From the Institut National de la Santé et de la Recherche
Médicale, INSERM U 410, Neuroendocrinologie et Biologie
Cellulaire Digestives, Faculté de Médecine Xavier Bichat,
B. P. 416, 75870 Paris Cedex 18, France
The human vasoactive intestinal peptide (VIP) 1 receptor belongs to the new class II subfamily of G protein-coupled
receptors. Specific change by mutagenesis of a strictly conserved
histidine into arginine at position 178 of the human VIP1 receptor
resulted in its constitutive activation with respect to cAMP
production. Transfection of the H178R mutant into COS cells resulted in
a 3.5-fold increase in the cAMP level as compared with cells
transfected with the wild type receptor or the vector alone. This
increase was proportional to the amount of transfected cDNA. The
H178R mutant exhibited an otherwise normal cAMP response to VIP as well as a dissociation constant similar to that of the wild type receptor. Other mutants at position 178 such as H178K, H178A, and H178D were not
constitutively activated. They were otherwise expressed at the cell
surface of transfected nonpermeabilized cells. Double mutants were then
constructed in which the H178R mutation was associated with a point
mutation in the the N-terminal extracellular domain that totally
abolished VIP binding or VIP-stimulated cAMP production,
i.e. E36A or D68A. The corresponding double mutants H178R/E36A and H178R/D68A were no longer constitutively activated. A
control double mutant (H178R/D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP was still constitutively activated. Our findings demonstrate that constitutive activation of the
VIP1 receptor by mutation of His178 into R requires the
functional integrity of the N-terminal extracellular VIP binding
domain. They might provide interesting generalities about the
activation process of G protein-coupled receptors.
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INTRODUCTION |
G protein-coupled receptors with seven transmembrane domains,
which constitute a large multigene family of eucaryotic proteins, interact with alkaloids, biogenic amines, peptides, glycoprotein hormones, light, and odorants (1). During the past few years, a
subfamily of the superfamily of G protein-coupled receptors has emerged
that shares the seven-membrane-spanning domain topography but has a low
overall amino acid sequence homology (<20%) with other members of the
superfamily (2, 3). This subfamily, now referred to as the class II G
protein-coupled receptor family, comprises receptors for a family of
structurally related peptides that includes vasoactive intestinal
peptide (VIP),1 pituitary
adenylate cyclase-activating polypeptide, glucagon, secretin,
glucagon-like peptide 1, gastric inhibitory polypeptide, and growth
hormone-releasing peptide and more unexpectedly also comprises
receptors for parathyroid hormone (PTH) and calcitonin (2-4). Recent
studies have extended this subfamily (3) with the discovery of subtypes
of the above mentioned receptors as well as two new members having an
extraordinary long N-terminal domain: the putative EGF
module-containing, mucin-like hormone receptor EMR1 (5) and the
leukocyte activation antigen CD97 (6).
Class II G protein-coupled receptors for peptides have homologies
ranging between 30 and 50% and among several common structural properties have a large N-terminal extracellular domain (>120 amino
acid residues) that contains highly conserved amino acids, including
numerous cysteine residues and several potential N-linked glycosylation sites (2, 3). Taking the human VIP1 receptor (7), which
activates adenylyl cyclase via stimulatory Gs proteins (8), as a
prototype of class II G protein-coupled receptors, we recently provided
evidence for an important role of the N-terminal domain for ligand
binding with several crucial residues (9) probably positioned in a
tertiary structure maintained by multiple disulfide bonds (10). We also
demonstrated the mandatory role of two glycosylation sites in this
domain for correct delivery of the receptor to the plasma membrane
(11). Other functional domains for ligand recognition do exist, since
we showed, by constructing receptor chimeras, that a structural
determinant for peptide selectivity was made of three nonadjacent amino
acid residues in the first extracellular loop and third transmembrane
domain (12). The role of extracellular domains in natural ligand
binding has now been documented for several members of class II G
protein-coupled receptors such as rat VIP1 and secretin (13, 14),
pituitary adenylate cyclase-activating polypeptide (15), calcitonin
(16), PTH (17, 18), glucagon (19, 20), glucagon-like peptide 1 (21,
22), or growth hormone-releasing peptide (23) receptors.
