13 for control, n = 24 for PDZ domains
1 + 2) and the percentage of responses to amylbutyrate decaying to basal levels within the given time-frames. (E) Transient expression of a
truncated version of OR2AG1 missing the last eight amino acids (trunc8). Bar chart showing the rise time (10–90%) for Hana3A cells
expressing the truncated receptor (n = 12 for control, n = 17 for OR2AG1-trunc8). (F) Decay of response (90–10%) (n = 12 for control,
n = 17 for OR2AG1-trunc8) and the percentage of responses to amylbutyrate decaying to basal levels within the given time-frames. Error
bars show the SEM. **P < 0.01, ***P < 0.001.
R. Dooley et al. PDZ proteins interact with olfactory receptors
FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7285
(Fig. 4C), although the decay was prolonged signifi-
cantly (Fig. 4D). Cells transfected with a scrambled
version of the siRNA did not show any significant
differences in the kinetics of the OR-elicited
Ca
2+

response compared to nontransfected cells
(Fig. 4E, F).
Interaction with MUPP1 is important for
controlling OR-mediated Ca
2+
signaling
Because PDZ domains 1 + 2 interact with MUPP1
(Fig. 2), we generated a cell line over-expressing these
two PDZ domains (Fig. 5A). Interestingly, the pro-
longed signal decay observed in the siRNA experi-
ments was mirrored in the OR-dependent responses of
Hana3A cells stably over-expressing PDZ domains
1 + 2 of MUPP1 (Fig. 5B). When monitoring the
response of transiently transfected OR2AG1 to amy-
lbutyrate, we found that the OR-elicited response did
not decay within the normal average time-frame in
cells over-expressing PDZ domains 1 + 2 (26.2 ±
1.8 s) compared to control cells (10.2 ± 1.9 s)
(Fig. 5D). As in siRNA-treated cells, the rise time after
odorant stimulation was almost indistinguishable in
cells over-expressing PDZ domains 1 + 2 (4.87 ±
0.2 s) compared to control cells (4.71 ± 0.69 s)
(Fig. 5C). In summary, when the interaction between
MUPP1 and OR2AG1 is inhibited by over-expressing
the interacting PDZ domains, the resulting response of
OR2AG1 to odorant is modified in that the rapid
decay of signal is impaired.
We finally examined the effect of deletion of the
components of the PDZ binding motif on the signal-
ing properties of the receptor in Ca

2+
imaging
experiments. Truncation of the receptor from the
final eight amino acids (amino acids 309–316)
resulted in a prolonged signal decay, similar to that
observed in the siRNA experiments and in the cells
over-expressing PDZ domains 1 + 2, whereas the
rise time of the signals was again not affected
(Fig. 5E, F).
Discussion
Until now, the involvement of PDZ domain scaffold-
ing proteins in olfactory signal transduction has gone
unstudied. It has previously been suggested that such
scaffolding networks or ‘olfactosomes’ may exist [9,10]
but, to date, no evidence for this phenomenon has
been outlined. In the present study, we have uncovered
a PDZ protein as a novel interaction partner of olfac-
tory receptors and have elucidated the molecular
details of this interaction.
The primary source of olfactory signaling and the
sites of expression of ORs are the ciliary structures of
the OSNs. Interestingly, we found MUPP1 to be pre-
dominantly expressed in the apical compartment of
OSNs and enriched in the cilia fraction of a prepara-
tion of whole murine olfactory epithelium. We hypoth-
esize that MUPP1, through its multivalent capabilities,
could play a key role as a central nucleator of olfac-
tory signal transduction. With its thirteen PDZ
domains, each diverse in its amino acid sequence,
MUPP1 holds great potential for organizing signal

transduction molecules into defined protein networks
and thereby regulating signaling events.
MUPP1 has previously been found to interact with
a diverse array of molecules, including G protein-cou-
pled receptors such as the 5-hydroxytryptamine recep-
tor type 2C [18,27] and the GABA
B
receptor [19]. We
postulated that MUPP1 could directly interact with
the olfactory receptor itself. We scanned the entire
human olfactory receptor repertoire and discovered
that up to 30% of receptors contain putative PDZ
binding motifs in their C-termini, following the previ-
ously outlined rules of binding [16]. PDZ domains
1 + 2 of MUPP1 indeed showed direct interaction
with OR C-terminal petides. Moreover, upon
co-expression of ORs containing classical PDZ bind-
ing motifs in their C-termini, MUPP1-GFP exhibited
a translocation from the cytosol to the plasma mem-
brane in the heterologous expression system, suggest-
ing a physical association between both proteins
within the cell. We found this translocation to be
dependent on the final amino acids of the receptor
protein. Similar to the other PDZ domain interactions
that have been shown to be abolished by mutating
amino acids at position 0 and )2 from the C-termi-
nus [16], we found that binding of the hOR2AG1 C-
terminus to PDZ domains 1 + 2 did not occur when
positions 0 and )2 were mutated to tryptophans,
which are not present in the PDZ binding motifs of

