From the Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky 40536
Received for publication, June 21, 2000, and in revised form, October 19, 2000
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ABSTRACT |
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Olfactory receptors are difficult to functionally
express in heterologous cells. They are typically retained in the
endoplasmic reticulum of cells commonly used for functional expression
studies and are only released to the plasma membrane in mature cells of the olfactory receptor neuron lineage. A recently developed olfactory cell line, odora, traffics olfactory receptors to the
plasma membrane when differentiated. We found that undifferentiated
odora cells do not traffic olfactory receptors to their
surface, even though they release the receptors to the Golgi apparatus
and endosomes. This behavior differs from other cell lines tested thus
far. Differentiated odora cells also properly traffic
vomeronasal receptors of the VN1 type, which lack sequence similarity
to olfactory receptors. ODR-4, a protein that is necessary for plasma
membrane trafficking of a chemosensory receptor in nematodes,
facilitates trafficking of rat olfactory receptor U131 in
odora and Chinese hamster ovary cells. Olfactory receptor
trafficking from the endoplasmic reticulum to the plasma membrane
involves at least two steps whose regulation depends on the maturation
state of cells in the olfactory receptor neuron lineage. These
results also indicate that some components of the regulatory mechanism
are conserved.
The olfactory receptors
(ORs)1 are a large and
diverse family of proteins belonging to the superfamily of G
protein-coupled receptors (GPCRs). Since the initial isolation of
cDNAs encoding rat ORs (1), ORs have been cloned from several
taxonomic groups including mouse, dog, human, fish, chicken (reviewed
in Ref. 2), nematodes, and Drosophila (3-5). The GPCR
superfamily also includes other chemoreceptors, two divergent types of
mammalian vomeronasal receptors and taste receptors in rodents and
Drosophila (6-10). Given the large evolutionary distance
between these organisms, the varied environments they inhabit, and the
evidence that hundreds of OR genes exist in a single species, it is not
surprising that chemosensory receptors are diverse sequences that
appear to have little in common besides general topology (2).
The capacity to traffic ORs to the plasma membrane efficiently appears
to be specific to mature ORNs. ORs do not traffic well to the plasma
membrane when expressed in common cultured cell lines, in neuron-like
cell lines, and even cell lines derived from the olfactory epithelium
(11, 12). This is consistent with the presumption that plasma membrane
insertion is necessary for OR function and explains why ORs are
difficult to functionally express in heterologous systems. Evidence
that mature ORNs traffic ORs to the plasma membrane comes from
immunocytochemical detection of ORs in olfactory cilia (13), which
appear to lack internal membranes, and from the successful functional
expression of an OR in ORNs in vivo (14). In all other
cells, we find that ORs are retained in the endoplasmic reticulum (ER)
and traffic poorly, if at all, to the Golgi apparatus (12). This
appears to be the typical behavior of an OR in a heterologous
expression system, although exceptions are known among nonmammalian ORs
(15, 16) and other families of chemosensory receptors (17). In
addition, plasma membrane trafficking of ORs in heterologous cells can
be improved by extending the N terminus of ORs (18, 19). These discoveries serve to underscore our ignorance about the mechanisms regulating trafficking of ORs. Only in Caenorhabditis
elegans is the identity of a protein that regulates OR trafficking
known. The odr-4 gene, which is expressed specifically in
chemosensory neurons, is necessary for trafficking of some ORs to
sensory neuron cilia (20). No mammalian homolog of the ODR-4 protein
has yet been identified. However, the number of accessory proteins that regulate the trafficking of other types of mammalian GPCRs continues to
grow (21-29).
Access to a cell line that mimics the ability of ORNs to traffic ORs
would aid the study of OR trafficking. The recently developed odora cells (30) may provide such a model cell line.
Differentiated odora cells express neuronal and olfactory
markers, including components of the olfactory transduction pathway,
and traffic exogenous olfactory receptors to their surface. We found
that differentiation is necessary for plasma membrane expression of ORs
in odora cells. In undifferentiated odora cells,
ORs were not released to the surface but were present in the Golgi
apparatus and endosomes. This suggests a two-step model for the
regulation of OR trafficking. We also found that differentiated
odora cells were able to traffic VN1-type vomeronasal
receptors to their plasma membrane and that coexpression of ODR-4 with
rat OR U131 facilitated trafficking of U131 to the plasma membrane.
These findings are evidence that mechanisms regulating OR trafficking
involve conserved functions.
Materials--
Suppliers of affinity-purified antibodies were as
follows: rabbit anti-KDEL was from Stressgen, mouse monoclonal
antibodies and rabbit polyclonal antisera against GFP were from
CLONTECH, mouse monoclonal anti-human transferrin
receptor (TfR) was from Zymed Laboratories Inc., mouse
monoclonal (16B12) antibodies (mAbs) against the hemagglutinin epitope
(HA-1) were from Berkeley Antibody Company, and all secondary
antibodies were from Jackson Immunoresearch Laboratories. Fluorescently
labeled wheat germ agglutinin was from Sigma. Sources of clones were as
follows: Drosophila dor64 (5) was a kind gift of
Dr. L. Vosshall, rat taste receptor TR2 (9) was a kind gift of
Drs. N. Ryba and C. Zuker, rat OR I7 (1) was a kind gift of
Dr. L. Buck, VN1 and VN7 (6) were kind gifts of Dr. C. Dulac, odr4
(20) was a kind gift of Dr. C. Bargmann, and pcDNA3.FLAG.hAIP1
(hAIP1 accession number AF151793) was a kind gift of
Dr. T. Shioda.
Cell Culture and Transfection--
Odora cells were
the kind gift of Dr. D. Hunter. They were cultured as described (30),
using Dulbecco's modified Eagle's medium from Bio-Whittaker, 10%
fetal bovine serum from Hyclone, 100 units/ml penicillin, and 100 µg/ml streptomycin (the latter two from Life Technologies, Inc.) at
33 °C and 7% CO2 in a humidified incubator. For
transfection, undifferentiated cells were plated in 8-well Lab-Tek
Permanox chamber slides (Nalge Nunc International, catalog number
177445) at a density of 18,750 cells per well, incubated at 33 °C
and 7% CO2 (undifferentiated conditions) and transfected
the next day with FuGENE-6 reagent (Roche Molecular Biochemicals)
according to the manufacturer's protocol. For differentiation, odora cells were plated in 8-well chamber slides at a
density of 6,250 per well, and the next day they were moved into
differentiation conditions (Dulbecco's modified Eagle's medium
containing 1 µg/ml insulin, 20 µM dopamine, and 100 µM ascorbate at 39 °C and 7% CO2). After
5 days, differentiated cells were transfected using TransFast (Promega)
as recommended by the manufacturer. In both cases, the cells were
analyzed 3 days after transfection. CHO cells (line ldl A-7)
were kindly provided by Dr. M. Krieger. They were maintained at
37 °C and 5% CO2 in a humidified incubator in F12
medium (Cellgro) with 5% fetal bovine serum, 100 units/ml penicillin,
and 100 µg/ml streptomycin. For transfection, CHO cells were plated
on 8-well chamber slides at a density of 12,500 cells per well,
transfected using FuGENE-6 reagent 24 h after plating, and
subjected to analysis 3 days after transfection.