Constitutively active mutants of several G protein-coupled receptors
have been characterized experimentally by site-directed mutagenesis
(24) and have been also described as disease-causing in humans (25,
26). A constitutively active mutant of a class II G protein receptor
has been reported for the PTH-PTH-related peptide receptor in
Jansen-type metaphyseal chondrodysplasia affecting a strictly conserved
histidine residue in the first intracellular loop of this class of
receptor (27).
In view of the fact that class II G protein-coupled receptors display
an original structure-function relationship with respect to ligand
recognition and very low sequence homology with other G protein-coupled
receptors (2, 3), we further investigated their constitutive activation
in the human VIP1 receptor whose structure-function relationship has
been previously documented (3). In this paper, we demonstrate that
specific mutation of histidine 178 in the VIP receptor causes its
constitutive activation. Moreover, we take advantage of the fact that
the VIP binding site is located, at least in part, in the N-terminal
extracellular domain to construct double mutants in which the
histidine-to-arginine mutation critical for constitutive activation has
been associated with point mutations in the N-terminal domain that
prevent VIP binding. This new approach allowed us to show that the
agonist-independent constitutive activation of the
histidine-to-arginine mutant appears to require the integrity of the
natural ligand binding site. These data provide new insight into the
constitutive activation of G protein-coupled receptors.
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EXPERIMENTAL PROCEDURES |
Materials--
Enzymes for cloning, sequencing, and
oligonucleotide-directed mutagenesis were obtained from Promega
(Madison, WI) or Life Technologies, Inc. (Cergy-Pontoise, France), and
synthetic oligonucleotides were from Eurogentec (Seraing, Belgium).
[
-35S]dATP (1000 Ci/mmol) and other radioactive
reagents were obtained from Amersham (Buckinghamshire, United Kingdom).
Synthetic porcine VIP was purchased from Neosystem (Strasbourg,
France), and culture medium and horse fetal serum from Life
Technologies. [125I]VIP was prepared and purified as
described (28). The monoclonal anti-Flag antibodies were obtained from
Eastman Kodak Co., and 125I-labeled antimouse IgG whole
antibody from goat was purchased from NEN Life Science Products. All
other chemicals of the highest quality commercially available were
purchased from Sigma (Saint-Quentin Fallavier, France).
Site-directed Mutagenesis--
The 1.4-kilobase EcoRI
fragment containing the entire coding sequence of the human VIP1
receptor (7) was subcloned into the EcoRI site of the
pAlter-1 vector, and single-stranded DNA ((+)-strand) was produced in
Escherichia coli JM109. Full-length VIP receptor mutants
were generated by oligonucleotide-directed mutagenesis as described
(11). Identification of the desired mutations was obtained by direct
double-stranded sequencing of the regions encompassing mutations.
Inserts encoding mutant sequences were subcloned in the eucaryote
expression vector pCDNA3. The wild type and mutant receptors were
all tagged in the N-terminal extracellular domain by inserting the
marker octapeptide DYKDDDDK (Flag) between Ala30 and
Ala31. The Flag sequence was inserted by
oligonucleotide-directed mutagenesis as described above using
5'-GGGCCGGCGGGCGGCCAGGCGGACTACAAGGACGACGATGACAAGGCCAGGCTGCAGGAG-3' oligonucleotide. This site of insertion was selected because it is localized between the end of the putative signal peptide (3, 7) and
Glu36 which has been shown to be the first crucial amino
acid residue for human VIP1 receptor's functional properties (29). It
was verified that insertion of the Flag octapeptide sequence between Ala30 and Ala31 did not modify the dissociation
constant for VIP or the dose response of VIP in stimulating cAMP
production, as compared with the native human VIP1 receptor.