other membrane proteins [17]. We also found interac-
tion of PDZ domains 1 + 2 with ORs showing no
classical PDZ binding motifs, indicating that the
understanding of the exact molecular rules of OR
PDZ interaction will require further analysis. Previous
work with other proteins has already indicated that it
is highly likely that a large number of PDZ domain
interactions will not fit into the confined class defini-
tions and that PDZ domains may have been opti-
mized across the proteome in order to minimize
cross-reactivity [17].
We observed that a reduction of MUPP1 resulted in
a significant increase in the duration of Ca
2+
responses
evoked by the activation of recombinantly expressed
PDZ proteins interact with olfactory receptors R. Dooley et al.
7286 FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS
hOR2AG1. Similarly, over-expression of the OR-inter-
acting PDZ domains 1 + 2 of MUPP1 also resulted in
odorant-evoked Ca
2+
responses that persisted longer
than those in control cells. When over-expressed, PDZ
domains 1 + 2 may bind to the C-terminus of
hOR2AG1, thus having a blocking effect on the bind-
ing of the less abundant endogenous MUPP1. A trun-
cated receptor no longer containing a PDZ motif in
the C-terminus showed the same effect of prolonged
signal duration. Thus, all of the experiments revealed

that the association of hOR2AG1 with MUPP1 regu-
lates signal duration. To a certain extent, the impaired
signal desensitization resembles the effect of the
absence of the multi-PDZ domain protein INAD in
the Drosophila visual signal transduction cascade. Flies
lacking INAD exhibit a profound reduction of the
light response [28]. Interestingly, INAD is also
required for normal deactivation of visual signaling by
positioning eye protein kinase C in close proximity to
TRP to facilitate its phosphorylation, ultimately result-
ing in deactivation of the channel [12,29,30]. On the
other hand, in contrast to the findings of the present
study, MUPP1 was shown to prolong the duration of
GABA
B
receptor signaling and increase the stability of
the receptor [19]. However, we must note that the situ-
ation in the recombinant expression system is different
from that in the olfactory neurons, where alternative
binding partners of MUPP1 presumably exist. It is
therefore possible that MUPP1 could exhibit alterna-
tive effects on the dynamics of calcium responses
induced by ORs in the neurons compared to those
induced by heterologously expressed ORs. The
observed effects can therefore only be taken as proof
of the functional significance of the observed interac-
tion. Further studies are necessary to shed light on the
function of this interaction in the in vivo situation.
By influencing the duration of the Ca
2+

signal of
ORs in the cilia of the sensory neurons, MUPP1 could
have a strong influence on the olfactory signaling path-
way. An interesting interaction partner of MUPP1 out-
lined to date is CamKII, which is known to play an
important role in olfactory adaptation [20]. By phos-
phorylation of adenylyl cyclase 3 in OSNs, CamKII
provides an important mechanism for the attenuation
of odorant-stimulated cAMP increases [31]. Alterna-
tively, because different pathways, such as those
involving phosphoinositide 3-kinase [32], are ultimately
engaged after OR stimulation, MUPP1 may control
OR activity by acting as a scaffold to link different
signaling pathways.
In conclusion, we have outlined a novel aspect of the
olfactory signal transduction cascade by uncovering a
previously unknown interaction partner of olfactory
receptors and a putative regulator of signaling
processes in OSNs. It is tempting to speculate that a
so-called ‘olfactosome’ exists in the cilia of olfactory
sensory neurons, organizing the vast array of signaling
molecules and ensuring the specificity of signaling.
How exactly MUPP1 could carry out such an impor-
tant task remains to be elucidated, although the answer
may lie in the remaining and as yet unidentified interac-
tion partners of MUPP1 in the olfactory sensory cell.
Experimental procedures
DNA constructs and primers
pCDNA3_MUPP1-GFP and in vitro translation tandem
PDZ domain constructs in vector pBAT were provided