Recombinant DNA Methods--
Several of the constructs used were
described previously (11); the OR constructs were OR5-GFP, U131-GFP,
HA-OR5, and HA-U131, and the human Immunocytochemistry and Image Analysis--
Procedures described
previously for other cell types (11, 12) were used for
immunocytochemistry of odora cells. For cell staining prior
to fixation, cells were rinsed with PBS (pH 7.3), incubated with
primary antibodies (16B12) diluted at 1:300 in 1% BSA in PBS (solution
B), then fixed with 3.7% paraformaldehyde, washed in PBS and incubated
in solution B and with secondary antibodies (Cy3-conjugated anti-mouse,
1:300 in solution B). All steps through fixation were performed on ice
to prevent receptor internalization. For permeabilized cell staining,
including dual labeling, cells were washed in PBS, fixed as above,
permeabilized for 2 min with cold methanol, and blocked in solution B
prior to incubation in primary antibodies (anti-GFP, 1:500; anti-KDEL
mAbs, 1:350; anti-TfR mAbs, 1:500; all in solution B), followed by
consecutive washes in PBS and solution B and incubation with
fluorescently labeled secondary antibodies (fluorescein
isothiocyanate-conjugated anti-rabbit, 1:350; Cy3-conjugated
anti-mouse; 1:350) or rhodamine-conjugated wheat germ agglutinin
(1:2000 in solution B). When two primary antibodies were used, they
were applied simultaneously. The following combinations of primary and
secondary antibodies were used individually or in combination: for
detection of the HA-1 epitope, 16B12 mouse mAbs and Cy3-conjugated goat
anti-mouse; for GFP, rabbit polyclonal anti-GFP and fluorescein
isothiocyanate-conjugated sheep anti-rabbit; for the ER detection,
anti-KDEL mouse mAbs and Cy3-conjugated goat anti-mouse; for endosome
detection, mouse anti-human TfR mAbs and Cy3-conjugated goat
anti-mouse. To mark the Golgi apparatus, rhodamine-conjugated wheat
germ agglutinin was used. Laser scanning confocal microscopy was
performed on a Leica TCS confocal system at the University of Kentucky
Imaging Facility. The signal from two fluorophores was detected
simultaneously. The numbers of pixels with signal from either or both
fluorophores was determined on a Macintosh computer using the public
domain NIH Image program (developed at the National Institutes of
Health and available on the Internet) as described previously
(12). For analysis of intensity of staining, images were collected with
a monochrome digital camera (Eastman Kodak Co. Megaplus 1.4) using the
same optical settings and exposure time for all images in the
experimental series. The number and the grayscale value of pixels in
the image were integrated using NIH Image, and the resulting intensity
values reflect both area of the staining and its intensity. The
intensity values for each condition were compared only to conditions
within the experiments performed that day. All quantitation was
performed on unenhanced digital images. For presentation purposes, the
monochrome images shown have been subjected to contrast enhancement
after all the parts of figures were assembled so that differences in staining intensity between the images were not distorted.
Undifferentiated odora Cells Do Not Traffic ORs to the Plasma
Membrane--
The first question we addressed was whether
undifferentiated odora cells traffic ORs to their plasma
membrane as do the differentiated cells. As a positive control for
receptor expression and trafficking, we used
HA- In Undifferentiated odora Cells, ORs Are Present in Endosomes and
the Golgi Apparatus--
We have previously shown that in cells of
several nonneuronal types (CHO, human embryonic kidney 293, Xenopus fibroblasts, and melanophores), inability to express
ORs on the plasma membrane is explained by the retention of the
receptors in the ER. In contrast, the Differentiated odora Cells Traffic to the Plasma Membrane
Chemosensory Receptors from Unrelated Families--
We further
investigated whether differentiated odora cells are capable
of plasma membrane expression of other families of chemosensory
receptors. We made HA-1-tagged versions of rat OR I7, rat vomeronasal
receptors VN1 and VN7, Drosophila OR dor64, and rat taste
receptor TR2. These groups of chemosensory receptors do not share
significant sequence homology but are thought to share the general GPCR
topology. As shown in Fig. 3, HA-I7,
HA-VN1, and HA-VN7 were detected at the cell surface as punctate or
concentrated regions of immunostaining done on living cells. The
appearance of this surface staining differed greatly from the
intracellular distribution of receptors in these cells. The
intracellular distribution was a network that extended throughout the
cell, often with high-intensity regions near the nucleus. This is shown
in Fig. 3 as the GFP fluorescence of HA- Odr-4 Facilitates Surface Expression of U131--
If diverse
chemosensory receptors share trafficking behavior in heterologous
expression systems, as our data indicate, they should also be expected
to share the ability to interact with proteins that regulate their
trafficking. Thus far, the only protein known to regulate trafficking
of an OR is ODR-4, a unique protein expressed in chemosensory neurons
of C. elegans, where it is necessary for the surface
expression of chemosensory receptor ODR-10. In odr-4
mutants, chemosensory receptor ODR-10 is confined to an intracellular
network in the cell body of the receptor neurons (20). When coexpressed
with HA-U131 in undifferentiated odora cells, ODR-4 caused
an increase in surface expression of HA-U131 in a small (0.3-0.5%)
population of cells (Fig. 4A).