Transfection of Cells--
Wild type and mutant VIP receptors
were transfected into COS-7 cells or CHO cells. Cells were grown in
medium (Dulbecco's modified Eagle's medium for COS-7 or Ham's F-12
for CHO) supplemented with 10% (v/v) heat-inactivated fetal bovine
serum, 100 IU/ml penicillin, 100 mg/ml streptomycin in a humidified
atmosphere of 95% air and 5% CO2 at 37 °C. Cells were
transfected by the electroporation method using an Electropor II
apparatus (Invitrogen). Briefly, 4 × 106 cells were
preincubated in ice for 5 min with 15 µg of salmon sperm DNA used as
carrier and 15 µg of wild type or mutant receptor cDNA constructs
in phosphate-buffered saline. After electroporation (330 V, 500 microfarads for COS-7 cells or 1,000 microfarads for CHO cells,
infinite resistance), cells were put on ice for 5 min and then
transferred into culture medium containing 10% (v/v) heat-inactivated
fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin
before seeding in Petri dishes for binding assay, 12-well trays for the
cAMP assay or 24-well trays for antibody binding experiments, or on
glass slides in 24-well trays for immunofluorescence studies. The
culture medium was changed 16-18 h after transfection, and cells were
used 48 h after transfection.
Ligand Binding Assay--
The functional properties of wild type
and mutant VIP receptors were analyzed by [125I]VIP
binding to transfected cell membranes. Transfected COS-7 cells were
washed twice with cold phosphate-buffered saline. Then they were
harvested with a rubber policeman and centrifuged at 3,000 rpm for 5 min at 4 °C, and the cell pellets were incubated for 30 min on ice
in a hypotonic 5 mM HEPES buffer, pH 7.4. Thereafter, cells
were homogenized as described (28), and the homogenate was centrifuged
at 11,000 rpm for 15 min at 4 °C. The pellet was washed with 20 mM HEPES buffer and stored at
80 °C until use. This
pellet was referred to as the membrane preparation. Membranes (200 µg
of protein/ml) were incubated for 60 min at 30 °C in 20 mM HEPES buffer, pH 7.4, containing 2% (w/v) bovine serum
albumin, 0.1% (w/v) bacitracin, 0.05 nM
125I-VIP in the presence of increasing concentrations of
unlabeled VIP. The reaction was stopped as described (28). Specific
binding was calculated as the difference between the amount of
125I-VIP bound in the absence and the presence of 1 µM unlabeled VIP. Binding data were analyzed using the
LIGAND computer program (30). Protein content in membrane preparations
was evaluated by the procedure of Bradford (31) with bovine serum
albumin as standard.
cAMP Experiments--
Transfected COS-7 cells or CHO cells were
grown in 12-well trays as described above. The culture medium was
discarded, and attached cells were gently rinsed with
phosphate-buffered saline (pH 7). They were then incubated with or
without VIP under continuous agitation in 0.5 ml of phosphate-buffered
saline containing 2% (w/v) bovine serum albumin, 0.1% (w/v)
bacitracin, 0.01 mg/ml aprotinin, and 1 mM
3-isobutyl-1-methylxanthine as described (7). At the end of the
incubation (30 min at room temperature), the medium was removed, and
cells were lysed by 1 M perchloric acid. The cAMP present
in the lysate was measured by radioimmunoassay as described (32). Cell
number was determined in parallel wells, and data are reported as pmol
of cAMP/106 cells.
Confocal Laser Scanning Microscopy--
Transfected cells were
grown on 12-mm glass coverslips for 48 h as described above. After
they were washed with phosphate-buffered saline (PBS), nonpermeabilized
cells were incubated for 60 min at room temperature with the mouse
monoclonal anti-Flag antibodies diluted 1:50 in PBS containing 1%
(w/v) bovine serum albumin. The cells were then washed three times with
PBS and exposed for 60 min to the secondary antibody (FITC-goat
anti-mouse IgG (Fab-specific) at a 1:250 dilution). Cells were then
fixed for 5 min in PBS containing 2% (w/v) paraformaldehyde. The
coverslips were mounted in 50% (v/v) glycerol in PBS, and selected
fields were scanned using a Leica TCS 4D true confocal scanner composed
of a Leica Diaplan inverted microscope equipped with an argon-crypton
ion laser (488 nm) with an output power of 2-50 milliwatts and a VME
bus MC 68020/68881 computer system coupled to an optical disc for image
storage (Leica Lasertchnik GmbH). The emitted light was collected
through a long pass filter on the target of the photo multiplier. Each
sample was treated with a kalman filter to increase the ratio signal versus background. All image generating and processing
operations were carried out using the Leica CLSM software package.
Screen images were taken on Kodak Ektachrome film using a 35-mm
camera.
Assessment of Cell Surface Expression of Mutated
Receptors--
Cell surface expression of mutated receptors was
assessed using the mouse monoclonal anti-Flag antibodies as described
(33) with modifications. Transfected cells grown in 24-well trays (see above) were rinsed twice with 50 mM Tris-HCl (pH 7.7), 100 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 5% heat-inactivated horse serum, and 0.5%
heat-inactivated fetal bovine serum (binding buffer), incubated for
4 h at room temperature with anti-Flag antibodies diluted 1:50 in
binding buffer. Cells were then washed three times with binding buffer
and exposed for 2 h at room temperature to the radiolabeled
(400,000 cpm/well) second antibodies (125I-labeled goat
anti-mouse IgG). Cells were rinsed again four times with binding buffer
and then lysed with 250 µl of 0.5 M NaOH, and the
radioactivity of the lysate was counted. Nonspecific binding was
determined with cells that were incubated only with the
125I-labeled second antibody. Binding of anti-Flag
antibodies to epitope-tagged mutant receptors was given as a percentage
of anti-Flag antibodies binding to epitope-tagged wild type
receptor.
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RESULTS |
Fig. 1 shows a schematic
representation of the wild type human VIP1 receptor, pointing out the
amino acid residues that have been changed by site-directed mutagenesis
in the present study. We first mutated histidine 178, which is highly
conserved in class II G protein-coupled receptors. The
histidine-to-arginine mutant (H178R) of the human VIP1 receptor, which
mimics the situation found in constitutively activated PTH receptors
(27), was transfected into monkey kidney COS-7 cells, and intracellular
cAMP was measured. The basal cAMP level was 3.5-fold higher than the
basal cAMP level measured after transfection of the wild type human
VIP1 receptor (Fig. 2) or the vector
alone (not shown). This latter observation suggests that the wild type
receptor was not constitutively activated. Similar constitutive
activity of the H178R mutant was observed upon transfection of the CHO
cell line derived from Chinese hamster ovary (not shown). Indeed, basal
cAMP level in CHO cells transfected with the H178R mutant was 5 times
higher than that observed for the wild type receptor, i.e.
19.5 ± 5.6 and 3.7 ± 1.4 pmol/106 cells (three
experiments), respectively. Therefore, constitutive activation does not
appear to be dependent on the nature of the cell line expressing the
mutated receptor cDNA. Fig. 3 shows
the cAMP response in COS-7 cells transfected by H178R mutant and wild type receptors upon stimulation by VIP. Maximal cAMP responses were
identical. Half-maximal stimulations above basal level were obtained
for similar concentrations of VIP in cells transfected with the H178R
receptor or the wild type receptor, i.e. 1.5 ± 0.5 × 10
10 M and 0.4 ± 0.1 × 10
10 M, respectively. Scatchard analysis
of VIP binding to COS-7 cell membranes indicated that the H178R
receptor mutant bound VIP with a similar dissociation constant as
compared with the wild type receptor (Table
I). In these experiments, the
concentration of VIP binding sites was higher in cells transfected with
the wild type receptor than in cells transfected with the H178R
receptor mutant (Table I). This observation did not favor the
hypothesis that the higher basal cAMP level in cells transfected with
H178R could be merely related to a higher expression of the receptor as
compared with cells transfected with the wild type receptor inasmuch as
the wild type receptor is not constitutively activated by itself (see
above). However, to document this issue, we transfected COS cells with
increasing concentrations of cDNA encoding the H178R mutant or wild
type receptor. For the receptor mutant, it was observed that basal cAMP
levels increased when increasing amounts of cDNA were transfected
in COS cells (Fig. 4). In sharp contrast,
the basal cAMP level in COS cells transfected with the wild type
receptor was constant regardless of the amount of cDNA transfected
(Fig. 4). This latter observation further argued against constitutive
activity of the wild type human VIP1 receptor itself. For both the
mutated and wild type receptors, we verified that VIP
(10
6 M)-stimulated cAMP levels increased with
the amount of transfected cDNA (Fig. 4). Likewise, the ligand
binding assay showed that the amount of 125I-VIP
specifically bound to transfected cell membranes increased with the
amounts of transfected cDNA for both the mutated and wild type
receptors (not shown).

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Fig. 1.
Putative seven-transmembrane-segment
topography of the human VIP1 receptor showing residues that have been
mutated in this study. This includes histidine 178, glutamate 36, aspartate 68, and aspartate 132.
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Fig. 2.
Basal (A) and VIP-stimulated
(B) cAMP levels in COS cells expressing wild type
(WT) human VIP1 receptor or His178 mutants of
the receptor. Cells were incubated for 30 min at room temperature
without (A) or with 10 6 M VIP
(B). The intracellular cAMP was measured as described under "Experimental Procedures." Data are means ± S.E. of three
experiments.
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Fig. 3.
Dose response of VIP in stimulating cAMP
accumulation in COS cells expressing the wild type human VIP1 receptor
( ) or the H178R receptor mutant ( ). Cells were incubated for
30 min at room temperature in the presence of various concentrations of
VIP as indicated. The cAMP was measured as described under "Experimental Procedures." Data are means ± S.E. of three
experiments.
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Table I
Binding parameters of wild type and mutated human VIP receptors after
transfection of cDNAs into COS-7 cells
Parameters were determined by Scatchard analysis of binding data as
described under "Experimental Procedures." Results are means ± S.E. of at least three experiments.
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Fig. 4.
Basal (A) and VIP-stimulated
(B) cAMP accumulation in COS cells transfected with
increasing concentrations of the plasmid DNA encoding the wild type
human VIP1 receptor ( ) or the H178R receptor mutant ( ).
Cells were incubated for 30 min at room temperature without
(A) or with (B) 10 6 M
VIP. Data are the mean ± S.E. of three experiments.
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Further experiments were carried out to determine whether mutation of
His178 into residues other than arginine also resulted in
constitutive activation of the human VIP1 receptor. We mutated H178
into a neutral residue (H178A), an acidic one (H178D), or a basic one (H178K). Among these mutants, none was constitutively activated after
transfection in COS-7 cells (Fig. 2). Moreover, these mutants were
unable to mediate VIP-stimulated cAMP production (Fig. 2), probably
because they no longer bound VIP (Table I). To determine the pattern of
expression of these inactive mutants in transfected COS-7 cells,
immunofluorescence studies and antibody binding experiments were
performed. No antibodies to native human VIP1 receptors are currently
available. However, the Flag sequence DYKDDDDK could be inserted
between Ala30 and Ala31 in the N-terminal
extracellular domain of the human VIP1 receptor without altering its
phenotype with regard to VIP binding and VIP-stimulated cAMP production
(see "Experimental Procedures"), indicating that insertion of the
Flag epitope had no impact on the receptor's functional properties.
Insertion of this extracellular epitope enabled us to perform
immunofluorescence studies with the wild type and mutated receptors in
nonpermeabilized transfected COS cells and also to assess cell surface
expression of receptors by anti-Flag antibody binding to
nonpermeabilized transfected COS cells. Confocal laser microscopy of
nonpermeabilized COS cells expressing the epitope-tagged wild type
receptor revealed intense fluorescence when incubated with the
anti-Flag antibodies and subsequently with an FITC-labeled antimouse
antibody (Fig. 5). This observation
supported delivery of the receptor protein at the cell surface as
expected for the wild type receptor. Untransfected COS cells that were
incubated with both antibodies or COS cells expressing the
epitope-tagged wild type receptor that were incubated only with the
FITC-labeled antimouse antibodies showed no fluorescence (Fig. 5).
Next, we examined all epitope-tagged mutants including H178R,
H178A, H178D, and H178K mutants. It appeared that COS cells transfected with these mutants also showed intense
fluorescence, indicating that mutants were expressed at the
cell surface. Since immunofluorescence techniques are not quantitative,
we measured cell surface expression of mutated receptors by antibody
binding to cells expressing mutants and wild type receptor (Table
II). It appeared that mutation of
His178 into Arg or Lys caused no alteration in cell surface
expression as compared with that of wild type receptor, whereas
mutations into Asp or Ala caused significant although moderate
reductions in cell surface expression (42 and 54% of wild type
receptor, respectively). Altogether, these data indicated that the
absence of VIP binding (Table I) and VIP-stimulated cAMP production
(Fig. 2) observed for the H178A, H178D, and H178K mutants was not
related to absence of expression at cell surface and was probably due to a drastic loss of intrinsic binding activity, contrasting with the
constitutive activity and high VIP binding affinity of the H178R
mutant.

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Fig. 5.
Confocal laser scanning microscopic detection
after transfection in COS cells of the epitope-tagged wild type
receptor and receptor mutants. Nonpermeabilized cells were
incubated with the mouse monoclonal anti-Flag antibodies, washed,
incubated with antimouse immunoglobulin G conjugated to FITC, and then
fixed as described under "Experimental Procedures." The following
receptor constructs were shown: wild type receptor (C),
H178R mutant (D), H178A mutant (E), H178D mutant
(F), H178K mutant (G), H178R/E36A mutant
(H), H178R/D68A mutant (I), and H178R/D132A
mutant (J). Controls were carried out with untransfected COS
cells (A) and COS cells expressing the epitope-tagged wild
type receptor that were incubated only with the FITC-labeled antimouse
antibody (B).
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Table II
Cell surface expression of mutated human VIP1 receptors after
transfection of cDNAs into COS-7 cells
Nonpermeabilized transfected cells were incubated with anti-Flag
antibodies and then exposed to the radiolabeled second antibodies. Cells were rinsed, and the radioactivity of cell lysates was counted as
described under "Experimental Procedures." Nonspecific binding was
determined with cells that were incubated only with the
125I-labeled second antibody. It represented 0.1% of total
radioactivity added. Binding of anti-Flag antibodies to epitope-tagged
mutant receptors is given as a percentage of anti-Flag antibodies
binding to epitope-tagged wild type receptor (mean ± S.E.). The
number of experiments for each receptor is indicated in parentheses.
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The fact that constitutive activation of the human VIP1 receptor by
mutation of His178 into arginine did not happen with
mutations into alanine, aspartate, and even lysine, and that such
mutations also abolished VIP binding suggests that such constitutive
activation might be dependent on the integrity of the VIP binding site.
To investigate this issue, we constructed double mutants in which the
mutation resulting in the constitutive activation of the receptor
(H178R) was associated with point mutations in the N-terminal
extracellular domain of the receptor, which abolished VIP binding as
shown previously (9, 29). First, we chose a mutation affecting an amino
acid residue highly conserved in the class II G protein-coupled
receptors, i.e. D68A (9). As expected, the H178R/D68A mutant
no longer bound VIP like the single mutant D68A (Table I); nor did it
mediate the stimulation of cAMP production by VIP (Fig.
6). Fig. 6 further shows that this double
mutant is no longer constitutively activated when expressed in COS
cells. It was verified that the epitope-tagged H178R/D68A mutant like
the D68A mutant (not shown) exhibited a cell surface expression similar
to that of the wild type receptor as assessed by confocal microscopy
(Fig. 5) and antibody binding experiments (Table II). Next, we chose an
amino acid residue that was not conserved in the class II G
protein-coupled receptors but the mutation of which abolished VIP
binding i.e. E36A (29). Like the single mutant E36A, the
H178R/E36A mutant no longer bound VIP (Table I) and did not mediate the
stimulation of cAMP production by VIP (Fig. 6). The H178R-E36A double
mutant was not constitutively activated when expressed in COS cells
(Fig. 6). Again, it was verified that the epitope-tagged H178R/E36A
mutant, like the E36A mutant (not shown), exhibited a cell surface
expression similar to that of the wild type receptor as assessed by
confocal microscopy (Fig. 5) and antibody binding experiments (Table
II). As a control, we developed another double mutant in which H178R
was associated with a point mutation (D132A) in the N-terminal
extracellular domain, which was previously shown not to alter VIP
binding (29). As expected, the H178R/D132A mutant, like the single
mutant D132A, bound VIP with a dissociation constant similar to that of
the wild type receptor (Table I). This mutant also mediated
VIP-stimulated cAMP production (Fig. 6) and exhibited cell surface
expression similar to that of wild type receptor in nonpermeabilized
transfected cells as assessed by confocal microscopy (Fig. 5) and
antibody binding experiments (Table II). Most interestingly, the double mutant H178R/D132A did exhibit constitutive activation upon
transfection in COS cells (Fig. 6).