by H. Lu
¨
bbert (Ruhr-University, Bochum, Germany).
pCDNA3_OR2AG1 was cloned as described previously
[33]. C-terminal mutant constructs of OR2AG1 [^PDZ_
pCDNA3 (S314A, L316A), trunc4_pCDNA3 (amino acids
313–316) and trunc8_pCDNA3 (amino acids 309–316)],
were obtained by PCR using varying 3¢ primers and
pCDNA3_OR2AG1 as a template. GST fusion constructs
of the C-terminus of OR2AG1, and mutant thereof (trunc8)
were created by cloning the region between amino acids
293 and 316 from the receptor into pGEX-3X vector
(Amersham Pharmacia Biotech, Piscataway, NJ, USA),
using varying reverse primers. PDZ domains 1 + 2 were
cloned using pBAT_1 + 2_HA as a template. The stable
cell line construct, pCMV ⁄ Bsd_PDZ1 + 2_HA, was cloned
using pCDNA3_MUPP1-GFP as a template. All constructs
were verified by sequencing. For RT-PCR, mRNA was
extracted from adult mouse olfactory epithelium, and the
primers were used were: 5¢-CAAAACGCTCTACAGGC
TCC-3¢,5¢-GAAGAGCTGGACAGAGGTGG-3¢ (ZO-1),
5¢-TTATGGGCCACCGGATATTA-3¢,5¢-GGAGAGTCA
CTGAAGGCTGG-3¢ (DLG-2), 5¢-AAGCTAAGAGGCA
CGGAACA-3¢,5¢-TCCTTATTGCCAGCGAGACT-3¢ (Patj),
5¢-TTGCAGACGGAAGAGGTTCT-3¢,5¢-GGCCACTT
TCAGCATCAAAT-3¢ (Erbin), 5¢-GCGGATCCGCAT
GTTGGAAACCATAGAC-3¢ and 5¢-GCGAATTCGA
CATTTTTAGTGAGTTCCAC-3¢ (MUPP1).
Antibodies
Primary antibodies used were: anti-adenylyl cyclase 3, rab-

bit polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA,
USA), directly labeled using DyLightÔ549 Microscale
Antibody Labeling Kit (Pierce, Rockford, IL, USA); anti-
MUPP1, rabbit polyclonal (provided by H. Lu
¨
bbert, Ruhr-
University); anti-GFP, rabbit polyclonal (#ab290-50;
Abcam, Cambridge, MA, USA); a-HA antibody, mouse
monoclonal (#H9658; Sigma, St Louis, MO, USA). Second-
ary antibodies used were goat anti-rabbit Alexa546nm
R. Dooley et al. PDZ proteins interact with olfactory receptors
FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7287
(Molecular Probes, Carlsbad, CA, USA) and horseradish
peroxidase (HRP) coupled goat anti-mouse and goat anti-
rabbit IgGs (Bio-Rad, Hercules, CA, USA).
Cell culture and transfection
All tissue culture media and related reagents were purchased
from Invitrogen (Carlsbad, CA, USA). Hana3A cells [23]
(provided by H. Matsunami, Duke University Medical Cen-
ter, Durham, NC, USA), were maintained in DMEM plus
10% fetal bovine serum and 1% penicillin ⁄ streptomycin, at
37 °C and 5% CO
2
, and transfections were carried out using
a standard calcium phosphate precipitation technique.
MUPP1-GFP and OR plasmid DNAs were transfected in a
ratio of 1 : 10, with approximately 2 lg of total DNA per
dish. All images were acquired using a Zeiss LSM 510 Meta
confocal microscope (Carl Zeiss, Oberkochen, Germany).
Hana3A cells were stably transfected with pCMV ⁄ Bsd plas-

mid (Invitrogen) containing tandem PDZ domains 1 + 2
along with an HA tag. Positive clones were selected for
using blasticidin (10 lgÆmL
)1
) and stable transfection was
confirmed by immunocytochemistry.
Cell membrane preparation and western blotting
The olfactory epithelium of CD1 mice was fractionated by
mechanical agitation as described previously [21]. Equal
amounts of protein from each fraction were loaded on an
SDS gel and subjected to immunoblotting on poly(vinyli-
dene difluoride) membrane (Millipore, Billerica, MA, USA)
and Coomassie staining. Detection was performed using the
ECL western blotting detection system (GE Healthcare,
Milwaukee, WI, USA). For co-immunoprecipitation,
Hana3A cells were transfected with OR2AG1-HA and
MUPP1_pCDNA3 or untransfected. The nucleus-free cell
lysates were incubated with biotinylated a-HA antibody
and precipitated protein collected using Dynabeads (Invi-
trogen) and DynaMag (Invitrogen). Any interaction was
detected using MUPP1 antibodies.
Immunohistochemistry
Mice were raised and maintained according to governmen-
tal and institutional care instructions. Immunohistochemis-
try was carried out on 14 lm horizontal sections, and
fluorescence images were obtained with a confocal micro-
scope (Zeiss LSM 510 Meta) with ·40 objective. Control
experiments in the absence of any primary antibody
revealed a very low level of background staining. For odor-
ant exposure experiments, OMP-GFP mice were exposed to

