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J Biol Chem, Vol. 274, Issue 33, 23191-23197, August 13, 1999
From Recently, we reported that the mutation of
His207 to Phe located in the second extracellular
loop of the cholecystokinin B receptor strongly affected
cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and
Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To
characterize the functional group in CCK that interacts with His207, we first substituted His207 to Ala.
This mutation decreased the affinity and the potency of CCK to produce
total inositol phosphates 302-fold and 456-fold without affecting the
expression of the mutant receptor. The screening of
L-alanine-modified CCK peptides to bind and activate the
wild type and mutant receptors allowed the identification of the
interaction of the C-terminal Asp8 of CCK with
His207. The H207A-CCKBR mutant, unlike the wild type
receptor, was insensitive to substitution of Asp8 of CCK to
other amino acid residues. This interaction was further confirmed by
mutating His207 to Asp. The affinity of CCK for the
H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant,
consistent with an electrostatic repulsion between the negative charges
of the two interacting aspartic acids. Peptides with neutral amino
acids in position eight of CCK reversed this effect and displayed a
gain of affinity for the H207D mutant compared with CCK. To date, this
is the first report concerning the identification of a direct contact
point between the CCKB receptor and CCK.
Cholecystokinin (CCK)1
is found throughout the gastrointestinal tract and the central nervous
system where it functions both as a hormone and a neurotransmitter (1).
CCK exists physiologically in multiple forms processed from a 115-amino
acid preprohormone. Post-translational processing of CCK involved
sulfation of tyrosine at position seven from the C terminus and
amidation of the C-terminal phenylalanine. In the gastrointestinal
system, CCK regulates motility, pancreatic exocrine secretions and
growth, gastric emptying, and inhibition of gastric acid secretion. In
the nervous system, CCK is implicated in anxiogenesis, satiety,
analgesia, and regulation of dopamine release. The actions of CCK are
mediated by two pharmacologically distinct receptor subtypes, the CCKA
and the CCKB receptors. The longer sulfated forms (CCK-58, CCK-39,
CCK-33, and CCK-8) bind to the CCKAR and CCKBR with similar nanomolar
affinities. The nonsulfated peptides as well as shorter C-terminal
fragments like CCK-4 and CCK-5 still bind with nanomolar affinities the
CCKBR but display very low affinities for the CCKAR. Gastrin, a related family peptide that shares with CCK the C-terminal pentapeptide also
discriminates the two subtypes by presenting similar affinity as CCK-8
for the CCKBR but micromolar affinity for CCKAR. Studies using
synthesized CCK fragments have shown that the C-terminal sulfated and
amidated octapeptide
Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2 retains the full spectrum of biological activity (2-5). The cloning of
the cDNA coding for these receptors has shown that CCKA and CCKB
receptors belong to the superfamily of seven transmembrane G-protein-coupled receptor and that they have approximately 50% homology (6, 7). Due to the important physiological roles of CCK acting
through these two receptor subtypes and, therefore, its possible
implication in associated disorders, there has been considerable
interest in the identification of ligands that selectively activate or
inhibit the CCKAR and CCKBR. However, to date none of these compounds
have been used as a therapeutic agent (8-13). The characterization of
the interactions between the key pharmacophores of CCK and their
partners in the receptor would certainly help in a more rational
approach to the design of new molecules or the modification of
preexisting molecules to optimize their properties. Despite numerous
studies concerning the identification of amino acids involved in the
binding site of agonist and antagonist ligands for the different
classes of G-protein-coupled receptors (14, 15), very few studies
concerned the identification of the functional groups in the ligand
that directly interact with the identified amino acid in the receptor.
However such studies are essential to model the binding pocket
accurately. One way to address this issue is through the use of
complementary substitutions in the ligand and the receptor as
previously reported for G protein-coupled receptors for
neurotransmitters and peptide receptors (16-20). We successfully used
this approach for the CCKA receptor subtype to demonstrate that two
amino acids Trp39 and Gln40, located in the
N-terminal domain of the CCKAR, interact with the residues
Arg1 and Asp2 of CCK (21). Recently, we
identified another interaction between Met195, located in
the second extracellular loop of CCKAR, and the aromatic ring of
sulfated tyrosine of CCK (Tyr3), an important pharmacophore
for conferring high affinity and activity of the peptide (22). To date,
no such study has been reported for the CCKBR. We demonstrated by
mutational analysis of the CCKBR that the extracellular domains of the
CCKBR are also implicated in the high affinity binding of CCK and
gastrin (23, 24). We showed that the mutation of the residue
His207, located in the second extracellular loop of the
CCKBR induced a dramatic decrease in CCK binding (24). Regarding the
important role of the second extracellular loop for the high affinity
binding of CCK in the CCKAR, the present study was undertaken to
determine the amino acid within CCK that interacts with
His207 in the CCKBR. The screening of modified CCK peptides
for their binding and biological activity toward the wild type receptor and a variety of His207-CCKB receptor mutants permitted us
to demonstrate that His207 directly interacts with the
C-terminal aspartic acid of CCK, an essential residue for this family
of peptides.
Material--
The sulfated C-terminal nonapeptide of CCK
[Thr28, Nle31]-CCK25-33 ((Thr,Nle)CCK1-9s),
the tetrapeptide CCK-30-33 (CCK6-9), and the sulfated heptapeptide
[Nle28,31]-CCK27-33 (CCK3-9s) were synthesized as
described previously (25). The sulfated and nonsulfated C-terminal
octapeptide (CCK2-9s and CCK2-9) were from Neosystem, Strasbourg,
France. 125INa was from Amersham Pharmacia Biotech.
(Thr28,Nle31)-CCK25-33 was conjugated with
Bolton-Hunter reagent, purified, radioiodinated as described previously
(26), and referred to as 125I-BH-(Thr,Nle)CCK1-9s.
Synthesis of Modified CCK Peptides--
The
L-alanine-modified peptides were synthesized by solid phase
synthesis on a Pioneer Perseptive Biosystems apparatus using Fmoc-peptide amide linker-polyethylene glycol flow continuous resin.
Fmoc amino acid side chains were protected with the acid-labile protecting groups (Asp: t-butyl; Tyr: t-butyl;
Trp, t-butyloxycarbonyl). Coupling reactions were carried
out with a 4-fold excess of Fmoc-protected amino acid residues, reagent
HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate), and diisopropylethylamine for 1 h. At the end of the synthesis, the Fmoc-protecting group was removed, and the
peptidyl resin was treated at room temperature for 2 h with 10 ml
of reagent K solution (trifluoroacetic
acid/water/phenol/thioanisole/ethanedithiol 82.5/5/5/5/2.5). The
peptides were isolated by precipitation with diethyl ether, solubilized
in a mixture of acetonitrile/water/trifluoroacetic acid (50/50/0.1,
v/v/v), and lyophilized. These peptides were purified by reverse phase
HPLC using a Waters Delta Prep 4000 instrument on a Delta-Pak C18
column (15 µm, 100 × 150 mm) with UV detection at 214 nm at a
flow rate of 50 ml/min of water/trifluoroacetic acid (0.1%) and
acetonitrile/trifluoroacetic acid (0.1%). The purity of the peptides
was checked by analytical reverse phase HPLC with a Beckman instrument
on a Delta-Pak C18 analytical column. The peptide structure was
assessed by mass spectroscopy on a Platform II (Micromass, Manchester,
UK) quadrupole mass spectrometer fitted with an electrospray interface.
Construction of Mutant Receptor cDNAs--
Mutant receptor
cDNAs were constructed by oligonucleotide-directed mutagenesis
(QuickChangeTM site-directed mutagenesis kit, Stratagene,
France) using the rat CCKBR cDNAs as template. Oligonucleotides
were designed to include a silent restriction site to facilitate
analysis of mutant constructs by restriction endonuclease digestion.
Mutations were confirmed by DNA sequencing using an automated sequencer
(Applied Biosystems).
Transfection of Wild Type and Mutant Receptor cDNAs into
Mammalian Cells--
COS-7 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 5% fetal calf serum. Two µg of the
CCKBR and mutant receptor cDNAs subcloned in pCDL-SRa were
transiently transfected into COS-7 cells using the DEAE/dextran method
as described previously (23). 24 h after transfection, the
transfected cells were transferred to 24-well culture plates, seeded at
a density of approximately 1 × 105 cells/well, and
assayed for 125I-BH-CCK-9 binding or inositol phosphate hydrolysis.
Binding of 125I-BH-CCK-9 and
[3H]L-365,260 to Transfected COS-7
Cells--
Twenty-four h after the transfer of transfected cells to
24-well plates, the cells were washed once with cold phosphate-buffered saline, pH 7.4, containing 0.1% bovine serum albumin and incubated in
Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin for 60 min at 37 °C with either 50 pM (WT-CCKBR)
or 500 pM (H207A-CCKBR) of
125I-BH-(Thr,Nle)CCK1-9s with and without increasing
concentrations of unlabeled peptides. Cell-associated
125I-BH-(Thr,Nle)CCK1-9s was separated from free
radioligand by washing 2× with phosphate-buffered saline containing
2% bovine serum albumin. Cell-associated
125I-BH-(Thr,Nle)CCK1-9s was collected with 0.5 ml of 0.1 N NaOH added to each well, and radioactivity was detected
in a Measurement of Total Inositol Phosphate
Accumulation--
Twenty-four h after COS-7 cell transfection, the
transfected cells were transferred to 24-well culture plates and
incubated overnight in Dulbecco's modified Eagle's medium with 2 µCi/well of myo-2-[3H]inositol (18.6 Ci/mmol)( NEN Life Science Products). After the aspiration of the
medium containing the myo-[3H]inositol, the
cells were incubated at 37 °C for 20 min with 1 ml of Dulbecco's
modified Eagle's medium containing 20 mM LiCl. The cells
were washed with PI buffer. pH 7.45 (20 mM Hepes, 135 mM NaCl, 2 mM CaCl2, 1.2 mM MgSO4, 1 mM EGTA, 10 mM LiCl, 11.1 mM glucose, and 0.5% bovine
serum albumin), incubated 1 h at 37 °C with PI buffer
containing the indicated concentrations of peptides. The reaction was
stopped with 1 ml of methanol/HCl added to each well, and the content
was transferred to a AG 1-X8 (formate form) column (Bio-Rad). Each
column was washed twice with 3 ml of water followed by 2 ml of 5 mM sodium tetraborate, 60 mM sodium formate. Total inositol phosphates were eluted from the column with 2 ml of 1 M ammonium formate, 100 mM formic acid.
myo-[3H]inositol phosphate Effect of the H2O7A Mutation on Binding and Biological Activity of
the CCKBR--
In a recent work, we have shown (24) that the mutation
of His207 in the CCKBR to phenylalanine (the equivalent
amino acid in the CCKAR) resulted in a marked decrease in CCK affinity.
Upon transient expression of the H207F-CCKBR mutant into COS cells,
there was no detectable binding of radiolabeled CCK, and there was a
3,044-fold reduction in CCK-stimulated inositol phosphate production
compared with the WT-CCKBR. All together these results suggested that
His207 was crucial for conferring high affinity to CCK
(24). The present study was undertaken to identify the amino acid
residue within CCK that interacts with His207.
We first tried a potentially less drastic exchange by mutating
His207 to Ala. This mutation resulted in undetectable
binding of 125I-BH-(Thr,Nle)CCK1-9s to the H207A-CCKBR
mutant transiently expressed in COS-7 cells using standard radioligand
concentration (50 pM). By contrast, significant
125I-BH-(Thr,Nle)CCK1-9s binding was observed in the
presence of 500 pM radioligand. Under these conditions,
Scatchard analysis of CCK octapeptide (CCK2-9s) competition binding to
the H207A-CCKBR mutant revealed a single class of binding sites with an
affinity of 232 (±12) nM and a maximal binding capacity of
4.35 (±1.52) pmol/106 cells. Compared with the WT-CCKBR,
which had an affinity of 0.51 (±0.15) nM and a maximal
binding capacity of 2.13 (±1.62) pmol/106 cells, the
H207A-CCKBR mutant displayed a decrease in CCK2-9s affinity of 456-fold
despite a similar level of cell surface expression. This mutant also
retained similar efficacy compared with WT-CCKBR (11-fold over basal)
for CCK2-9s-stimulated increase in total IP production, although the
EC50 was 302-fold higher than the WT-CCKBR
(EC50: 145 (±79) nM versus 0.48 (±0.3) nM). The decrease in CCK2-9s potency to stimulate
IP production correlates well with the decrease in CCK2-9s affinity
observed for the H207A-CCKBR mutant (302-fold versus
456-fold). On the basis of the marked shift observed in CCK2-9s
affinity and potency when His207 was mutated to Ala, we
hypothesized that His207 might interact with a crucial
amino acid of CCK.
Structure-Function Studies of CCK in COS Cells Expressing the
WT-CCKBR--
Since no previous CCK structure-function studies have
been reported for the CCKBR in any expression systems, we first
determined to what extent each amino acid of CCK contributes to its
affinity and potency for stimulation of total IP in the WT-CCKBR
transiently expressed in COS-7 cells. For that, we tested different
fragments of CCK and synthesized CCK octapeptides modified by replacing each single amino acid by an L-alanine. We expected similar
affinity and potency decreases resulting from alanine substitutions in the receptor and in the ligand. In this first screening, the
L-alanine-modified peptides were synthesized in a
nonsulfated form for the convenience of the synthesis. As shown in
Table I, the N-terminal extension of CCK
has a small effect on the affinity and activity of CCK peptides.
CCK1-9s and CCK3-9s have similar affinities and potencies for the
WT-CCKBR than CCK2-9s, indicating that Arg1 and
Asp2 do not contribute to the activity and affinity of CCK.
The absence of sulfate on the Tyr3 decreases by 5.6-fold
the affinity and by 4-fold the potency of CCK2-9, whereas the complete
exchange of the sulfated Tyr3 by an Ala induces a 28-fold
and 16-fold decrease in affinity and potency, respectively. These data
are close to that found with the C-terminal tetrapeptide (CCK6-9),
which displays a 16-fold and 12-fold decrease in affinity and potency,
respectively, compared with CCK2-9s. In addition, the exchange of
Thr4 and Gly5 to Ala had no further additional
effect on affinity and potency, indicating that only the presence of
the sulfated Tyr3 slightly increases the affinity and
potency of CCK peptides compared with CCK6-9. Together these data
suggest that the important shift observed in both affinity and potency
of CCK2-9s for the H207A-CCKBR mutant could not be due to an
interaction of His207 with one of the N-terminal amino
acids, 1 to 5, of CCK. We then tested the contribution of the last four
C-terminal amino acids. As shown in Table I, the exchange of
Trp6 by Ala induces a dramatic decrease in both the
affinity and potency. It was difficult to determine an accurate
Ki because [Ala6]CCK2-9 bound the
WT-CCKBR with a very low affinity. Consistent with this result, the
EC50 of [Ala6]CCK2-9 was reduced 13,975-fold.
These results are not in favor of an interaction of Trp6
with His207, because Trp6 substitution results
in a large decrease in the affinity and potency of the modified peptide
for the WT-CCKBR, which does not correlate with that of CCK2-9s for the
H207A-CCKBR mutant. The affinities of [Ala7]CCK2-9 and
[Ala9]CCK2-9 were reduced 5,224- and 5,008-fold,
respectively, which correlated with the 4,629- and 6,837-fold decrease
in potencies observed for these two peptides, whereas the exchange of
Asp8 by Ala induces a less important decrease in the
affinity and potency of [Ala8]CCK2-9 with a shift of
1,191- and 935-fold, respectively (Table I). Considering that these
peptides are not sulfated, the decrease in the affinities and potencies
of [Ala7]CCK2-9, [Ala8]CCK2-9, and
[Ala9]CCK2-9 for the WT-CCKBR are closed to the 456- and
302-fold reduction in CCK2-9s affinity and potency observed for the
H207A-CCKBR mutant, suggesting that these residues could interact with
His207.
Demonstration That His207 of the CCKBR Interacts with
Asp8 of CCK--
We subsequently tested the equivalent
sulfated compounds [Ala7]CCK2-9s,
[Ala8]CCK2-9s, and [Ala9]CCK2-9s on the
WT-CCKBR and H207A-CCKBR to determine if these residues interact with
His207. We hypothesized that removing the functional group
in CCK that interacts with His207 should not further affect
the affinity and potency of the modified peptide for the H207A-CCKBR
mutant compared with CCK2-9s, because the mutation should have already
disrupted the interaction. On the contrary, exchanging a functional
group that does not interact with His207 should induce an
additional decrease in the affinity and potency of the modified peptide
for the H207A-CCKBR mutant. As shown in Table
II, [Ala7]CCK2-9s displayed
a 321- and 268-fold lower affinity and potency for the H207A-CCKBR
mutant compared with CCK2-9s. The decrease in the affinity and potency
of [Ala7]CCK2-9s for the WT-CCKBR was in the same range,
686- and 650-fold, respectively. No inhibition of
125I-BH-(Thr,Nle)CCK1-9s binding and no production of IP at
a concentration of 10
We subsequently tested the effect of exchanging Asp8 of
CCK2-9s with other amino acid residues on the ability of the modified peptide to bind and stimulate total IP production to both wild type and
mutated receptor (Table III). For the
WT-CCKBR, the exchange of Asp8 to Glu reduced the affinity
and the potency of [Glu8]CCK2-9s, 199- and 148-fold,
respectively. These values are similar to that obtained when
Asp8 was exchanged to Ala, indicating that the length of
the side chain at this position is as crucial as the presence of the
carboxylic group. Similarly, the exchange of Asp8 by Phe in
CCK2-9s reduced the affinity and the potency of the modified peptide
[Phe8]CCK2-9s to the same extent (Table III). CCK peptide
with an Asp8 to Leu substitution
([Leu8]CCK2-9s) bound to the WT-CCKBR with an affinity
lower than the other peptides modified at position 8. Moreover, this
peptide did not stimulate IP production, even at a concentration of
10
All these modified peptides had similar affinities and potencies
compared with CCK2-9s for the H207A-CCKBR mutant (except [Leu8]CCK2-9s, which did not stimulate IP even at
10
Because we have previously shown that the sulfated Tyr3 of
CCK interacts with Met195, located near His207
in the second extracellular loop of the CCKA receptor (22), we tested a
CCK peptide modified at position 3, [Ala3]CCK2-9, to
further demonstrate that the effect of substituting His207
on CCK affinity and potency was specific for Asp8. The
effect of mutating His207 and substituting Tyr3
of CCK was additive. [Ala3]CCK2-9 displayed an affinity
and a potency for the H207A-CCKBR mutant that were further reduced 11- and 13-fold compared with CCK2-9s. Moreover, the affinity and potency
of [Ala3]CCK2-9 was reduced 100- and 246-fold compared
with the WT-CCKBR (Table III).
To further demonstrate the interaction of Asp8 of CCK with
His207 in the CCKBR, we mutated His207 to Asp.
If Asp8 interacts with the residue 207 in the receptor, the
introduction of a negative charge at position 207 should further
decrease CCK2-9s affinity and potency compared with the H207A mutant
receptor because of the repulsive effect between the two negatively
charged residues. After transient expression of the H207D-CCKBR mutant
in COS cells, 125I-BH-(Thr,Nle)CCK1-9s binding was
undetectable even with 500 pM radioligand. However,
Scatchard analysis of the CCKBR nonpeptide antagonist,
[3H]L365,260, binding on transfected COS-7 membranes
revealed a similar level of expression of the H207D-CCKBR mutant
(Bmax = 3.9 ± 1.3 fmol/µg of protein)
compared with WT-CCKBR (Bmax = 2.4 ± 1.5 fmol/µg of protein). These data suggest that the loss of 125I-BH-(Thr,Nle)CCK1-9s binding was caused by an important
decrease in CCK affinity and was not due to a loss in receptor
expression. Consistent with the important shift in CCK affinity
observed, the potency of CCK2-9s for the H207D-CCKBR mutant was further decreased 112-fold (maximal efficacy of 67%) compared with the H207A-CCKBR mutant (Table IV and Fig.
2). Moreover, the
[Glu8]CCK2-9s peptide weakly stimulated total IP
production at 10 Use of mutational studies to identify receptor amino acids
important for ligand binding cannot by themselves determine whether the
mutated amino acids have a direct versus an indirect effect. The effect of a mutation may directly influence the binding pocket by
substituting a residue that directly binds the ligand. Alternatively, the mutation may indirectly affect the binding pocket by causing a
conformational change that disrupts a distal binding site. One way to
address this question is to characterize the functional group in the
ligand that interacts with the mutated receptor amino acid by using
modified ligands. We have recently shown by mutational analysis of the
CCKBR that one residue, His207, located in the second
extracellular loop of the CCKBR when mutated, affects CCK2-9s high
affinity binding (24). Regarding the direct role of the second
extracellular loop of the CCKAR for the high affinity binding of CCK
(22), the aim of the present study was to characterize the functional
group in CCK that interacts with His207 in the CCKBR and
thus to determine if His207 is likely to be directly
involved in the CCK binding pocket.
We first showed that the mutation of His207 to Ala
decreased 456-fold the affinity of CCK2-9s, and 302-fold, its potency
to stimulate total IP production compared with the wild type CCKB
receptor. The similar expression of the mutated receptor and wild type
receptor at the cell surface as well as the similar biological efficacy of CCK2-9s for the two receptors indicated that the mutation did not
introduce a gross conformational change in the receptor. The mutation
of His207 to Ala, albeit less drastic than to Phe, still
affected the affinity and potency of CCK2-9s, suggesting that this
residue might interact with an important amino acid of CCK. In a
preliminary approach to identifying the putative amino acid of CCK
interacting with His207, we first screened
L-alanine-substituted analogues of CCK octapeptide at each
position for their ability to bind and stimulate IP production at the
wild type receptor. We expected similar affinity and potency decreases
resulting from Ala substitutions in the receptor and in the ligand.
This strategy permitted us to identify three residues at the C terminus
of CCK, Met7, Asp8, and Phe9, as
being putative partners of His207, since the affinities and
potencies of the corresponding modified peptides for the WT-CCKBR were
close to that of CCK2-9s for the H207A-CCKBR mutant. Among these
residues, Asp8 was identified as the only residue
interacting with His207, since the H207A-CCKBR mutant
displayed similar affinities and potencies for
[Ala8]CCK2-9s and CCK2-9s, whereas its affinity and
potency for [Ala7]CCK2-9s and [Phe9]CCK2-9s
were importantly decreased. The interaction between Asp8
and His207 was confirmed by the fact that the H207A-CCKBR
mutant was unable to distinguish between the native ligand and a series
of analogues modified at position 8, whereas the WT-CCKBR was highly
sensitive to changes introduced at this position. Moreover the peptides modified at position 8 displayed similar affinities and potencies for
the wild type and mutated receptor, whereas the analogues modified at
positions 7 and 9 bound to and activated H207A-CCKBR mutant with
decreased affinities and potencies relative to the wild type receptor.
Further support for an interaction of Asp8 with
His207 was brought by mutating His207 to Asp.
The drastic effect observed on CCK2-9s affinity and potency when
His207 was substituted to Asp compared with Ala (100-fold)
is consistent with an electrostatic repulsion between the negative
charges of the Asp8 of the ligand and the one introduced at
position 207 in the receptor. In addition, the severe loss of affinity
and potency of [Glu8]CCK2-9s for the H207D-CCKBR mutant
is in accordance with an increase of the repulsive effect due to an
increase of the length of the side chain while maintaining the
carboxylic function in position 8. In agreement with this result, when
the repulsive force was removed by substituting Asp8 of the
ligand for a neutral amino acid such as Ala or Phe, the potency of the
two modified peptides for the mutant H207D were increased accordingly
12- and 27-fold compared with CCK2-9s. This gain of function is a
strong evidence for the direct interaction between His207
of the CCKBR and Asp8 of CCK. Moreover, since the affinity
and potency of the two peptides [Ala8]CCK2-9s and
[Phe8]CCK2-9s for the H207A and H207D-CCKBR mutants were
similar, it is likely that the repulsive effect observed was caused by
the direct repulsion of the negative charges of the aspartic residues of the ligand and the receptor and is not due to a nonspecific interaction of Asp207 with another residue of the receptor.
All together these results argue that the changes introduced in the
mutated receptor and in the ligand affect the same bond.
However, the present work does not allow one to conclude about the
nature of this interaction. Imidazole-carboxylate interactions such as
the one found here between the carboxyl side chain of CCK and
His207 of the CCKBR are often found as an important
stabilizing element in protein structures (31-33), enzyme reactions
(34-36), and ligand-receptor recognition (37, 38). Such interactions
can be due to a salt bridge between imidazolium and carboxylate ions or
to a network of hydrogen bonds linking the imidazole and carboxylate
side chain together. Such an interaction might be considered as the
initialization step of a proton pumping mechanism, which could be
hypothetized in the activation process of G protein-coupled receptors
(37). The implication of Asp8 of CCK in CCKBR activation,
in addition to its role in ligand binding, was previously reported for
the stimulation of acid secretion from dispersed gastric cells
(39). Consistent with this result, we observed in the present paper
that the substitution of Asp8 for Leu completely abolished
total inositol phosphate production both in the wild type CCKBR
and in the H207A-CCKBR mutant while maintaining micromolar affinities
for both receptors.
Direct interactions of peptides with G protein-coupled receptors have
been documented in a limited number of cases. However, it appears that
the direct involvement of the extracellular domains in the peptide
binding pocket is not unique to the CCKA and CCKB receptors subtypes. A
few reports have indicated that the extracellular domains of the
peptide receptors are direct contact sites for the peptide ligands. For
example, it has been reported that in the AT1 angiotensin receptor,
Asp281 of the extracellular loop 3 and His183
of the extracellular loop 2, respectively, bind Arg2 and
Asp1 of angiotensin II (19). Similarly, Tyr115
of the extracellular loop 1 of the vasopressin V1A receptor interacts with Arg8 of arginine-vasopressin (40).
Despite the fact that CCK presents a similar high affinity for both the
CCKA and the CCKB receptor subtypes, the mutagenesis data obtained in
this study clearly indicate a different anchoring of CCK in the CCKBR
compared with the CCKAR (21, 22). These results are also consistent
with a recent study in which we showed that the reciprocal mutation in
the CCKBR of the two residues corresponding to Trp39
and Gln40 in the CCKAR (21) that directly interact with the
N terminus of CCK2-9s have no effect on CCK2-9s binding in the
CCKB receptor (24).
In conclusion, using site-directed mutagenesis of the CCKB receptor
together with the analysis of the binding and biological potency of
different modified CCK analogues, we have shown in this study that
His207 is directly involved in the binding pocket of CCK by
interacting with the penultimate Asp8 residue of CCK. This
study represents an important step in the molecular characterization of
the CCK binding site especially since this is the first report
concerning the identification of a direct contact point between the
CCKB receptor and CCK. The importance of this study is reinforced by
the fact that it involves an important amino acid of CCK.
*
This work was supported in part by Association pour la
Recherche sur le Cancer Grant ARC 6234 and Region
Midi-Pyrénées Grant 9507618.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.
§
This author is in charge of research for the CNRS. To whom
correspondence should be addressed: INSERM U151, CHU Rangueil, Bat L3,
31403 Toulouse Cedex, France. Tel.: 33 5 61 32 30 57; Fax: 33 5 61 32 24 03; E-mail:Sandrine.Poirot@rangueil.inserm.fr.
The abbreviations used are:
CCK, cholecystokinin;
CCKAR, CCK receptor type A;
CCKBR, CCK receptor type
B;
WT, wild type;
BH, Bolton-Hunter;
IP, inositol phosphate;
Nle, norleucine;
Fmoc, N-(9-fluorenyl)methoxycarbonyl;
HPLC, high performance liquid chromatography.
Evidence for a Direct Interaction between the Penultimate
Aspartic Acid of Cholecystokinin and Histidine 207, Located in the
Second Extracellular Loop of the Cholecystokinin B Receptor*
§,,,
,,,,, and
INSERM U 151, Institute Louis Bugnart, CHU
Rangueil, Bat L3, 31403 Toulouse Cedex, France, ¶ CNRS, UMR
5810, Faculté de Pharmacie, 34060 Montpellier, France,
Laboratoire de Chimie Théorique,
Université de Nancy, 54506 Vandoeuvre, Nancy, France, ** Digestive
Diseases Branch, NIDDK, National Institutes of Health, Bethesda,
Maryland 20892-1804, and Max Planck Institute für
Biochemie, 82143 Martinsried, Federal Republic of Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
counter. Nonspecific binding (determined in presence of 1 µM CCK-8) was always less than 10% that of total
binding. Plasma membranes from transfected COS cells were prepared as
described in Kennedy et al. (21) and incubated (50-100
µg) with 500 pM [3H]L-365,260 for 45 mn at
37 °C in the presence of increasing L-365,260 concentrations (27).
After incubation, the membranes were washed and filtered through
Whatman GF/C glass fiber filters over a vacuum-filtering manifold.
Filters were counted in a 
counter. Binding assays were performed in
duplicate in at least three separate experiments. Binding data were
determined using the nonlinear, least squares curve-fitting computer
program, Ligand (28) and GraphPad Prism Program (Software).
Ki values were calculated as Ki = IC50/(1 + [labeled ligand]/Kd of
labeled ligand).
radioactivity
was detected in a liquid scintillation counter (Packard Instrument
Co.). EC50 was calculated using GraphPad Prism program software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Affinities and potencies of CCK peptides, CCK fragments, and
L-alanine-modified CCK peptides for the WT-CCKBR
transiently expressed in COS cells
6 M
CCK2-9s and referred as Emax. ND, not determined.
4 M were observed with
[Ala9]CCK2-9s on the H207A-CCKBR mutant, indicating a
dramatic shift in the affinity and potency. This peptide displayed a
4897- and 5025-fold lower affinity and potency than CCK2-9s on the
WT-CCKBR. Together, these results indicate that Met7 and
Phe9 modifications of CCK and H207A mutation effects are
additive and support that Met7 and Phe9 do not
interact with His207. In contrast to that observed with
[Ala7]CCK2-9s and [Ala9]CCK2-9s, the
affinity and potency of [Ala8]CCK2-9s for the H207A-CCKBR
mutant were similar to that CCK2-9s but were decreased 460- and
151-fold, respectively, for the WT-CCKBR (Fig.
1, right, and Table II). Thus,
[Ala8]CCK2-9s, unlike CCK2-9s,
[Ala7]CCK2-9s, and [Ala9] CCK2-9s, is
insensitive to the H207A mutation. These results and the fact that
[Ala8]CCK2-9s had a similar affinity and potency for the
H207A-CCKBR mutant and the WT-CCKBR suggest a direct interaction
between Asp8 of CCK and His207 (Fig. 1 and
Table II). To confirm this interaction, these data were analyzed
according to the mutant cycle methodology of Hidalgo and MacKinnon (29)
and Schreiber and Fersht (30). 
values, defined as = Ki (WT:WT) × Ki
(mut:mut)/Ki (WT:mut) × Ki
(mut:WT), determined if two modified or mutated residues are
independent of one another ( will be unity) or if these residues
interact ( will deviate from unity). As shown in Table II, the large
value for the [Ala8]-CCK2-9s/H207A-CCKBR pair
supports that Asp8 interacts specifically with
His207. This contrasts with the value for the
[Ala7]-CCK2-9s/H207A-CCKBR pair that is near unity,
suggesting that Met7 does not interact with
His207. Similarly, our inability to determine value for
the [Ala9]-CCK2-9s/H207A-CCKBR pair is compatible with
their independence. Together these results provide further support for
a direct and specific interaction of Asp8 with
His207.
Affinities and potencies of CCK2-9s and modified CCK2-9s peptides for
the WT-CCKBR and H207A-CCKBR mutant transiently expressed in COS
cells
6 M
CCK2-9s on the WT-CCKBR and referred to as Emax.
The FWT factor represents the effect of the mutation
on the affinity or on the potency of the peptides tested relative to
the wild type receptor. ND, not determined. The factor was
calculated according to the equation described under "Results." For
values less than unity, reciprocals are given for ease of comparison.
View larger version (14K):
[in a new window]
Fig. 1.
Competition of
125I-BH-(Thr,Nle)CCK1-9s binding (left)
and stimulation of total IP production (right) by
CCK2-9s ( 
) and [Ala8]CCK2-9s (
) on the WT-CCKBR
(broken line) and H207A-CCKBR (solid
line) mutant transiently expressed in COS cells.
Binding is expressed as the percent of specifically bound
125I-BH-(Thr,Nle)CCK1-9s. Inositol phosphate production is
expressed as percent of maximum stimulation. The data represent the
mean of three to five experiments performed in duplicate.
4 M, indicating that the substitution of
Asp8 by Leu affects the agonist activity of the peptide.
This result is in accordance with the importance of the length of the
side chain at this position.
Affinities and potencies of CCK2-9s and modified CCK2-9s peptides for
the WT-CCKBR and H207A-CCKBR mutant transiently expressed in COS
cells
6 M
CCK2-9s on the WT-CCKBR and referred to as Emax.
ND, not determined.
4 M as with the WT-CCKBR), indicating that
unlike the WT-CCKBR, the H207A-CCKBR mutant is insensitive to the
changes introduced at position 8 of CCK (Table III). It should be noted
that all these modified peptides had a 1.6- to 2-fold lower efficacy
for stimulating total IP production in the H207A-CCKBR mutant compared
with the WT-CCKBR. This is most likely due to the additive effects of
the changes introduced both in the mutated receptor and in the modified peptides, whereas CCK2-9s had the same efficacy on both wild type and
mutated receptor.
4 M (around 9%) at the
H207D-CCKBR mutant, whereas [Glu8]CCK2-9s maintained
similar affinity and potency on the H207A-CCKBR mutant than CCK2-9s
(Table IV). When [Ala8]CCK2-9s was tested for its ability
to stimulate total IP production through the H207D-CCKBR mutant, we
observed a 12-fold increase in [Ala8]CCK2-9s potency
relative to CCK2-9s (Table IV and Fig. 2). A similar increase (27-fold)
in potency was also observed with [Phe8]CCK2-9s (Table
IV). Thus, a gain of function was observed when the repulsive force
between Asp207 and Asp8 was removed by
replacing Asp8 by a neutral amino acid.
Potencies of CCK2-9s and modified CCK2-9s peptides for the
H207A-CCKBR and H207D-CCKBR mutants transiently expressed in COS cells
6 M
CCK2-9s on the WT-CCKBR and referred to as Emax.
ND, not determined.
View larger version (19K):
[in a new window]
Fig. 2.
Stimulation of total IP production by CCK2-9s
( ) and [Ala8]CCK2-9s () on the H207A-CCKBR and
H207D-CCKBR mutants transiently expressed in COS cells. Inositol
phosphate production is expressed as percent of maximum stimulation.
The data represent the mean of three to five experiments performed in
duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Rehfeld, J. F.,
and van Solinge, W. W.
(1994)
Adv. Cancer Res.
63,
295-347[Medline]
[Order article via Infotrieve]
2.
Silvente-Poirot, S.,
Dufresne, M.,
Vaysse, N.,
and Fourmy, D.
(1993)
Eur. J. Biochem.
215,
513-529[Medline]
[Order article via Infotrieve]
3.
Crawley, J. N.,
and Corwin, R. L.
(1994)
Peptides
15,
731-755[CrossRef][Medline]
[Order article via Infotrieve]
4.
Wank, S. A.
(1995)
Am. J. Physiol.
269,
G628-G646 5.
Wank, S. A.
(1998)
Am. J. Physiol.
274,
G607-G613
6.
Wank, S. A.,
Pisegna, J. R.,
and de Weerth, A.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
8691-8695 7.
Wank, S. A.,
Harkins, R.,
Jensen, R. T.,
Shapira, H.,
de Weerth, A.,
and Slattery, T.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
3125-3129 8.
Woodruff, G. N.,
and Hughes, J.
(1991)
Annu. Rev. Pharmacol. Toxicol.
31,
469-501[CrossRef][Medline]
[Order article via Infotrieve]
9.
Iversen, L. L.,
Dourish, C. T.,
and Iversen, S. D.
(1991)
Biochem. Soc. Trans.
19,
913-915[Medline]
[Order article via Infotrieve]
10.
Schiantarelli, P.
(1993)
Pharmacol. Res.
28,
1-9[CrossRef][Medline]
[Order article via Infotrieve]
11.
Betancur, C.,
Azzi, M.,
and Rostene, W.
(1997)
Trends Pharmacol. Sci.
18,
372-386[Medline]
[Order article via Infotrieve]
12.
Horwell, D. C.
(1996)
Bioorg. Med. Chem.
4,
1573-1576[CrossRef][Medline]
[Order article via Infotrieve]
13.
Pritchard, M. C.,
Raphy, J.,
and Singh, L.
(1997)
Exp. Opin. Invest. Drugs
6,
349-365[CrossRef]
14.
Schwartz, T. W.
(1994)
Curr. Opin. Biotechnol.
5,
434-444[CrossRef][Medline]
[Order article via Infotrieve]
15.
Strader, C. D.,
Fong, T. M.,
Graziano, M. P.,
and Tota, M. R.
(1995)
FASEB J.
9,
745-754[Abstract]
16.
Strader, C. D.,
Sigal, I. S.,
Candelore, M. R.,
Rands, E.,
Hill, W. S.,
and Dixon, R. A.
(1988)
J. Biol. Chem.
263,
10267-10271 17.
Strader, C. D.,
Candelore, M. R.,
Hill, W. S.,
Sigal, I. S.,
and Dixon, R. A.
(1989)
J. Biol. Chem.
264,
13572-13578 18.
Fong, T. M.,
Cascieri, M. A., Yu, H.,
Bansal, A.,
Swain, C.,
and Strader, C. D.
(1993)
Nature
362,
350-353[CrossRef][Medline]
[Order article via Infotrieve]
19.
Feng, Y. H.,
Noda, K.,
Saad, Y.,
Liu, X.,
Husain, A.,
and Karnik, S. S.
(1995)
J. Biol. Chem.
270,
12846-12850 20.
Noda, K.,
Saad, Y.,
Kinoshita, A.,
Boyle, T. P.,
Graham, R. M.,
Husain, A.,
and Karnik, S. S.
(1995)
J. Biol. Chem.
270,
2284-2289 21.
Kennedy, K.,
Gigoux, V.,
Escrieut, C.,
Maigret, B.,
Martinez, J.,
Moroder, L.,
Frehel, D.,
Gully, D.,
Vaysse, N.,
and Fourmy, D.
(1997)
J. Biol. Chem.
272,
2920-2926 22.
Gigoux, V.,
Escrieut, C.,
Silvente-Poirot, S.,
Maigret, B.,
Gouilleux, L.,
Fehrentz, J. A.,
Gully, D.,
Moroder, L.,
Vaysse, N.,
and Fourmy, D.
(1998)
J. Biol. Chem.
273,
14380-14386 23.
Silvente-Poirot, S.,
and Wank, S. A.
(1996)
J. Biol. Chem.
271,
14698-146706 24.
Silvente-Poirot, S.,
Escrieut, C.,
and Wank, S. A.
(1998)
Mol. Pharmacol.
54,
364-371 25.
Moroder, L.,
Wilschowitz, L.,
Gemeiner, M.,
Gohring, W.,
Knof, S.,
Scharf, R.,
Thamm, P.,
Gardner, J. D.,
Solomon, T. E.,
and Wunsch, E.
(1981)
Hoppe-Seyler's Z. Physiol. Chem.
362,
929-942[Medline]
[Order article via Infotrieve]
26.
Fourmy, D.,
Lopez, P.,
Poirot, S.,
Jimenez, J.,
Dufresne, M.,
Moroder, L.,
Powers, S. P.,
and Vaysse, N.
(1989)
Eur. J. Biochem.
185,
397-403[Medline]
[Order article via Infotrieve]
27.
Chang, R. S.,
Chen, T. B.,
Bock, M. G.,
Freidinger, R. M.,
Chen, R.,
Rosegay, A.,
and Lotti, V. J.
(1989)
Mol. Pharmacol.
35,
803-808[Abstract]
28.
Munson, P. J.,
and Rodbard, D.
(1980)
Anal. Biochem.
107,
220-239[CrossRef][Medline]
[Order article via Infotrieve]
29.
Hidalgo, P.,
and MacKinnon, R.
(1995)
Science
268,
307-310 30.
Schreiber, G.,
and Fersht, A. R.
(1995)
J. Mol. Biol.
248,
478-486[Medline]
[Order article via Infotrieve]
31.
Hefford, M. A.,
and Kaplan, H.
(1989)
Biochim. Biophys. Acta
998,
267-270[CrossRef][Medline]
[Order article via Infotrieve]
32.
Ippolito, J. A.,
Alexander, R. S.,
and Christianson, D. W.
(1990)
J. Mol. Biol.
215,
457-471[Medline]
[Order article via Infotrieve]
33.
Brocchieri, L.,
and Karlin, S.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9297-9301 34.
Menard, R.,
Khouri, H. E.,
Plouffe, C.,
Laflamme, P.,
Dupras, R.,
Vernet, T.,
Tessier, D. C.,
Thomas, D. Y.,
and Storer, A. C.
(1991)
Biochemistry
30,
5531-5538[CrossRef][Medline]
[Order article via Infotrieve]
35.
Bischoff, K. M.,
and Rodwell, V. W.
(1997)
Protein Sci.
6,
156-161[Abstract]
36.
Bragg, P. D.
(1998)
Biochim Biophys Acta
1365,
98-104[Medline]
[Order article via Infotrieve]
37.
Joseph, M. P.,
Maigret, B.,
Bonnafous, J. C.,
Marie, J.,
and Scheraga, H. A.
(1995)
J. Protein Chem.
14,
381-398[CrossRef][Medline]
[Order article via Infotrieve]
38.
Lu, D.,
Vage, D. I.,
and Cone, R. D.
(1998)
Mol. Endocrinol.
12,
592-604 39.
Magous, R.,
Martinez, J.,
Lignon, M. F.,
and Bali, J. P.
(1983)
Regul. Pept.
5,
327-332[CrossRef][Medline]
[Order article via Infotrieve]
40.
Chini, B.,
Mouillac, B.,
Ala, Y.,
Balestre, M. N.,
Trumpp-Kallmeyer, S.,
Hoflack, J.,
Elands, J.,
Hibert, M.,
Manning, M.,
Jard, S.,
and Barberis, C.
(1995)
EMBO J.
14,
2176-82[Medline]
[Order article via Infotrieve]
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