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Fig. 6.
Basal (A) and VIP-stimulated
(B) cAMP levels in COS cells expressing the wild type human
VIP1 receptor or single or double mutants of the receptor as
indicated. Cells were incubated for 30 min at room temperature
without (A) or with 10 6 M VIP
(B). The intracellular cAMP was measured as described under "Experimental Procedures." Data are means ± S.E. of three
experiments.
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DISCUSSION |
The concept of ligand-independent or constitutive activation of G
protein-coupled receptors has emerged following site-directed mutagenesis of the
1-adrenergic receptor (34). It was
subsequently documented by the discovery of naturally occurring
mutations in diseases (25, 26) as well as experimental mutations (24) in various G protein-coupled receptors. Following the discovery of a
constitutively active mutant of the PTH-PTH-related peptide receptor in
Jansen-type metaphyseal chondrodysplasia (27), this paper is the first
to analyze constitutive activation of a member of the emerging class II
family of G protein-coupled receptors, e.g. the human VIP1
receptor.
This work demonstrates that replacement of histidine 178 by arginine in
the human VIP1 receptor results in constitutive activation of the
receptor with respect to cAMP production, which constitutes its
signaling pathway (35). In contrast, the wild type VIP receptor appears
to be truly silent in the absence of VIP. While mutation of histidine
178 into arginine results in constitutive activation of the receptor,
mutations into other residues such as alanine, aspartate, and even
lysine did not confer ligand-independent activation. This suggests that
the replacement by arginine evokes a subtle conformational change that
cannot be mimicked by lysine and is probably not simply related to the
electrical charge of arginine. This situation is quite different from
that described for the
1B-adrenergic receptor, in which
the 19 possible amino acid substitutions at a single site confer
constitutive activation, suggesting that this site may function to
constrain the G protein coupling of the receptor in the inactive form
in the wild type receptor (36). With regard to the human VIP1 receptor,
it may be suggested that the histidine to arginine exchange does not
simply remove some stabilizing conformational constraints as in
1B-adrenergic receptors (36) or
2-adrenergic receptors (37). In this context, it is
worth pointing out that the naturally occurring mutation of the
equivalent histidine in the PTH-PTHrP receptor in Jansen metaphyseal chondrodysplasia was also into arginine (27). The crucial importance of
histidine 178 in the human VIP1 receptor is further suggested by the
fact that substitutions other than arginine resulted in the absence of
VIP binding and of VIP-stimulatable cAMP production, supporting the
idea of an important structural change in these mutants. In contrast,
the histidine to arginine substitution did not change the dissociation
constant of the receptor, nor did it alter the potency of VIP in
stimulating cAMP production. This contrasts again with the
1B-adrenergic receptor, since all mutated receptors that
are constitutively activated demonstrate a higher affinity for the
agonist, a characteristic of the active conformation of G
protein-coupled receptors (36). Similar increased sensitivity to the
agonist was reported for constitutively active
2-adrenergic receptors (38) or muscarinic receptors
(39). However, such an increase in the apparent affinity of
constitutively activated receptors for agonists was not observed for
several other receptors such as the thyrotropin receptor (40) or the
luteinizing hormone receptor (41). A recent report indicates that
constitutive activation of the AT1A angiotensin II receptor
by mutation in the third transmembrane domain does not modify the
Kd and Kact values for
angiotensin (42). This suggests that differences regarding the
mechanism of constitutive activation may exist not only between amine
receptors and the class II family to which VIP receptors belong but
also within the class I family of receptors for amines, peptides, or glycoprotein hormones.
Constitutive activation evoked by replacement of histidine 178 by
arginine in the human VIP1 receptor is much less efficient than
activation induced by the natural agonist VIP in the H178R mutant
receptor or the wild type receptor. Nevertheless, the construction of
double mutants supports a close relationship between the
ligand-dependent activation triggered by VIP and the
ligand-independent one evoked by mutation of histidine 178 into
arginine. Indeed, when the H178R mutation at the junction of the first
intracellular loop and second transmembrane domain was associated with
mutation of aspartate 68 (D68A) in the N-terminal extracellular domain,
which abolishes VIP binding and VIP-stimulated cAMP production (9), the
resulting double mutant was no more constitutively activated and has
the phenotype of the D68A mutant. Since aspartate 68 is highly
conserved in the class II family of receptors (3), it may be argued
that its mutation profoundly affects the overall structure of the
receptor by disrupting the structure of the N-terminal extracellular
domain. We have thus considered another residue within the N-terminal extracellular domain that is not conserved in the class II family of
receptors but whose mutation abolishes VIP binding and VIP-stimulated cAMP production i.e. glutamate 36 (29). Again, the double
mutant H178R/E36A was no longer constitutively activated and displayed the same phenotype as the single mutant E36A. These observations suggested that the integrity of the VIP binding site at the N-terminal extracellular domain should be maintained for constitutive activation by the H178R mutation. This was further supported by a control double
mutant affecting a residue in the N-terminal extracellular domain whose
mutation was previously shown not to affect the phenotype of the
receptor i.e. aspartate 132 (29). The resulting double mutant H178R/D132A clearly displayed constitutive activation. From
these data, it could be hypothesized that the H178R mutation mimics
what happens when VIP binds to the wild type receptor and thereby
triggers activation of cAMP production. Similar conclusions were
previously drawn from analysis of constitutive activation of other
receptors (43) such as thrombin receptors (44) or
2-adrenergic receptors (45). However, it is worth
pointing out that our original approach consisting of constructing
double mutants provides new arguments indicating that the
conformational change triggering constitutive activation in a receptor
mutant requires the functional integrity of the ligand binding site. This double mutation approach was feasible for the VIP receptor, because the binding site in the N-terminal extracellular domain is well
separated from the site of the mutation evoking constitutive activation. To the best of our knowledge, such a double mutation approach has not been described previously.
Many natural or experimental mutations resulting in constitutive
activation of G protein-coupled receptors have been reported in the
third intracellular loop, most probably because this loop is involved
in G protein activation by the receptor (4). Such mutations have been
also described for several receptors in other domains including
transmembrane segments and extracellular loops (4). Mutation in the
N-terminal extracellular domain of the thrombin receptor (44) or
deletion of a portion of this domain in the thyrotropin receptor (46)
can also cause ligand-independent transmembrane signaling. To our
knowledge, constitutively activating mutations at the junction of the
first intracellular loop and second transmembrane domain have been only
described for the VIP1 receptor (this paper) and the PTH-PTHrP receptor
(27). Whether such atypical localization is related to a unique
characteristic of the class II family of G protein-coupled receptors
with respect to coupling to Gs proteins and their mechanism
of transmembrane signaling remains unclear. What is clear is that the
third intracellular loop of members of class II family of receptors
such as the glucagon-like peptide 1 receptor (47) and the PTH-PTHrP
receptor (48) is critical for coupling to the cAMP signal transduction
pathway as has been observed for other G protein-coupled receptors (4). At present, nothing is known about the domain(s) involved in the coupling of VIP1 receptors with Gs proteins.
Since histidine 178 in the human VIP1 receptor is strictly conserved in
all members of the class II family of receptors, it may be of general
functional importance in these G protein-coupled receptors. Whether
mutation of the equivalent histidine in other peptide receptor of this
family results in ligand-independent activation has been only reported
for the PTH receptor (27). In this context, it is worth pointing out
that two members of this family are receptors having an extraordinary
long N-terminal extracellular domain with unique features: the putative
EGF module-containing, mucin-like hormone receptor EMR1 (5) and the
leukocyte activation antigen CD97 (6). Since the signaling pathway(s)
are not known, experimental mutation of the equivalent histidine in
these two receptors could be instrumental in demonstrating their
possible coupling with adenylyl cyclase.
In conclusion, this study represents the first analysis of a
constitutively active receptor within the class II G protein-coupled receptor family. Our results underscore the importance of the N-terminal extracellular agonist binding domain in the mechanism of
ligand-independent constitutive activation of the VIP1 receptor. Whether the extracellular ligand binding domain is also important in
the mechanism of constitutive activation of other class II G
protein-coupled receptors remains to be established.