a mixture of 100 different odorant molecules (Henkel
KGaA, Du
¨
sseldorf, Germany) for specified amounts of
time. Control mice were housed in a separate room free
from artificial odorant stimulation. All mice were held in
standard cages at room temperature. Each cage was sur-
rounded by a Perspex chamber with ventilation suction to
maintain a constant air-flow.
GST fusion peptides and in vitro interaction
assays
The C-terminal region of OR2AG1 was found to lie between
amino acids 293 and 316, as predicted by tmhmm, a trans-
membrane helices prediction program based on a hidden
Markov model [34]. OR2AG1 C-terminus GST fusion pro-
teins and mutant construct thereof (trunc8-GST) were pro-
duced in Escherichia coli XL1 blue and purified on
glutathione sepharose beads (Becton-Dickinson Biosciences,
Franklin Lakes, NJ, USA). PDZ domains were in vitro trans-
lated using the TNTÒT3 Coupled Reticulocyte Lysate Sys-
tem (Promega, Madison, WI, USA). Interaction assays were
carried out by incubating 10 lLofin vitro translation prod-
uct with 50 lL of GST fusion peptide bead slurry for 2 h at
4 °C with gentle shaking. After a series of washing steps using
Buffer S (20 mm Hepes, 100 mm KCl, 0.5 mm EDTA, 1 mm
dithiothreitol, pH 7.9), specific interactions were assessed via
immunoblotting. GST alone was used as a negative control.
Peptide microarray
CelluSpotsÔ Peptide Arrays (Intavis AG, Cologne,
Germany) were blocked for 2 h at room temperature with

5% skimmed milk in NaCl ⁄ Tris ⁄ Tween. The arrays were
incubated with a PDZ1 + 2_HA fusion protein (produced
as described in E. coli) overnight at 4 °C. CelluSpotsÔ were
incubated with the a-HA antibody for 4 h at room temper-
ature. Detection was performed with HRP coupled second-
ary antibody and using ECL western blotting detection
reagent (GE Healthcare).
Mupp1 siRNA
Pre-synthesized and tested Mupp1 siRNA (identification 1
#107246 and 2 #216971) and a custom designed scrambled
version of Mupp1 (CUGACUGUGUAUCGAACGGtt)
were purchased from Ambion (Austin, TX, USA). siRNA
was transfected 72 h prior to calcium imaging using Lipo-
fectamine 2000 (Invitrogen), in serum-free medium
(Opti-Mem; Invitrogen). Forty-eight hours prior to calcium
imaging, 2 lg of OR2AG1 plasmid DNA were transfected
per dish using ExGen 500 transfection reagent (Fermentas,
Glen Burnie, MD, USA). Medium was exchanged for fresh
DMEM 24 h post-transfection.
Ratiometric Ca
2+
imaging in heterologous cells
Stably ⁄ transiently transfected Hana3A cells were incubated
with 7.5 lm FURA-2 AM (Invitrogen). Ratiometric
PDZ proteins interact with olfactory receptors R. Dooley et al.
7288 FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS
calcium imaging was performed as described previously [25]
using a Zeiss inverted microscope equipped for ratiometric
imaging. Cells were exposed to 100 lm amylbutyrate (Hen-
kel GmbH), which is a typical odorant concentration used

for heterologously expressed ORs [23,35,36], employing a
specialized microcapillary application system. The rise time
was calculated as the time in seconds from 10% of peak
response, starting from the average baseline value, to 90%
of peak amplitude. The response decay duration was calcu-
lated as the time in seconds between 90% and 10% of the
maximum amplitude.
Acknowledgements
We thank H. Bartel and J. Gerkrath for their excellent
technical assistance; H. Matsunami (Duke University
Medical Center, Durham, NC, USA) for the donation
of Hana3A cells; P. Mombaerts (MPI Biophysics,
Frankfurt, Germany) for the donation of OMP-GFP
transgenic mice; and H. Lu
¨
bbert ⁄ X. Zhu (Ruhr Uni-
versity, Bochum, Germany) for the donation of
MUPP1 antibodies and constructs. This work was sup-
ported by the International Max-Planck Research
School in Chemical Biology (IMPRS-CB), the Stud-
ienstiftung des deutschen Volkes, the Heinrich und
Anna Vogelsang Stiftung and the Deutsche Fors-
chungsgemeinschaft (SFB 642).
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Supporting information
The following supplementary material is available:
Table S1. Peptide microarray.
This supplementary material can be found in the
online version of this article.
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PDZ proteins interact with olfactory receptors R. Dooley et al.
7290 FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS