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INTRODUCTION |
Certain ligands can assume distinct functions in different
tissues. For example, platelet-activating factor
(PAF)1 is involved in
embryogenesis as well as in modulation of a variety of functions of the
immune and central nervous systems (1-3). With respect to PAF, for
which the only known receptor is a member of the G-protein-coupled
receptor (GPCR) family, the diversity of responses generated is likely
a result of the receptor coupling to different signaling pathways in
the target cells (3, 4). On the other hand, for other ligands such as
adrenaline and dopamine, the cell can also determine the specificity of
its response by the differential expression of receptor subtypes with
distinct characteristics of binding, coupling, and desensitization (5). Subtypes of a given receptor can be derived from distinct genes or
generated by alternative splicing. Divergence between receptor isoforms, as for the prostaglandin (EP) and the MCP-1 (CCR2) receptors, is most often limited to the carboxyl-terminal cytoplasmic tail, a
region that is potentially involved in G-protein coupling,
internalization, and down-regulation of the receptors (6, 7).
Alternative splicing can also lead to the formation of nonfunctional
receptors or receptors with certain functions that are greatly modified
(8, 9). Individually, these receptors are not involved in signaling,
but some of them can show dominant-negative properties when
co-expressed with a functional subtype. It has recently been
demonstrated that the expression of a truncated isoform of the human
gonadotropin-releasing hormone receptor could affect the extent of
agonist-specific cellular response by inhibiting the cell surface
expression of the functional isoform (10). A similar effect, that of
confining the WT receptor intracellularly, was also reported for a
deletion mutant (
32) of the CCR5 receptor, conferring a certain
level of resistance to human immunodeficiency virus infection and
delayed onset of acquired immune deficiency syndrome to heterozygous
individuals (11). All the evidence of the dominant-negative effect of
these truncated receptors points toward their capacity to form
heterocomplexes with their respective WT functional receptors (8, 10,
11). The ability of an EP1 receptor isoform to inhibit signaling by EP1
as well as EP4 receptors suggests an association between the different
receptor subtypes (8). The formation of heterocomplexes between
different receptor subtypes may be an efficient mechanism to control
the cellular response. For example, using immunoprecipitation and immunohistochemical studies, hetero-oligomerization between isoforms of
the rat D3 dopaminergic receptor has been shown to occur in vivo (12).
Multimer formation has been shown or suggested for several other
members of the GPCR family (13-20), including the human PAF receptor
(hPAFR) (21, 22) and
2-adrenergic receptor (23), but he role of
homo-oligomerization is still ill-defined. However, a study on the
opioid receptor suggested that the transition between the oligomeric
and monomeric forms of the receptor could be an important step in the
internalization process (24). In addition, Herbert et al.
(23) have suggested that oligomers constitute the active form of the
2-adrenergic receptor. They also demonstrated that exposure to an
inverse agonist promoted the monomeric form. Thus, GPCRs could show
similarities with the growth factor and cytokine receptors, whose
activation depends on their transition from monomers to oligomers (25,
26).
The GPCRs are involved in a multitude of fundamental processes in
higher eukaryotes such as development, homeostasis, and the transfer
and integration of information (27). Several hereditary diseases have
been linked to mutations within the coding regions of GPCRs (28-36).
The dominance phenomenon observed with some receptor isoforms and the
evidence of a connection between oligomerization and activation raise
the possibility of the existence of genetic diseases caused by GPCRs
with dominant properties. In an attempt to verify whether some
mutations could generate a dominant phenotype, two members of the GPCR
family, the hPAFR and a splice variant (hCCR2b) of the human MCP-1
receptor, were co-expressed with mutant receptors incapable of
undergoing an agonist-stimulated response or altered in other
functions. Cell surface expression, ligand binding, and signaling
characteristics were then evaluated. For the hPAFR, the WT receptor was
co-transfected with the substitution mutants N285I (37), D289A (37),
Y293A (38), and D63N (39) and the deletion mutant receptor K298stop
(38). The N285I (37) and K298stop (38) mutant receptors do not show any
specific binding of PAF (a phospholipid) or the antagonist WEB2086 (a
benzodiazepine). Substitutions of a highly conserved aspartate in the
second transmembrane domain (D63N) or in the conserved motif
D/NPXXY of the seventh transmembrane domain (D289A) lead to
a receptor with reduced internalization capacity but with an increased
affinity for PAF (37-39). However, the substitution Y293A, in the same
motif, did not significantly affect these properties (38). We also
co-expressed the WT hCCR2b and a carboxyl terminus deletion mutant
(K311stop), which did not show any detectable 125I-MCP-1
binding capacity, to evaluate whether our observations for the hPAFR
could be extended to another member of the GPCR family.
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MATERIALS AND METHODS |
Construction of c-myc-tagged WT and Deletion Mutant
Receptors--
The WT hPAFR cDNA was a generous gift from Dr.
Richard Ye (The Scripps Research Institute, La Jolla, CA). The mutant
hPAFR-encoding cDNAs were constructed by polymerase chain reaction
(PCR) and characterized as described previously (37-39). The WT hPAFR
was subcloned into the pJ3M (tagged with c-myc) and pJ3H (tagged with hemagglutinin) expression vectors (kindly provided by Dr. J. Chernoff, Fox Chase Cancer Center, Philadelphia, PA). hCCR2b WT
encoding cDNA was amplified from MonoMac1 total RNA by
reverse transcription-PCR using oligonucleotides CKR2-FWD
(5'-CGGGATCCCTGTCCACATCTCGTTCTTGGTTTATCAGA-3') and CKR2b-RVS
(5'-GGGGTACCTTATAAACCAGCCGAGACTTCCTGCTCCCC-3'). The PCR product was
digested with BamHI-Acc65I and subcloned into the
pJ3M expression vector. The carboxyl-terminal deletion mutant K311stop
was constructed by PCR using the CCR2-FWD primer and oligonucleotide
5'-CGGGGTACCTCGAGTCGCGACTACTCCCCRACRAACGCGTAGATGATGGG-3', and the
PCR product was subcloned into BamHI-Acc65I of
pJ3M. All constructions were sequenced (University of Calgary, Alberta, Canada).
Cell Culture and Transfections--
COS-7 and CHO cells were
grown in Dulbecco's modified Eagle's medium high glucose and
Dulbecco's modified Eagle's medium F12 (Life Technologies, Inc.),
respectively, supplemented with 10% fetal bovine serum. Cells were
plated in 30-mm dishes (2.0 × 105 COS-7 cells/dish or
3.0 × 105 CHO cells/dish) and transiently transfected
with the constructions encoding WT and mutant receptors or dynamin Ia
K44A (a kind gift from M. G. Caron, Duke University, Durham, NC)
using 4 µl of LipofectAMINE (Life Technologies, Inc.) and the
indicated quantity of cDNA for each clone per dish. Cells were
harvested 48 h after transfection. For each experiment, the
transfected cDNA quantities were adjusted with the expression
vector pCDNA3 (Invitrogen).
Radioligand Binding Assay--
Competition binding curves were
done on CHO cells co-expressing the WT and mutant receptors. Cells
expressing hPAFR were harvested and washed twice in Hepes-Tyrode's
buffer containing 0.1% (w/v) bovine serum albumin. Preparation of
membrane fractions and 3H-WEB2086 (DuPont New England
Nuclear) binding on membranes or intact cells were carried out as
described previously (39). For 125I-MCP-1 (Amersham Corp.)
binding, cells expressing hCCR2b were harvested and washed once with
phosphate-buffered saline. Binding reactions were carried out on 5 × 105 cells in a total volume of 100 µl of Hepes binding
buffer (1 mM CaCl2, 5 mM
MgCl2, 0.5% bovine serum albumin, and 50 mM
Hepes, pH 7.2) containing 100 pM 125I-MCP-1 and
increasing concentrations of nonradioactive MCP-1 for 90 min at
25 °C. The free radioactivity was separated from the cells by
centrifugation through Ficoll-Hypaque. The radioactivity contained in
the cell pellet was counted on a LKB
counter.
Inositol Phosphate Determination--
COS-7 cells were
co-transfected with WT and mutant receptor cDNA and, in the case of
hCCR2b, also with the G-protein subunit G
14 (a generous
gift from Dr. Melvin I. Simon (California Institute of Technology,
Pasadena, CA)). Cells were labeled the following day for 18-24 h with
myo-[3H]inositol (Amersham Corp.) at 3 µCi/ml in Dulbecco's modified Eagle's medium (high glucose, without
inositol; Life Technologies, Inc.). After labeling, cells were washed
and preincubated for 5 min in phosphate-buffered saline at 37 °C.
The phosphate-buffered saline was removed, and cells were incubated in
Dulbecco's modified Eagle's medium containing 0.1% bovine serum
albumin and 20 mM LiCl for 5 min (hPAFR) or in Dulbecco's
modified Eagle's medium containing only 10 mM LiCl for 10 min (hCCR2b). Cells were then stimulated for 1 min with the indicated
concentrations of PAF or for 30 min with MCP-1. The reactions were
terminated by the addition of perchloric acid. Inositol phosphates were
extracted and separated on Dowex AG1-X8 (Bio-Rad) columns. Total
labeled inositol phosphates were then counted by liquid scintillation.
Flow Cytometry Studies--
CHO and COS-7 cells co-transfected
with c-myc-tagged hPAFR WT receptor and N285I or D63N constructions
were harvested between 48 and 72 h after transfection, and
2.5 × 105 cells were subjected to flow cytometry
analysis. For receptor expression studies (surface staining), labeling
with anti-c-myc antibody (9E10 hybridoma; American Type Culture
Collection) was performed as described previously (37). For
internalization inhibitor studies, at 48 h after transfection,
cells were pretreated with the internalization inhibitors concanavalin
A (0.25 mg/ml, 20 min), sucrose (0.45 M, 20 min), and
NH4Cl (10 mM, 10 min) or not pretreated. Cell
surface receptor expression was evaluated with an anti-c-myc antibody
30 min after pretreatment. For phophotyrosine staining (40), cells were
fixed with paraformaldehyde (0.5% in phosphate-buffered saline) for 30 min at 4 °C, permeabilized with 0.1% saponin, and then labeled
overnight with mouse monoclonal anti-phophotyrosine antibody (pTyr-01;
a generous gift of Drs. P. Angelisova and V. Horejsi, Prague, Czech
Republic). The secondary antibody used in these studies was a
fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Bio/Can
Scientific, Mississauga, Ontario, Canada), and labeling was as
performed as described previously (37). All measures were performed on
a FACScan flow cytometer (Becton Dickinson).
Western Blot--
COS-7 cells co-transfected with WT and
pCDNA3 (WT) or WT and D63N (WT+D63N) in a 1:3 ratio were cultivated
as described above and lysed at 4 °C for 30 min in a buffer
containing 50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40,
0.25% sodium deoxycholate, 150 mM NaCl, 1 mM
EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 mM
Na3V04, 1 mM
Na2P207, 1 mM NaF, and
10 mg/ml of the protease inhibitors aprotinin and leupeptin. After the
removal of debris by centrifugation, whole cell extracts were separated
by SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride membranes. Proteins phosphorylated on
tyrosine were revealed with anti-pTyr-01 and the ECL detection system (Amersham).
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RESULTS |
Receptor Distribution and Affinity for WEB2086 (Antagonist) in
Cells Co-expressing WT and Mutant Receptors--
Ligand binding was
evaluated after co-transfection of WT hPAFR cDNA and different
mutant constructs in CHO cells. 3H-WEB2086 binding
characteristics were determined by competition with WEB2086. Affinity
was not affected by co-expression of the D63N, D289A, or Y293A mutant
receptors when compared with cells expressing only the WT (Fig.
1A; Table
I). In contrast, the N285I mutant
receptor was efficient at preventing WEB2086 binding to either whole
cells or membrane preparations (Fig. 1B). In Fig. 1B, results obtained for 3H-WEB2086 binding and
cell surface expression (assessed by flow cytometry) were relative to
those of cells expressing only the WT receptor (defined as 100%).
Co-expression of the N285I mutant with the WT receptor also caused a
significant decrease of cell surface expression of both receptors (Fig.
1B). Although WEB2086 binding was almost completely
inhibited (3 ± 1%, residual binding) at a 1:1 ratio, the loss of
surface expression of these receptors was only partial (23 ± 8%,
residual expression). These results suggested the existence of
receptors on the cell surface that are incapable of binding WEB2086. In
addition, it is of interest to note that the cell surface expression of
the co-expressed receptors was significantly lower (23 ± 8%)
than that of the WT (defined as 100%) or of the N285I (65 ± 15%) mutant receptor expressed alone (Fig. 1B). Confocal
microscopy showed that the hemagglutinin-tagged WT and the c-myc-tagged
N285I mutant receptors were efficiently expressed on the cell surface.
The N285I mutant receptors, however, were not as evenly distributed and
seemed to aggregate. When co-expressed, the distribution of these
receptors changed to being localized intracellularly, for the most part
(results not illustrated). The decreased cell surface expression was
not due to an increased internalization rate of the co-transfected
receptors because inhibitors of internalization failed to modify the
distribution of WT+N285I complexes. No accumulation of receptors on the
cell surface was observed by FACScan analysis (WT+N285I untreated,
29 ± 12%; NH4Cl, 31 ± 1.8%; sucrose, 31 ± 8%; dynamin K44A, 15 ± 8%; the percentages are in relation
to WT expressed alone after undergoing the same treatment and set at
100%), although these inhibitors have been shown to efficiently block
the internalization of hPAFR (38).

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Fig. 1.
Cell surface expression and
3H-WEB2086 binding of hPAFR on CHO cells co-expressing WT
and mutant receptors. A, competition binding isotherms of
3H-WEB2086 by WEB2086. Binding was measured on cells that
were transiently co-transfected with the WT (1 µg) and either the
D63N, D289A, or Y293A mutant receptor-encoding cDNA (1 µg). The
results are the means of five independent experiments done in
triplicate. B, CHO cells were transiently co-transfected
with different ratios (1:0, 0:1, and 1:1 to 1:2 5) of
c-myc-tagged WT (1 µg) and c-myc-N285I mutant (1 µg to
2 5 µg) receptor-encoding cDNA. Cell surface
expression of hPAFR was then evaluated by FACScan analysis using an
anti-c-myc antibody, and specific 3H-WEB2086 binding was
carried out on intact cells or membrane preparations. Results are
relative to those of the WT (ratio, 1:0) and are expressed as a
percentage of fluorescent cells and a percentage of specific
3H-WEB2086 binding (in cpm %) and are the means ± S.E. of three independent experiments done in triplicate.
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Table I
Ligand binding parameters of the co-expressed WT and mutant hPAFRs
Binding parameters were determined as described under "Materials and
Methods" on transiently transfected COS-7 cells. Receptor densities
(Bmax) are indicated in receptor/cell, and
dissociation constants (Ki) are indicated in nM. The
results are the means ± S.E. of three independent determinations.
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The influence of the K298stop mutant was comparable to N285I, and the
extent of inhibition of expression and 3H-WEB2086 binding
was dependent on the amount of co-transfected mutant cDNA, even to
membrane preparations (data not shown). The similar effects of these
two mutants suggest a comparable conformational change induced by the
N285I substitution and the deletion of the last 44 amino acids of the
carboxyl terminus (K298stop) in the seventh transmembrane domain.
Receptor Affinity for PAF in Cells Co-expressing WT and Mutant
Receptors--
We next characterized the binding and cellular response
to PAF in cells co-expressing the same combination of constructs, as
illustrated in Fig. 3. Affinity for the agonist was assessed by
competition of 3H-WEB2086 binding by PAF (Fig.
2A). Transfection conditions
were such that high receptor expression levels were attained, allowing for the measurement of binding properties independent of G-protein coupling (41). Binding isotherms were all uniphasic and were indicative
of only one detectable class of binding sites, even in cells
co-expressing receptors with different affinities (Fig. 2A).
For instance, the D63N mutant has an ~6.7-fold higher affinity for
PAF than the WT receptor when expressed individually (39), but after
co-expression with the WT receptor, the apparent affinity was only
~1.9-fold higher than that of the WT alone (Table I). Similar results
were obtained with D289A, which displays a ~2.8-fold higher affinity
than the WT when expressed alone (37) and an apparent increase in
affinity of 1.3-fold over the WT when these two receptors are
co-expressed. These results suggest that a large proportion of the cell
surface receptors, which bind WEB2086 and PAF, could be in an
associated form of mutant/WT receptors. Co-expression of the WT and
Y293A receptors resulted in an apparent affinity very similar to the
one displayed by the WT alone. Evaluation of PAF affinity after
co-expression of WT and N285I or K298stop receptors could not be done
using 3H-WEB2086 because the level of specific binding was
negligible. High levels of nonspecific 3H-PAF binding on
membrane preparations also prevented an accurate evaluation of PAF
binding (data not shown).

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Fig. 2.
PAF binding and response in IP accumulation
of cells co-expressing WT and mutant hPAFRs. A, competition
binding isotherms of 3H-WEB2086 by PAF. Binding was
measured on CHO cells that were transiently co-transfected with the WT
(1 µg) and either the D63N, D289A, or Y293A mutant receptor-encoding
cDNA (1 µg). B and C, IP accumulation in
response to graded concentrations of PAF. Total IP was measured after a
1-min stimulation with the indicated PAF concentrations of COS-7 cells
co-transfected with a 1:1 (B) and a 1:3 (C) ratio
of the WT (300 ng in B and 150 ng in C) and
either D63N, N285I, D289A, or Y293A mutant receptor-encoding cDNA
or the pCDNA3 vector (150 ng in B and 450 ng in
C). The results are representative of five independent
experiments, each done in triplicate. D, baseline IP
accumulations of the experiments in B and C are
presented, in the respective ratio, as relative values of WT:pCDNA
and are the means ± S.E. of five independent experiments done in
triplicate.
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The numbers of cell surface receptors for the co-expressed WT+D289A and
WT+Y293A were equivalent to the WT alone (Table I). When the WT was
co-expressed with D63N in a 1:1 proportion, the cell surface receptor
expression was slightly reduced (~25%) (Table I), but no further
reduction was seen at a 1:3 proportion of WT:D63N in FACScan analysis
(data not shown).
The Effect of WT and Mutant hPAFR Co-expression on PAF-induced
Response--
None of the mutant receptors used in this study is
capable of inducing IP production in response to PAF stimulation when
expressed individually (37-39). We thus proceeded to look at the
effect of co-expressing these mutants with the WT receptor on the
PAF-induced IP response. COS-7 cells were co-transfected with the WT
and either an equal (Fig. 2B) or three times greater amount
of mutant receptor cDNA (Fig. 2C). IP accumulation was
evaluated after a 1-min stimulation with PAF. Compared with COS-7 cells
expressing only the WT receptor, the co-transfection of the Y293A or
D289A mutants in a 1:1 proportion with the WT did not affect the
dose-dependent response to PAF. EC50 (median
effective concentration) values could not be calculated because a
plateau of response was not reached, even at the highest concentrations
of PAF. Concentrations higher than 10
6 M PAF
have a detergent-like effect on the membrane and thus cannot be used.
Co-transfection of N285I and WT receptor cDNA generated a slightly
lower maximal IP response than that seen with the WT alone (Fig.
2B). A decrease (approximately 3-fold) was also observed when these two receptors were co-expressed in a 1:3 proportion of
WT:N285I (Fig. 2C). It is of interest to note that the
response levels (Fig. 2, B and C), cell surface
expression, and 3H-WEB2086 binding (Fig. 1B)
were all inversely proportional to the quantity of transfected N285I
mutant cDNA. However, PAF-induced IP accumulation was inhibited by
WEB2086 in the same manner in cells expressing the WT alone
(IC50 = 4.1 ± 0.5 µM) or in the presence of N285I (IC50 = 4.5 ± 0.9 µM)
mutant receptor (data not shown).
Co-expression of the D63N mutant with the WT receptor seemed to impart
a constitutively active phenotype (Fig. 2D; Fig.
3, A and B). When
co-transfected in a 1:3 proportion of WT:D63N, the baseline IP levels
(308 ± 75%) were ~3-fold higher than those in cells expressing
only the WT receptor (Fig. 2D). This increase represented
about 85% of the level attained after stimulation of WT-transfected
cells with 1 µM PAF. In these transfected cells (1:3
proportion of WT:D63N), there was no augmentation of IP levels after a
stimulation, even with 1 µM PAF (Fig. 2C).
However, when the proportion of WT:mutant was 1:1, there was no
inhibition of PAF-induced responsiveness; on the contrary, enhanced IP
production could be observed (Fig. 2B), even with a baseline
level (187 ± 26%) ~2-fold higher than the WT (defined as
100%) (Fig. 2D).

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Fig. 3.
Basal tyrosine kinase activity in cells
co-expressing the WT and D63N mutant hPAFRs and the impact on IP
accumulation. A, comparative analysis of phosphotyrosine
levels in COS-7 cells co-expressing 1:3 WT:D63N or the WT receptor (1:3
WT:pCDNA3). After transfection, cells were cultivated for 3 days
with 1% fetal bovine serum and analyzed with an anti-phosphotyrosine
antibody as described under "Materials and Methods." The results
are representative of seven independent experiments. B,
Western blot. The cells were transfected and cultivated as described
above. They were then lysed, separated on SDS-polyacrylamide gel
electrophoresis, and immunoblotted with an anti-phosphotyrosine
antibody as described under "Materials and Methods." Results are
representative of four independent experiments. C,
evaluation of the effect of tyrosine phosphorylation on basal IP
accumulation in COS-7 cells co-expressing the WT and D63N mutant
receptors (1:3 WT:D63N). Cells were incubated at 37 °C with 250 µM genistein (a tyrosine kinase inhibitor) or
Me2SO (DMSO; diluent; defined as 100%) for 20 min. IP quantification was performed as described under "Materials
and Methods." Values are relative to those obtained with
Me2SO and are the means ± S.E. of three independent
experiments done in duplicate.
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The co-transfection of WT+D63N also led to a constitutive increase of
phosphotyrosine content as compared with WT-transfected cells (Fig.
3A). Western blotting indicated increased phosphorylation of
bands at approximately 33, 40, and 43 kDa (Fig. 3B). There was also increased phosphotyrosine content in the region of higher molecular masses (89-205 kDa). When the blots were significantly underexposed, a major phosphorylated band was found at approximately 190 kDa in the WT+D63N lysate. The increase in cellular phosphotyrosine levels could also directly or indirectly participate in the basal IP
production because genistein (a tyrosine kinase inhibitor) significantly decreased IP accumulation in the cells co-expressing the
WT+D63N (1:3) complex (Fig. 3C).
Not all inactive mutant receptors were capable of affecting the
apparent phenotype of cells expressing the WT hPAFR. The uncoupled mutant receptor Y293A had no effect on WT distribution, binding characteristics, or response. Other mutant receptors (C90A, C90S, C173A, and C173S) with altered cell surface expression and binding characteristics (41) did not modulate the WT receptor phenotype (data
not shown).
The Effect of the Co-transfection of hCCR2b and K311stop on IP
Production and MCP-1 Binding--
To verify whether our observations
with the hPAFR could apply to another GPCR, we performed experiments
with the hCCR2b receptor. A modification in the binding characteristics
and response was seen for this receptor when co-transfected with the
deletion mutant receptor K311stop (Fig.
4A). Cells transfected with
this mutant receptor cDNA (and G
14) did not show any
response to MCP-1 or specific 125I-MCP-1 binding, even to
membrane preparations. However, by co-expressing this mutant with the
WT receptor, a shift in affinity from a Kd of
320 ± 20 to 610 ± 35 pM was observed (Fig.
4A). It also had a significant inhibitory effect on the
response to MCP-1, as measured by IP production (Fig. 4B).
This inhibition may be due to the decrease in affinity for the agonist
induced by the co-expression of the mutant and WT receptors.

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Fig. 4.
Characterization of cells co-expressing WT
and deletion mutant cDNAs of hCCR2b. A, competition
binding isotherms of 125I-MCP-1 by MCP-1.
125I-MCP-1 binding was measured as indicated under
"Materials and Methods" on CHO cells that were transiently
co-transfected with the WT (1 µg) and K311stop mutant
receptor-encoding cDNAs (1 µg). B, inositol phosphate
accumulation in response to graded concentrations of MCP-1 (0.1, 1, 5, 20, and 80 nM). Total IPs were measured after a 30-min
stimulation with the indicated MCP-1 concentrations of COS-7 cells
co-transfected with 150 ng of the G-protein subunit G 14
cDNA and either hCCR2b WT or K311stop receptor cDNA, alone (150 ng) or in combination (1:1 ratio (150 ng WT:150 ng K311stop) or 1:3
ratio (150 ng WT:450 ng K311stop)). IP quantification was as described
under "Materials and Methods." The results are representative of
three independent experiments done in duplicate.
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DISCUSSION |
This study demonstrates the possibility of using the co-expression
of certain point substitution or deletion mutants to modulate the
response and expression of the WT GPCR. The resultant response, after
co-transfection, is not necessarily the sum of the individual characteristics of each receptor because certain mutants induced a
dominant-negative effect such as the inhibition of surface expression (N285I and K298stop) or generated a new phenotype such as constitutive activation (D63N). Selective modulation of binding characteristics for
the agonist (D289A, D63N, and K311stop) and decreased cell surface
expression (N285I and K298stop) have also been observed.
It is of interest to note that the cell surface expression of the
co-expressed receptors was significantly lower (23 ± 8%) than
that of the WT (defined as 100%) or of the N285I (65 ± 15%) mutant receptor expressed alone. In addition, confocal microscopy indicated that co-expression of the WT and mutant receptors leads to
receptor redistribution. This effect was not due to an increase in
constitutive internalization because inhibitors of hPAFR
internalization did not alter the cell surface expression of the
mutant:WT receptors. Rather, the receptor redistribution may be the
result of faulty membrane targeting. Interestingly, at a 1:1 ratio of
WT:N285I, there was only a partial loss of surface receptor protein
expression (23 ± 8%, residual expression), but WEB2086 binding
was almost completely inhibited (3 ± 1%, residual binding).
These results suggest the existence of cell surface receptors whose
capacity of binding WEB2086 had been altered by the co-expression of
the N285I mutant. This particular population does not seem to
participate in PAF-induced IP production, because this response is
sensitive to WEB2086 inhibition in the same manner as in cells
expressing only the WT receptor (results not shown). The lower
PAF-induced maximal response observed with these co-transfected cells
could also be attributed to the substantial loss of functional
receptors on the cell surface.
Receptor expression, binding capacity, and IP responses were all
inversely proportional to the quantity of mutant cDNA (N285I and
K311stop) co-transfected with the WT of hPAFR and CCR2b. These results
indicate an interdependence between the WT:mutant receptor ratio and
the resulting phenotype. This was also shown for the human
gonadotropin-releasing hormone receptor and a splice variant that
inhibits cell surface expression and thus reduces the maximal ligand-dependent response (10). These data could indicate
that GPCRs form complexes that may be composed of two or more receptor molecules and that these complexes define the properties of the receptor. This is supported by: 1) the fact that binding curves were
all uniphasic for co-expressed receptors with different affinities, 2)
a shift in affinity for MCP-1 was observed with the co-expression of
the hCCR2 and K311stop mutant, and 3) the efficiency of the N285I
mutant in inhibiting WEB2086 binding to hPAFR. Our hypothesis is also
supported by the results of other groups, which show not only dimeric
receptors but also complexes of much higher molecular weight on
SDS-polyacrylamide gel electrophoresis for the hPAFR (20, 22) and other
members of the GPCRs (12-14, 42). One must also consider that these
results could be the result of a greater predisposition toward the
formation of heterocomplexes than homocomplexes. This type of
interaction has been reported for the dopamine D3 receptor
and a nonfunctional splice variant (12). Our data also showed that
different ratios of D63N:WT modulate the phenotype of the complex. A
1:3 WT:mutant ratio drives the complex toward a more constitutively
active conformation than a 1:1 ratio and abolishes responsiveness to
PAF.