Similar observations were made in CHO cells (Fig. 4C). No
staining above background (nontransfected cells) was seen in the cells
transfected with ODR-4 alone or HA-U131 alone (Fig. 4, A and
C). Coexpression with ODR-4 did not have any effect on the
surface expression of HA-OR5 (not shown). The staining intensity of the
cells expressing HA-U131 on their surface varied. To estimate the
effect of ODR-4 in odora cells, we acquired for each
transfection condition the first 12 to 20 images of the cells that were
visually perceived as being above the background in both transfected
and control cells. We quantified staining in unmodified images of a
standard size by digitally integrating pixel values between grayscale
levels 40 and 254 using the integration function of NIH Image.
Representative images from an odora cell experiment are
shown in Fig. 4A, and the intensity values are presented on
Fig. 4B. For this experiment, the mean intensity values were
as follows: 1782 ± 1981 for ODR-4 coexpression with HA-U131, and
135 ± 177 for HA-U131 alone (mean ± S.D.; arbitrary units;
the data represent 4 independent transfections). In another experiment
we transfected, stained, and collected the intensity data for HA-U131 + ODR-4 (329 ± 320; mean ± S.D.; four independent
transfections) and HA-U131 (49 ± 55), HA-OR5 + ODR-4 (81 ± 50), and HA- The ability to traffic ORs to the plasma membrane appears to be
limited to ORNs. When expressed in nonolfactory cell lines, ORs are
retained in the ER (12). Odora cells are an exception to
these generalities, perhaps because they resemble ORNs (30). First,
they traffic ORs to the plasma membrane when in the differentiated state. Second, in the undifferentiated state they show a unique pattern
of OR trafficking; ORs traffic into the Golgi apparatus and endosomes
but not to the plasma membrane. These data predict that as progenitor
cells progress through differentiation to become mature ORNs they may
acquire stepwise changes in their ability to move ORs toward the plasma
membrane. Our results further indicate that the mechanisms regulating
OR trafficking have conserved features. First, trafficking of the VN1
type of vomeronasal receptors depends on the differentiation of
odora cells just as do ORs. Second, the ability of a
C. elegans protein, ODR-4, to improve plasma membrane
trafficking of a mammalian OR is similar to its ability to facilitate
trafficking of a C. elegans OR.
Our results support a model of OR trafficking that has two regulatory
checkpoints (Fig. 5). The first
checkpoint is exit from the ER. Because undifferentiated
odora cells, unlike other cell lines, can traffic ORs past
the ER, the mechanism allowing ORs to cross the ER checkpoint may be
expressed relatively early in the differentiation of ORNs. Because ORs
are not present in the plasma membrane of undifferentiated
odora cells, but are present there after differentiation, a
second checkpoint may occur at a post-Golgi compartment. We do not yet
have evidence of whether ORs traffic directly to endosomes from the
Golgi apparatus or arrive there by rapid internalization from the
plasma membrane. The proteins that regulate these checkpoints remain
obscure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic
receptor constructs were
2-adrenergic receptor-GFP and HA-
2-adrenergic receptor.
Epitope-tagged receptors contain the hemagglutinin epitope tag (HA-1)
at their N termini; the GFP constructs have receptors fused at their C
termini to hGFP-S65T (obtained from CLONTECH; see
Ref. 31). To generate PCR products for subcloning, we used
high-fidelity polymerases Pfu Turbo (Stratagene) or Platinum
Pfx (Life Technologies, Inc.). To generate HA-1-tagged versions of
chemosensory receptors, we first made a fusion plasmid, pcDNA3.HA.
Using HA-
2-adrenergic receptor as a PCR template
(11), T7 as the forward primer, and 5'-ATTTTACTCGAGAGCGTAATCTGG as the
reverse primer, a PCR fragment (98 base pairs) was generated
containing (5' to 3') a BamHI restriction site, an ATG codon
in an optimal Kozak context, the HA-1 epitope coding sequence, and an
XhoI restriction site. This fragment was cloned into a
BamHI/XhoI digest of pcDNA3.1(+)/Hygro
(Invitrogen). The resulting construct includes XhoI,
XbaI, and ApaI restriction sites downstream of
the HA-1 coding sequence. To fuse receptor open reading frames with the
HA-1 epitope reading frame, receptors were cloned into an
XhoI/XbaI digest of pcDNA3.HA. In all cases, fusion at the XhoI site resulted in Leu-Glu as a spacer
between HA-1 and the receptor protein. The following primers were used (all from 5'): for VN1, forward ATACTCGAGATGATGAATAAGAACAGC, reverse CCGTCTAGATCAAGGAATATTCAAAC; for VN7, forward ATACTCGAGATGTTCTTTGAAGAG, reverse GAGTCTAGATCAAGTATTTACTATTCTG; for TR2, forward
ATTTATCTCGAGCAGGCAAGGACACTC, reverse CTGCTTCTAGATTAGCTCTTCCTCATGGTG
(note that this results in the deletion of the first three amino acid
residues of TR2); for I7, forward ATACTCGAGATGGAGCGAAGGAAC, reverse
ATTTCTAGACTAACCAATTTTGCTGCC; for dor64, forward
ATAACTCGAGAAACTCAGCGAAACCC, reverse ATAATCTAGACTAAGAACCCAGGCCATC. To allow for mammalian expression, odr-4 was subcloned
into a BamHI/XhoI digest of
pcDNA3.1(+)/Hygro. All constructs were sequenced across the
junction points to ensure that no frameshifts were introduced.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor or
2-adrenergic
receptor-GFP. In both differentiated and undifferentiated
odora cells, HA-
2-adrenergic receptor was
present on the plasma membrane (Fig. 1).
As previously reported (30), HA-U131 (rat) was present on the plasma
membrane of differentiated odora cells, and the staining
pattern was punctate. In undifferentiated odora cells,
HA-U131 was not present on the surface. Two other rat ORs, HA-OR5 (not
shown) and HA-I7 (see Fig. 3), behaved in the same manner as HA-U131.
We conclude that whereas differentiated odora cells traffic
ORs to the plasma membrane, undifferentiated odora cells do
not.
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Fig. 1.
Olfactory receptors are absent from the
plasma membrane in undifferentiated odora cells.
Both differentiated and undifferentiated odora cells that
were transiently transfected with HA- 2-adrenergic
receptor expressed the HA-1 epitope on the cell surface.
Differentiated, but not undifferentiated, odora cells
expressed HA-U131 on the cell surface. The cells were immunostained
prior to fixation. Scale bars, 25 µm.
2-adrenergic
receptor is expressed in the ER, the Golgi apparatus, endosomes, and on
the plasma membrane (11, 12). Because undifferentiated odora
cells do not traffic ORs to the plasma membrane, we tested whether they
also retained ORs in the ER. We used C-terminal fusions of receptors to
hGFP-S65T for these colocalization studies; receptors fused to the HA-1
epitope tag or to GFP variants have the same intracellular distribution
(11, 12). In confocal images of undifferentiated odora cells
2-adrenergic receptor-GFP was distributed as expected
(Fig. 2), with 31 ± 11% (mean ± S.D.; n = 4) of pixels with GFP signal coinciding
with staining for an ER marker, 56 ± 16% (n = 3)
colocalized with the Golgi marker, and 19 ± 9%
(n = 6) with an endosome marker. Even though it is not
present on the plasma membrane, U131-GFP also colocalizes with the
Golgi marker (29 ± 12% of pixels with GFP signal,
n = 3) and the endosome marker (37 ± 22%;
n = 5), besides being present in the ER (45 ± 29%; n = 4). The percent overlap values for the three
compartments are not expected to sum to 100%, because each compartment
was tested in different cells, and other compartments that we did not
test (e.g. proteolytic compartments and transport
vesicles) presumably contain receptors. OR trafficking in
undifferentiated odora cells shows a pattern that differs
from nonneuronal cells and from mature ORNs. The ORs are released from
the ER and are present in the Golgi apparatus and in TfR-positive
endosomes but are not detected on the plasma membrane. Our previous
results (12) suggest that exit from the ER is a regulated process that depends on structural determinants in the C-terminal part of an OR.
These new data suggest a second regulatory process, occurring in a
post-Golgi compartment and sensitive to the differentiation state of
the ORN.
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Fig. 2.
Olfactory receptors are released from the ER
in undifferentiated odora cells. Confocal
images show that for both 2-adrenergic receptor-GFP
(a-c) and U131-GFP (d-f), the GFP signal
(green) overlaps with the red signal from markers
for the ER (KDEL; a and d), the Golgi apparatus
(wheat germ agglutinin; b and e), and endosomes
(TfR; c and f). The images show transiently
transfected cells that were permeabilized prior to immunostaining.
Scale bars, 25 µm.
2-GFP and
U131-GFP expressed in differentiated odora cells. Receptors
fused at the C termini to GFP show trafficking behavior identical to
the same receptors containing the N-terminal HA-1 epitope tag (12).
These data argue that HA-I7, HA-VN1, and HA-VN7 were present on the
plasma membrane of the differentiated odora cells. The
efficiency of expression we observed (0.5-2% of the cells) was
similar to that for HA-U131 and HA-OR5. As observed with HA-U131 and
HA-OR5, none of these chemosensory receptors were detected on the
plasma membrane of undifferentiated odora cells (not shown).
We did not detect surface staining in undifferentiated (not shown) or
differentiated odora cells transfected with HA-TR2 and
Drosophila HA-DOR64; the intensity of nonpermeabilized
staining in these cells was similar to very weak background staining
occurring in control cells. We conclude that differentiated
odora cells are capable of properly trafficking to the
plasma membrane at least two unrelated types of chemosensory receptors.
The underlying mechanisms must therefore recognize both OR and VN1
receptor families, suggesting that these receptors have some common
features.
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Fig. 3.
Differentiated odora cells
express unrelated chemosensory receptors on the plasma membrane.
Differentiated odora cells were transiently transfected with
receptors fused to the HA-1 epitope at their N termini. Transfected
cells were immunostained prior to fixation so that positive staining
would identify only the HA-1 epitope that was expressed on the plasma
membrane. The surface staining was apparent in cells transfected with
HA-I7, HA-VN1, and HA-VN7 but not in cells transfected with HA-DOR64 or
HA-TR2. To demonstrate the difference in appearance between surface
staining and intracellular staining, differentiated odora
cells were transiently transfected with a 2-adrenergic
receptor that was tagged with the HA-1 epitope at the N terminus and
GFP at the C terminus (HA-
2-GFP).
Two images of HA-
2-GFP in the same cell show the surface
staining obtained with immunostaining prior to fixation
versus the total distribution of the GFP fluorescence, much
of which is from receptors in intracellular membranes. The GFP
fluorescence of U131-GFP in a differentiated odora cell is
shown to demonstrate the network pattern typical of the intracellular
distribution of olfactory receptors. Scale bars, 25 µm.
wt, wild type.
2-adrenergic receptor (1396 ± 1557).
To test whether coexpression with any protein was sufficient to promote surface expression of HA-U131, other proteins were coexpressed with
HA-U131. We tested hAIP1 (fused to the FLAG epitope), which has no
sequence similarity to ODR-4, because it was detected in a yeast
two-hybrid screen as a possible binding partner of a fragment of
U131.2 Coexpression
with hAIP1 did not increase plasma membrane expression of HA-U131 (Fig.
4B, inset). Coexpression with hGFP-S65T gave identical negative results. ODR-4 had a significant effect
(p < 0.05, with averaged intensity values from two
separate sets of four transfections each, normalized to the mean value
of U131 + ODR-4 in each case) on the plasma membrane expression of
U131. The action of ODR-4 did not appear to be correlated with an
increased amount of receptor protein. Coexpression of U131-GFP and
ODR-4 in undifferentiated odora cells was compared with
cotransfection of U131-GFP plus the empty vector pJG3.6. The resulting
distributions of GFP fluorescence intensity values were similar, but
the peak intensity values from the cells transfected with U131-GFP plus pJG3.6 was 30% higher. Although ODR-4 worked in two cell lines, including a nonolfactory line, it is probably unsuitable for use in
functional expression screening of the odorant selectivity of ORs. Its
effects are relatively weak and appear to be limited to a subset of
mammalian ORs. Nevertheless, our data do suggest parallels between the
mechanisms of regulation of the plasma membrane expression of
chemosensory receptors in C. elegans and in mammals.
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Fig. 4.
Nematode protein ODR-4 facilitates plasma
membrane expression of rat receptor U131. A,
undifferentiated odora cells were transiently transfected
with plasmids encoding HA-U131, ODR-4, or both (U131 + ODR-4) and stained prior to fixation. Untransfected cells and
cells expressing ODR-4 or HA-U131 alone did not exhibit significant
membrane staining. Some of the cells transfected with both HA-U131 and
odr-4 exhibited HA-1 surface staining of widely varied
intensities. Images from a single representative experiment are shown.
The scale bar (25 µm) applies to all images in
A. B, area and intensity of staining in
individual cells was quantified from 8-bit digital images as described
under "Experimental Procedures." Bars represent
individual cells. Values are normalized to the average value of cells
transfected with HA-U131 alone. A representative sample from the same
experiment (4 independent transfections) as in A is shown.
Inset, coexpression with FLAG-hAIP1 did not increase the
HA-U131 surface expression. C, CHO ldl A-7 cells
were transiently transfected with HA-U131, odr-4, or both.
When immunostained prior to fixation, only cells cotransfected with
both odr-4 and HA-U131 exhibited surface immunostaining
against the HA-1 epitope above that of the untransfected cells or that
of cells transfected with HA-U131 or odr-4 alone. The
scale bar (25 µm) applies to all images in C. wt, wild type.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
A two-stage model of accessory
protein-dependent trafficking of olfactory receptors.
A, in the absence of specific accessory proteins, the ORs
are retained in the ER. B, an accessory protein
(X) is necessary for OR release from the ER into the Golgi
apparatus and endosomes. The OR could be either sent directly to
endosomes or rapidly recycled from the plasma membrane to endosomes
(dashed arrows). C, another accessory protein
(Y) is necessary for OR release to, or anchoring at, the
plasma membrane.
The model does make predictions, however, about the nature of these regulatory proteins. It predicts that the critical proteins have positive effects, either directly on the OR by activating other proteins to promote OR trafficking or by disinhibition of processes that suppress OR trafficking. There are examples of other GPCRs whose membrane trafficking in heterologous cells improves in the presence of specific accessory proteins (23-25). We have now shown that at least one OR, U131, behaves similarly when coexpressed with the C. elegans protein, ODR-4. Our model predicts that mammalian ORNs express proteins with functions analogous to ODR-4. Because ODR-4 does not traffic to the cilia of sensory neurons with its receptor in C. elegans, and because ODR-4 promotes plasma membrane trafficking of U131 in a cell line where U131 is normally retained in the ER, we suspect that ODR-4 and its predicted functional homologs in vertebrates are involved in the exit of ORs from the first checkpoint at the ER. Why ORs are retained in the ER in the first place is not yet clear, but misfolding or the lack of a partner for heteromeric proteins are common causes of active ER retention of plasma membrane proteins by ER quality control mechanisms (32, 33). The second, post-Golgi checkpoint is more mysterious. It may be specific to cells of the ORN lineage, but this has not been tested directly. The proteins regulating this checkpoint may interact with ORs either at the Golgi apparatus or endosomes to target the ORs onward to the plasma membrane. It is possible that they release ORs from the proteins that promoted exit of the ORs from the first checkpoint. Alternatively, the second checkpoint may consist of proteins that encounter ORs at the plasma membrane, trapping them there and slowing OR recycling to endosomes. The latter possibility is analogous to other GPCRs whose accumulation in certain subdomains of the plasma membrane is regulated by specific accessory proteins (for review see Refs. 34 and 35). A third alternative is that processing at the ER can generate receptor proteins with differing trafficking capacities, some capable of reaching endosomes and others capable of reaching the plasma membrane. This would be consistent with the ability of ODR-4 to promote plasma membrane trafficking in both CHO cells, where ORs are retained in the ER, and in undifferentiated odora cells, where ORs reach the Golgi apparatus and endosomes. Whatever the mechanism, the reason for such apparently complex regulation of OR trafficking is unknown. Perhaps it is related to the hypothesis that the olfactory cilium is a privileged plasma membrane domain containing a unique subset of proteins.
An alternative to our model is the possibility that mature olfactory receptor neurons lack an otherwise widely expressed factor that suppresses plasma membrane trafficking of ORs. We cannot exclude this possibility, but it is not consistent with the ability of ODR-4 to act as a positive factor or with analogies to positive factors that regulate the trafficking of other GPCRs (23-25). A more complex mechanism, such as depicted in our model, therefore seems justified. The model is not meant to suggest that negative factors do not play significant roles in regulating OR trafficking, however. For example, ER quality control mechanisms may play a significant inhibitory role if nascent ORs are misfolded or lacking a binding partner, something that is consistent with the effects of truncation on OR trafficking (12). An interdependence of positive and negative factors would be consistent with the apparent complexity of OR trafficking.
Given the paucity of conserved residues among ORs in particular, and
among chemoreceptor proteins in general, wide variability in
trafficking behavior in heterologous cells might be expected. Indeed,
there are examples of ORs that traffic well to the plasma membrane in
at least some heterologous cells (15, 16, 36). However, these are the
exceptions rather than the norm. Instead, our results emphasize the
conservation of the mechanisms regulating trafficking of chemoreceptor
proteins. First, the trafficking behavior of the VN1 type of
vomeronasal receptor mirrors that of ORs. They traffic to the plasma
membrane of odora cells only when these cells are
differentiated. Second, the ability of ODR-4 to increase the plasma
membrane trafficking of at least one vertebrate OR suggests
conservation of at least part of the mechanism regulating trafficking.
The absence of sequence similarity among the receptor proteins we
tested argues that the conserved functions we identified depend on
protein signal features more complex than the primary structure. This
is consistent with the view that a small number of accessory proteins
are responsible for regulating the trafficking of the entire OR repertoire.
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ACKNOWLEDGEMENTS |
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We thank those who shared materials and clones (detailed under "Experimental Procedures"). We thank Dr. P. Lakhlani for technical assistance. We thank Dr. D. Hunter, Dr. A. Y. Gimelbrant, and participants of the NIH Image discussion group for useful discussions, and Dr. S. Carlson for the loan of an incubator.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Award DC02736 (to T. M.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Thomas Skinner
Associate Professor of Physiology, Dept. of Physiology, University of
Kentucky, 800 Rose St., Lexington, KY 40536-0298. Tel.: 859-323-1083; Fax: 859-323-1070; E-mail: mcclint@pop.uky.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M005433200
2 T. McClintock, C. Josefowicz, S. C. Bose, and A. Gimelbrant, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: OR(s), olfactory receptor(s); CHO, Chinese hamster ovary; ER, endoplasmic reticulum; GFP, green fluorescent protein; GPCR(s), G protein-coupled receptor(s); mAbs, monoclonal antibodies; ORN(s), olfactory receptor neuron(s); TfR, transferrin receptor; HA, hemagglutinin; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
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