The concept of the formation of a hetero-oligomeric complex was first
introduced by the demonstration of the functional rescue of receptor
properties such as agonist binding, signaling, and cell surface
expression after co-expression of two distinct nonfunctional mutants or
chimeric receptors (15-18). Recently, it has been shown that
hetero-complexes formed between the CCR5 receptor and a deletion mutant, CCR5
32, retained the WT in the cellular compartment and thus
reduced cell surface expression of the functional receptor (11).
Similar results have also been obtained with a splice variant of the
human gonadotropin-releasing hormone receptor (10). The fact that some
mutant receptors, which are themselves only expressed intracellularly,
inhibit the expression of the WT on the cell surface suggests that the
oligomerization takes place very early after protein synthesis,
possibly in the endoplasmic reticulum, where the mutants seem trapped.
Adequate folding seems to be necessary for the association of the
receptors because mutants of the hPAFR, which are thought to lack
disulfide bonds (43), did not influence the WT phenotype when
co-expressed in CHO
cells.2
Our results expand these observations by demonstrating that
co-expression of receptors altered in certain functions could also be
used to modify specific characteristics of WT receptors such as the
affinity for ligand, the level of basal activity, and the extent of
cell surface expression, presumably by the occurrence of
hetero-oligomerization. This raises the possibility that naturally occurring splice variants, such as CCR2a and CCR2b, with distinct coupling, distribution, and desensitization characteristics could associate in a hetero-oligomeric complex and permit a subtle control of
the ligand-induced response by modulating the ratio of the two
receptors. Indeed, splice variants of the gonadotropin-releasing hormone receptor and the EP1 subtype of prostaglandin E2
receptor attenuate the action of the specific ligands (8, 10). This is
also the case for the carboxyl-terminal deletion mutant of CCR2b whose
presence modulates the ligand affinity and responsiveness to MCP-1.
This study showed that a simple substitution of an amino acid of a GPCR
could lead to a dominant-negative phenotype. We also demonstrate that
co-expression of an uncoupled mutant (D63N) with the WT receptor could
lead to an apparent constitutively active phenotype highly dependent on
receptor:mutant ratio. Interestingly, even though D63N does not seem
capable of G-protein coupling, it demonstrates a higher affinity for
its ligand, a characteristic that it shares with constitutively active
mutants. The co-expression of nonfunctional mutants, chimeric
receptors, and a recent study by Hebert et al. (44) have
shown that rescue of receptor function is achievable through
oligomerization (15-18, 44). It is therefore conceivable that
oligomerization between the WT and D63N receptors may result in a
complex that possesses both the capacity for G-protein coupling and
certain characteristics of constitutively active receptors. The fact
that the 1:3 WT:D63N was unresponsive to PAF could be due to the fact
that all receptor complexes are already in an active conformation. This
constitutive activation would also result in a certain quantity of
receptors being desensitized and internalized, which may also explain
the fact that the levels of basal IP accumulation (1:3 WT:D63N) are
lower than if one stimulates a population of WT receptors, of which
none are desensitized. The combination of WT and D63N also resulted in
an increase in tyrosine phosphoproteins as compared with cells
transfected only with WT. Several tyrosine kinases have been shown to
be stimulated by PAF in specific cell types (45). We are now pursuing
the identification of the proteins that were differentially
phosphorylated in the presence of D63N to compare the patterns with
ligand-induced phosphorylation.
Several diseases linked to altered functions of GPCRs have been
described (28-36), and many of these are attributed to an apparent constitutive activity (28, 33-36). A better understanding of dominant
phenotypes in this receptor family could ultimately lead to venues of
genetic therapy.