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J Biol Chem, Vol. 274, Issue 29, 20457-20464, July 16, 1999
From The cholecystokinin-A receptor (CCK-AR) is a G
protein-coupled receptor that mediates important central and peripheral
cholecystokinin actions. Residues of the CCK-AR binding site that
interact with the C-terminal part of CCK that is endowed with
biological activity are still unknown. Here we report on the
identification of Arg-336 and Asn-333 of CCK-AR, which interact with
the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively.
Identification of the two amino acids was achieved by dynamics-based
docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints, previous results that demonstrated that Trp-39/Gln-40 and
Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine
of CCK, respectively. Arg-336-Asp-8 and Asn-333-amide interactions were
pharmacologically assessed by mutational exchange of Arg-336 and
Asn-333 in the receptor or reciprocal elimination of the partner
chemical functions in CCK. This study also allowed us to demonstrate
that (i) the identified interactions are crucial for stabilizing the
high affinity phospholipase C-coupled state of the CCK-AR·CCK
complex, (ii) Arg-336 and Asn-333 are directly involved in interactions
with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336
but not Asn-333 is directly involved in the binding of the peptide
antagonist JMV 179 and the peptide partial agonist JMV 180. These data
will be used to obtain an integrated dynamic view of the molecular
processes that link agonist binding to receptor activation.
The cholecystokinin-A receptor
(CCK-AR)1 is a member of the
superfamily of G protein-coupled receptors and transduces CCK signals into target cells (1, 2). CCK-AR has important physiological functions
in the central nervous system (stimulation of satiety) and in
peripheral organs, particularly in the gut (stimulation of gall bladder
contraction, pancreatic secretions, and digestive motility) (3).
CCK-AR, like other G protein-coupled receptors, may be involved in the
development of certain pathologies in humans and as such is an
important target for therapeutic intervention involving the
pharmacological blockade or activation of the receptor using
appropriate ligands (4, 5).
The natural ligand of CCK-AR, CCK, is composed of several molecular
variants, the octapeptide (CCK-8) being the major fully active one (6).
Posttranslational processing of CCK involves sulfation of the tyrosine
at position seven from the C-terminal and Most of the available data regarding the topography of the agonist and
antagonist binding sites of G protein-coupled receptors were derived
from studies using site-directed mutagenesis. Data from these studies
led to the conclusion that G protein-coupled receptors for
neuropeptides have determinants of their agonist binding sites located
both in extracellular regions and within the transmembrane domains
(19-21). For CCK-AR, several lines of evidence support the view that
its binding site for CCK comprises amino acids located in extracellular
domains. Indeed, the removal of the first 43 amino acids of CCK-AR, and
more recently, the mutation of two residues located at the top of the
first transmembrane segment, (i.e. Trp-39 and Gln-40) were
found to decrease the affinity of the CCK agonist but not that of
nonpeptide antagonists (22, 23). In latter study, we showed that
receptors mutated at positions 39 and 40 exhibited decreased affinities
for CCK octa- and nonapeptide but did bind CCK heptapeptide with the
same affinity as the wild-type receptor. These data were interpreted as
providing a strong indication that Trp-39 and Gln-40 are part of the
CCK binding site by interacting directly the N-terminal portion of CCK.
Using these data and molecular dynamics-based ligand docking, two
additional residues, Met-195 and Arg-197, located in the second
extracellular loop, were recently identified as part of the agonist
binding site because they interact with the sulfated tyrosine of CCK,
which is crucial for binding and activity of CCK (24, 25).
Identification of amino acids of CCK-AR that interact with the
C-terminal moiety of CCK represents one of the prerequisites for the
understanding of how CCK signal is converted into receptor activation.
Here, we present important new data that identify residues Arg-336 and
Asn-333 as part of the CCK-AR agonist binding site. These amino acids,
which are located at the top of transmembrane segment VI, are
interacting with the aspartic acid carboxylate and the C-terminal amide
of CCK, respectively, which are crucial for binding and activity. In
addition, these amino acids are involved differentially in the binding
site of peptide and nonpeptide antagonists of CCK-AR, supporting the
existence of binding sites for individual agonists/antagonists, which
may or may not overlap.
Materials--
The C-terminal nonapeptide of CCK,
(Thr,Nle)-CCK-9, was synthesized by Luis Moroder (Max-Planck-Institut
für Biochimie, Martinsried, Germany). The other analogues of CCK,
namely (Ala-8)-CCK, (Arg-8)-CCK, (PheCH3)-CCK, JMV 180, and JMV 179, were synthesized by Jean Martinez's group.
(1-(2-(4-(2-Chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl)acetic acid)
(SR-27, 897) and its tritiated derivative, [3H]SR-27,897
(31 Ci/mmol), were donated by Sanofi Research (Toulouse, France) (26).
L-364,718,
3S( Computer Modeling of CCK-AR and the CCK-AR·CCK Complex--
A
CCK-AR model was built using the transmembrane helical arrangement
found in the bacteriorhodopsin crystal structure as starting point
(28). It was then modified according to the rhodopsin crystal structure
(29) and to the mutant data base "input/output" information scheme
defined in the Viseur program (30). Extracellular and intracellular
loops connecting the transmembrane helices were then added to the
preliminary 7-helix bundle and modeled with the use of simulated
annealing procedures. The entire system was finally relaxed and
submitted to 1 ns of molecular dynamics, with possible translation and
rotation movements of individual transmembrane helices taken into
account. For molecular dynamics-based docking of CCK into the CCK-AR
binding site, experimental data that identified contact points between
Trp-39/Gln-40 (23) and the N-terminal moiety of CCK and between
Met-195/Arg-197 and the sulfated tyrosine of CCK (24, 25) were taken
into account, as well as the molecular electrostatic potentials at the
top of the receptor groove. The resulting complex was submitted to
annealing molecular dynamics calculations. A program package (Insight
II, Discover, Homology, and Biopolymer) from Molecular Simulations Inc.
(San Diego, CA) was used.
Site-directed Mutagenesis--
Site-directed mutagenesis was
carried out using the Chameleon 228 double-stranded site-directed
mutagenesis kit (Stratagene) following the manufacturer's
instructions. The protocol is based on the method of mutagenesis by
unique site elimination. Mutations were introduced into the human
CCK-AR cDNA cloned into pRFENeo vector using mutagenic primer based
on the published human CCK-AR cDNA sequence (1, 2). Selection
primers mutated a unique SmaI restriction site to a unique
NruI site and vice versa. Plasmids were isolated from
individual colonies and screened for the incorporation of the desired
mutation by restriction site analysis. The presence of the desired
mutations and the absence of undesired mutations were confirmed by
automated sequencing (Applied Biosystems).
Transient Transfection of COS-7 Cells--
COS-7 cells (1.5 × 106) were plated onto 10-cm culture dishes and grown in
Dulbecco's modified Eagle's medium containing 5% fetal calf serum
(complete medium) in a 5% CO2 atmosphere at 37 °C.
After overnight incubation, cells were transfected with 2.5 µg/plate
of pRFENeo vectors containing the cDNA for the wild-type or mutated
CCK-A receptors, using a modified DEAE-dextran method. Approximately
24 h posttransfection, the cells were washed twice with
phosphate-buffered saline, pH 6.95, and then seeded onto 24-well dishes
in complete medium at a density of approximately 1 × 105 cells/well, for binding assays. For inositol phosphate
assay, the cells were resuspended in complete medium in presence of 2 µCi/ml myo-2-[3H]inositol (Amersham Pharmacia Biotech)
and incubated overnight in 24-well dishes.
Receptor Binding Assay--
Approximately 24 h after the
transfer of transfected cells to 24-well plates, the cells were washed
with phosphate-buffered saline, pH 6.95, 0.1% bovine serum albumin and
then incubated for 60 min at 37 °C in 0.5 ml of Dulbecco's modified
Eagle's medium, 0.1% bovine serum albumin with either 71 pM 125I-BH-(Thr,Nle)CCK-9, 71 pM
125IBH-JMV 179, or 1.83 nM
[3H]SR-27,897 in the presence or absence of competing
agonists or antagonists. The cells were washed twice with
phosphate-buffered saline, pH 6.95, containing 2% bovine serum
albumin, and cell-associated radioligand was collected with 0.1 N NaOH added to each well. The radioactivity was directly
counted in a gamma counter (Auto-Gamma, Packard Instrument Co.) or
added to scintillant and counted for the tritiated radioligand.
Nonspecific binding was always less than 10% of total binding.
Inositol Phosphate Assay--
Approximately 24 h after the
transfer to 24-wells plates and following overnight incubation in
complete medium containing 2 µCi/ml of
myo-2-[3H]inositol, the transfected cells were washed
with Dulbecco's modified Eagle's medium and then incubated for 30 min
in 2 ml/well Dulbecco's modified Eagle's medium containing 20 mM LiCl at 37 °C. The cells were washed with PI buffer
(phosphate-buffered saline containing 135 mM NaCl, 20 mM HEPES, 2 mM CaCl2, 1.2 mM MgSO4, 1 mM EGTA,10
mM LiCl, 11.1 mM glucose and 0.5% bovine serum
albumin) at pH 7.45. The cells were then incubated for 60 min at
37 °C in 0.5 ml of PI buffer with or without ligands at various
concentrations. The reaction was stopped by adding 1 ml of
methanol/chlorhydric acid to each well, and the content was transferred
to a column (Dowex AG 1-X8, formate form, Bio-Rad) for the extraction
of inositol phosphates. The columns were washed twice with 5 ml of
distilled water and twice more with 2 ml of 5 mM sodium
tetraborate/60 mM sodium formate. The content of each
column was eluted by addition of 2.5 ml of 1 M ammonium
formate/100 mM formic acid. 0.5 ml of the eluted fraction
was added to scintillant, and Docking of CCK into the CCK-AR Binding Site--
In order to
identify new residues of the CCK-AR binding site for CCK, molecular
dynamics-based docking of CCK into the three-dimensional model of the
receptor was performed using, as constraints, previous experimental
results that demonstrated interactions between the N terminus of CCK
and residues Trp-39/Gln-40 of CCK-AR and between the sulfated tyrosine
of CCK and residues Met-195/Arg-197 (Fig. 1). (23-25). We focused our attention on
interactions with residues and chemical functions of the C-terminal
part of CCK, which are crucial for its biological activity. The
C-terminal part of CCK was found to interact electrostatically with
residues located at the entrance of the transmembrane bundle of the
receptor. A first group of interactions involved the guanidium of
Arg-336 and the carboxylate of Asp-8 of CCK. A second point of contact attracted our attention because binding of Asn-333 side-chain to the
C-terminal amide of CCK was observed. This terminal Effects of R336M and N333A Mutations on Expression and Binding
Properties of CCK-AR--
Arg-336 and Asn-333 were exchanged for amino
acids lacking the chemical functions thought to be responsible for the
interactions with CCK (Fig. 1). We exchanged Arg-336 for a methionine
that possesses an hydrophobic side-chain but should not interact with the negatively charged Asp-8 carboxylate and exchanged Asn-333 for an
alanine. We first determined whether COS-7 cells expressing the mutated
receptors could bind the agonist radioligand
125I-BH-(Thr,Nle)-CCK-9. No agonist radioligand binding
could be demonstrated with either the (R336M)- or (N333A)-CCK-AR
mutants even when the concentration of the radioligand was increased up to 250 pM. This result suggests that Arg-336 and Asn-333
are involved in high affinity CCK binding. Alternatively, the mutations
could affect the expression of the receptor by COS-7 cells and/or
disrupt conformation of CCK-AR. We determined whether the mutants were expressed at the cell surface by performing binding of antagonist radioligands. The nonpeptide CCK-AR antagonist radioligand
[3H]SR-27,897 and the peptide antagonist
125I-BH-JMV 179 were used for this purpose. The
(R336M)-CCK-AR mutant bound [3H]SR-27,897 to a single
class of binding sites that exhibited a 8-fold lower affinity than the
wild-type receptor ((R336M)-CCK-AR: Kd, 20.1 ± 2.6 nM, n = 3; (WT)-CCK-AR:
Kd, 2.5 ± 0.2 nM,
n = 6, data not shown), and was expressed at the cell surface at similar levels to (WT)-CCK-AR as shown by maximal binding capacities ((R336M)-CCK-AR: Bmax, 2.7 ± 0.7 pmol/106 cells; (WT)-CCK-AR:
Bmax, 3.7 ± 0.7 pmol/106
cells, data not shown). The (N333A)-CCK-AR mutant did not bind [3H]SR-27,897; however, it did bind
125I-BH-JMV 179. Scatchard analysis of the binding
demonstrated a single class of binding sites having similar affinity to
(WT)-CCK-AR ((N333A)-CCK-AR: Kd, 9.1 ± 2.6 nM, Bmax, 0.7 ± 0.1 pmol/106 cells, n = 4; (WT)-CCK-AR:
Kd, 8.0 ± 0.2 nM,
Bmax, 3.6 ± 1.6 pmol/106
cells, n = 3, data not shown). Although the binding
capacity of this mutant was decreased by 5-fold, the inability of the
two receptor mutants to bind CCK is unlikely to be due to any
differences in expression levels.
Effects of R336M and N333A Mutations on Functional Coupling of
CCK-AR to Phospholipase C--
The functionality of the mutant
receptors was evaluated by determining inositol phosphate accumulation
in transfected COS-7 cells. The (R336M)- and (N333A)-CCK-AR mutants
mediated CCK-induced activation of phospholipase C with 9300- and
1351-fold lower potencies (Fmut) than that of
(WT)-CCK-AR, respectively (Fig. 2).
Maximal responses (efficacy) obtained with these mutants reached 81 and 60% of that achieved with (WT)-CCK-AR. When Arg-336 was exchanged for
an Ala instead of a Met, similar results were obtained (not illustrated). Thus, Arg-336 and Asn-333 likely play a critical role in
CCK-AR receptor activation by CCK.
Pharmacological and Functional Evidence for an Interaction between
Arg-336 of CCK-AR and the Asp-8 Carboxylate of CCK--
To verify
experimentally the predictions from molecular modeling that suggest
Arg-336 as the amino acid of CCK-AR that interacts with the Asp-8 of
CCK through salt bridge interactions, we first determined the affinity
of the (R336M)-CCK-AR mutant for several CCK-related peptides by
performing competition binding experiments using
[3H]SR-27,897 (Fig. 3). This antagonist radioligand
allows to detect binding of CCK to the very low affinity state of
(WT)-CCK-AR (24). The (R336M)-CCK-AR mutant bound sulfated CCK with a
117-fold lower affinity than (WT)-CCK-AR. In contrast, (R336M)-CCK-AR
bound (Ala-8)-CCK with only a 2-fold lower affinity than did
(WT)-CCK-AR (Table I). This result is
consistent with the view that Arg-336 of CCK-AR interacts with Asp-8 of
CCK.
We then exchanged Arg-336 for an aspartic acid and Asp-8 of CCK for an
arginine in order to reverse the charges on putative interacting
partners and analyzed the properties of the resulting receptor-ligand
complexes. Competition binding using [3H]SR-27,897 was
performed. As shown in Fig. 4,
A and B, although neither binding of unmodified
CCK to (R336D)-CCK-AR nor binding of (Arg-8)-CCK to (WT)-CCK-AR could
be detected (Ki
Exchange of Arg-336 of CCK-AR for an aspartic acid caused a 13,400-fold
decrease in the potency of the receptor to produce inositol phosphate
in response to native CCK, the efficacy of the mutant being 65% of
that of (WT)-CCK-AR (Fig. 4C). Similar to the substitution
of Arg-336 in CCK-AR, substitution of Asp-8 of CCK by an arginine led
to a 12,900-fold decrease in the potency at (WT)-CCK-AR to induce
inositol phosphate production (Fig. 4D). The efficacy of the
modified peptide (Arg-8)-CCK at (WT)-CCK-AR represented 35% of that of
native CCK. Surprisingly, the (R336D)-CCK-AR mutant did not produce any
detectable inositol phosphates in response to stimulation by
(Arg-8)-CCK. Therefore, although the individual exchange of Arg-336 of
CCK-AR for an Asp or Asp-8 of CCK for an Arg produced similar drops in
biological potency of the CCK-AR·CCK complexes, simultaneous
inversion of residues on both the receptor and CCK yielded a
nonfunctional, low affinity CCK-AR·CCK complex.
The fact that the (R336M)-CCK-AR mutant bound the nonpeptide antagonist
SR-27,897 with a slightly lower affinity (8-fold, Table I) than
(WT)-CCK-AR suggests the existence of interactions between Arg-336
side-chain and a negatively charged chemical function of SR-27,897. The
presence of an acetic acid moiety in SR-27,897 may account for the
existence of such an interaction (Fig.
3). In agreement with this hypothesis,
the (R336D)-CCK-AR mutant bound SR-27,897 with a 92-fold lower affinity
than (WT)-CCK-AR (Ki, 229 ± 81 nM,
n = 3, versus 2.5 ± 0.2 nM, n = 3, Fig.
5A). Therefore, exchange of
Arg-336 of CCK-AR for an aspartic acid instead of a methionine caused a
more pronounced loss in the affinity of CCK-AR for SR-27,897, as was
observed for CCK binding. L-364,718 is another highly selective and
potent nonpeptide CCK-AR antagonist that is of interest because it does
not possess a carboxylate function (Fig. 1). Competition binding
demonstrated that binding of this antagonist to the (R336D)-CCK-AR
mutant occurred with only a slightly lower affinity (5-fold) than to
(WT)-CCK-AR (Ki, 2.4 ± 0.4 nM,
n = 3, versus 0.5 ± 0.1 nM, n = 3, Fig. 5B). Competition binding experiments using [3H]SR-27,897 indicated that
the (R336M)-CCK-AR mutant binds CCK-related peptides JMV 179 and JMV
180 with 30- and 21-fold lower affinities than (WT)-CCK-AR,
respectively (Table I). All these results with antagonists fully
confirms the conclusion that Arg-336 interacts with the Asp-8
carboxylate of CCK. They also show that Arg-336 is a common determinant
for interactions with CCK, CCK-related peptides JMV 179 and 180, and
the nonpeptide antagonist SR-27,897, which possess a carboxylate
function.
Pharmacological and Functional Evidence for an Interaction between
Asn-333 of CCK-AR and the C-terminal Amide of CCK--
In order to
verify experimentally that Asn-333 of CCK-AR interacts with the
C-terminal amide of CCK, competition binding using the peptide
antagonist 125I-BH-JMV 179 was performed. The results
indicated that the (N333A)-CCK-AR mutant bound JMV 179 and JMV 180, which lack the C-terminal carboxy-amide, with the same affinity as
(WT)-CCK-AR (Table I). The mutant binds sulfated and nonsulfated CCK
with 10-fold decreased affinities and (PheCH3)-CCK with a 4.5-fold
decreased affinity relative to (WT)-CCK-AR (Table I).
We then evaluated the contribution of the amide to the ability of CCK
to stimulate inositol phosphate production in COS-7 cells expressing
(WT)-CCK-AR. As shown in Fig. 6, the CCK
analogue having the C-terminal amide substituted by a methyl carbonyl
((PheCH3)-CCK ) was 7021-fold less potent than unmodified
CCK. This value is of the same order as the 1350-fold decrease of
potency caused by exchange of Asn-333 of CCK-AR for an Ala (Fig. 6).
Moreover, in contrast to (WT)-CCK-AR, the (N333A)-CCK-AR mutant
responded to (PheCH3)-CCK with only a 11.4-fold lower
potency than to unmodified CCK, and the potency of the (N333A)-CCK-AR
mutant was only 2-fold lower than that of (WT)-CCK-AR when they were
stimulated by (PheCH3)-CCK (Fig. 6). All these data are in
favor of interactions between Asn-333 and the amide of CCK.
Because the (N333A)CCK-AR mutant failed to bind
[3H]SR-27,897, we postulated an involvement of Asn-333 of
CCK-AR in the binding of SR-27,897 antagonist. The aminocarbonyl
linking the chlorophenyl thiazol to the indol moieties of SR-27897 is a
chemical function that, like the amide of CCK, could interact with
Asn-333 of CCK-AR. Competition binding using 125I-BH-JMV
179 indicated that the (N333A)-CCK-AR mutant could bind SR-27,897, but
with a 136-fold lower affinity than (WT)-CCK-AR, supporting the
existence of interactions between Asn-333 and the nonpeptide antagonist
(Table I). To assess further the existence of such an interaction, we
determined the affinity of the (N333A)-CCK-AR mutant for L-364,718 that
is structurally distinct from SR-27,897 but also possesses an
aminocarbonyl moiety. As shown in Table I, (N333A)-CCK-AR mutant bound
the antagonist L-364,718 with a 13-fold lower affinity than
(WT)-CCK-AR. In agreement with the loss in affinity, SR-27,897 and
L-364,718 antagonists were 113- and 31-fold less potent in inhibiting
CCK-stimulated production of inositol phosphates in COS-7 cells
expressing the (N333A)-CCK-AR mutant relative to cells expressing
(WT)-CCK-AR (Fig. 7, A and B). In contrast, JMV 179 and JMV 180, which were recognized
with the same affinity by (N333A)- and (WT)-CCK-AR, inhibited
CCK-induced production of inositol phosphates with an equal potency on
cells expressing (N333A)- and (WT)-CCK-AR (Fig. 7, C and
D). Note that unlike SR-27,897 and L-364,718, JMV 180 and
JMV 179 lack the C-terminal amide (Fig. 3).
In the current study, we have identified two new residues, Arg-336
and Asn-333 of the human CCK-AR, that are crucial for binding and
biological activity of CCK. We have demonstrated that these two amino
acids interact with the Asp-8 side-chain and C-terminal amide of CCK,
respectively, and that they play a crucial role in both the binding and
functional properties of CCK-AR. In addition we have shown that, unlike
previously identified amino acids (Trp-39/Gln-40 and Met-195/Arg-197)
(23-25), the two newly identified residues are involved differentially
in the binding site of peptide and nonpeptide antagonists of
CCK-AR.
The identification of Arg-336 and Asn-333 was reached after
optimization of CCK docking into the three-dimensional model of CCK-AR.
For this optimization, we constrained the CCK peptide through
interactions between Trp-39 and Gln-40, located at the upper part of
the first transmembrane domain and the N-terminal moiety of CCK, and
between Met-195 and Arg-197, located within the second extracellular
loop and the sulfated tyrosine of CCK (23, 24). This procedure allowed
us to position the Asp-8 carboxylate side-chain of CCK in interaction
with the Arg-336 guanidium and the C-terminal amide of CCK in contact
with Asn-333. Exchange of Arg-336 and Asn-333 for amino acids lacking
the chemical functions expected to interact with CCK resulted in
dramatic decreases in both the affinity of CCK-AR for CCK and of its
potency to mediate CCK-stimulated production of inositol phosphates.
These experimental data suggested that Arg-336 and Asn-333 are involved
in the CCK-AR binding site for CCK. Because the observed losses in
function are not sufficient to distinguish between direct and indirect effects caused by the mutations, additional experiments were conducted.
Experimental data demonstrating the interaction between Arg-336 and the
Asp-8 carboxylate of CCK were obtained. First, binding experiments
revealed that exchange of Arg-336 for a Met strongly affected the
affinity of the CCK-AR mutant for CCK-related peptides containing an
Asp carboxylate, whereas affinity for (Ala8)-CCK remained nearly
constant. Second, exchange of Arg-336 of CCK-AR for an Asp caused a
decrease in the potency of the mutated receptor when stimulated by
unmodified CCK that was equal to that observed when the wild-type
CCK-AR was stimulated by the CCK analogue having Asp-8 substituted by
an Arg. This decrease in potency was larger than those obtained when
Arg-336 and Asp-8 were individually exchanged for noncharged residues,
clearly indicating that they result from repulsive forces that were
introduced in the CCK-AR·CCK complexes. Third, and more importantly,
the simultaneous double mutation Arg-336 Experimental data also support the fact that Asn-333 is the amino acid
of the CCK-AR binding site that interacts with the C-terminal amide of
CCK. Indeed, functional analysis of (N333A)-CCK-AR indicated that, in
contrast to (WT)-CCK-AR, the mutant poorly discriminated between
(PheCH3)-CCK and unmodified CCK. It also responded to
(PheCH3)-CCK with only a 2-fold lower potency than did
(WT)-CCK-AR, whereas it responded to unmodified CCK with 1350-fold lower potency than (WT)-CCK-AR. Moreover, (N333A)-CCK-AR bound 125I-BH-JMV 179, the structure of which is lacking the
C-terminal amide as (WT)-CCK-AR (Fig. 3). The binding properties of
(N333A)-CCK-AR toward nonpeptide antagonists that possess a
carboxyamide moiety are clearly in favor of an interaction between
Asn-333 and the carboxyamide of CCK ligands. Indeed, (N333A)-CCK-AR had
a decreased affinity for SR-27,897 as well as for L-364,718, suggesting
that Asn-333 is a crucial residue of the CCK-AR binding site shared by
CCK and the nonpeptide antagonists SR-27,897 and L-364,718.
To our knowledge, Arg-336 of CCK-AR has not been previously mutated by
others. In contrast, mutation of an Asn residue of the rat CCK-AR that
corresponds to Asn-333 in the human CCK-AR was reported to affect
receptor sensitivity to both agonist (CCK) and nonpeptide antagonist
(L-364,718). The authors suggested that the observed effects were due
to receptor expression default (33). The results from our study
confirmed that exchange of Asn-333 for an Ala diminishes receptor
expression at the cell surface. More importantly, they demonstrated
direct involvement of Asn-333 in ligand recognition. In the human
CCK-B/GR, exchange of Asn-353, homologous to Asn-333 of the human
CCK-AR, for an Ala decreased affinity of the receptor for CCK (34).
Mutagenesis data so far available on the CCK-B/GR indicate that the CCK
binding site of this receptor probably differs from that of CCK-AR,
although it presents some similarities. Other authors who have mutated
numerous amino acids in transmembrane domains of the CCK-B/GR have
demonstrated that these amino acids did not play a critical role in CCK
binding. In contrast, several amino acids within extracellular regions, particularly at the top of the first transmembrane domain and in the
first and second extracellular loops, were found to be critical for CCK
binding and activity (36). According to these different reports, the
major energetic contribution to CCK binding to CCK-B/GR is likely to be
conferred by extracellular residues. In CCK-AR, in addition to amino
acids from extracellular regions (Trp-39/Gln-40 and Met-195/Arg-197),
Asn-333 and Arg-336, which are located at the top of the sixth
transmembrane domain, strongly contribute to CCK recognition.
Pharmacological and functional data showing the importance of
Arg-336-Asp-8 and Asn-333-amide interactions are in agreement with the
first structure/function studies on CCK peptides using biological
models naturally expressing CCK-AR. Indeed, it was shown that
substitution of the Asp equivalent to Asp-8 of CCK-9 used in the
current work by an Ala, a Glu, or a Concerning the mechanisms whereby G protein-coupled receptors are
activated by their agonist ligands, the ternary complex model and the
analysis of constitutively active G protein-coupled receptors led us to
consider that receptors spontaneously interchange between several
conformations and that the agonist exerts its biological effect either
by selecting or by stabilizing and inducing (or both) an active
conformation (40). In other words, binding of the agonist allows for a
reduction in the energy barrier for a transition from an inactive to an
active state of the receptor. In order to better understand the
mechanisms that govern CCK-AR activation, additional studies, including
those that will identify residues that interact with the aromatic ring
of the C-terminal Phe and the Trp of CCK are necessary. However, in the
course of our work regarding mapping of CCK-AR agonist binding site, we have found that several residues of CCK, such as the sulfated tyrosine,
that are crucial for high affinity binding and biological activity, are
much less important for binding to low and very low affinity states of
CCK-AR (24). The results were interpreted as an indication that these
residues of CCK contribute to stabilization of the high affinity state
of the receptor-ligand complex. Our previous study, which analyzed the
pharmacological and the functional properties of the (M195L)-CCK-AR
mutant, extended this conclusion to amino acids of the receptor that
interact with these crucial residues of CCK (24). Results from the
current study, showing that the (R336D)-CCK-AR·(Arg-8)-CCK complex
had an affinity identical to that resulting from binding of CCK to the
very low affinity state of (WT)-CCK-AR but remained inactive, confirm
that correct positioning of pairing amino acids at the binding site is
a prerequisite for occurrence of an active CCK-AR·CCK complex. These
results illustrate how two-dimensional mutagenesis applied to putative peptide binding site of G protein-coupled receptors is a difficult task
in term of gaining function.
Direct interactions of peptide ligands with their receptors have only
been reported in a limited number of cases. Recently, in the secretin
receptor, interactions between two basic residues and secretin-Asp-3
were characterized using a strategy of two-dimensional site-directed
mutagenesis (41). In the AT1 angiotensin-II receptor, two extracellular
residues, His-183 of the second extracellular loop and Asp-281 of the
third extracellular loop were shown to interact with the N-terminal
residues Asp-1 and Arg-2 of angiotensin-II (42). In contrast, amino
acids of the receptor that interact with residues of the C-terminal
half of angiotensin-II, which is essential for binding and biological
activity, were identified within transmembrane domains III, V, and VI
(43). In the NK-1 neurokinin receptor, several extracellular residues
in addition to residues from the upper part of transmembrane domains
were shown to directly interact with the natural ligand, Substance P
(44-46). Interestingly, binding sites for nonpeptide antagonists on
these two receptors were localized in a pocket within transmembrane domains and appear therefore to be composed almost exclusively of amino
acids distinct from those of the binding site for peptide agonists (20,
47, 48). Such findings imply that in these receptors, nonpeptide
antagonists exclude the binding of agonist by allosteric regulation of
the receptor conformation (20). With respect to this point, our data on
CCK-AR showing that Asn-333 and to a lesser extent Arg-336 are likely
to be directly involved in the binding of nonpeptide antagonists
SR-27897 and L-364,718, as well as in that of CCK, are original and
indicate that no simple generalization can be made as to where ligands
bind to G protein-coupled receptors.
In conclusion, the amino acids identified in this study (Arg-336 and
Asn-333) together with those from previous studies (Trp-39, Gln-40,
Met-195, and Arg-197) (23-25) indicate that the agonist binding site
of CCK-AR is made up of several hydrophilic amino acids. Electrostatic
interactions are therefore likely to represent the critical driving
force for CCK binding to the high affinity active state of CCK-AR. Our
data will be used to obtain an integrated dynamic view of the molecular
processes that link agonist binding to receptor activation.
We thank Dr. Karen Kennedy for reading the manuscript.
*
This study was supported by Grants 6234 and 9257 from the
Association pour la Recherche sur le Cancer and Grant 9407555 from Région Midi-Pyrénées.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.
The abbreviations used are:
CCK-AR, cholecystokinin receptor-A;
WT, wild-type.
Arginine 336 and Asparagine 333 of the Human Cholecystokinin-A
Receptor Binding Site Interact with the Penultimate Aspartic Acid and
the C-terminal Amide of Cholecystokinin*
,,,
,, and
INSERM U151, Institut Louis Bugnard, Centre
Hospitalier Universitaire Rangueil, Bat. L3, 31403 Toulouse
Cedex 4, France, the ¶ Laboratoire de Chimie Théorique, Max-Planck-Institut für
Biochemie, 82143 Martinsried, Germany, and ** Sanofi-Recherche,
195 route d'Espagne, 31036 Toulouse Cedex, France
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amidation of the
C-terminal phenylalanine residue (7). Studies using synthetic fragments
have shown that this sulfation and the amidation of the octapeptide are
required for full biological activity and that the C-terminal
tetrapeptide Trp-Met-Asp-Phe-NH2 corresponds to the minimal
fragment endowed with biological activity (8). Elimination of the
carboxy-amidated phenylalanine in CCK switches the activity of the
molecule from a full agonist on pancreatic amylase secretion to a
partial agonist or an antagonist according to the animal species used
(9, 10). Some antagonistic molecules were synthetized on the basis of
the importance of the carboxy-amidated phenylalanine for full
biological activity. Two such molecules, JMV 180 and JMV 179, have been
used extensively for the characterization of CCK-AR (11-15). Evidence
exists to show that JMV 180 induces distinct pharmacological effects
and signal transduction pathways from those of the natural agonist CCK
through binding to CCK-AR (15). These studies suggest that interactions
between the C-terminal amidated phenylalanine of CCK and so far unknown
amino acid(s) within the CCK-AR agonist binding site are important for
full activation of the receptor. The tryptophan and aspartic acid
within the C-terminal tetrapeptide of CCK are also key amino acids (16, 17). The structure of several classes of CCK-AR antagonists and
agonists contain structural elements that resemble tryptophan and
aspartic acid side-chains, suggesting that all ligands may share
determinants of the CCK-AR binding site (3, 18).
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
)2,3-dihydro-1-methyl-2oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-1H-indole-2-carboxamide was donated by Merck Sharp and Dohme. Na125I was from
Amersham Pharmacia Biotech. (Thr,Nle)CCK-9 and JMV 179 were conjugated
with Bolton-Hunter reagent, purified, and radioiodinated as described
previously (27). The specific activity of radioiodinated peptide was
1600-2000 Ci/mmol. All other chemicals were obtained from commercial sources.
-radioactivity was counted.
RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
amide, which is
present in half of neuropeptides and peptide hormones (31), is
essential for CCK activity as demonstrated by the partial agonist/antagonist properties of CCK derivatives lacking the amide
(11, 12, 32).
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Fig. 1.
Simplified representation of the CCK-A
receptor and of its agonist binding site. a, serpentine
representation of the human CCK-AR with the amino acids involved in
binding and activity marked. The current study concerns identification
of amino acids Arg-336 and Asn-333 (red circles). Other
mentioned amino acids Trp-39/Gln-40 and Met-195 were previously
demonstrated to interact with the N-terminal moiety and the tyrosine
aromatic ring of CCK, respectively (23, 24). Arg-197 was demonstrated
to bound the sulfate group of CCK (25). b and c,
side views of the three-dimensional model of the active high affinity
CCK-AR·CCK complex. The model was built as described under
"Experimental Procedures" using a program package from Molecular
Simulations Inc.(San Diego, CA). For clarity, the detailed view
shows only identified amino acid side-chains in interaction with CCK in
the phospholipase C-coupled high affinity CCK-AR·CCK complex.
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[in a new window]
Fig. 2.
Loss of production of inositol phosphate
following mutation of residues Arg-336 and Asn-333 in CCK-AR.
Inositol production assays were conducted as described under
"Experimental Procedures." Results with all of the CCK-AR mutants
are expressed as percentage of maximal inositol phosphate production
obtained in COS-7 expressing the wild-type CCK-AR after stimulation by
unmodified CCK ((WT)/CCK complex). D50
(concentration of agonist producing 50% of maximal response) was
determined using the GraphPad Prism program and the mutation factors
(Fmut) were calculated as
D50 (mutated
receptor)/D50 ((WT)-CCK-AR).
View larger version (28K):
[in a new window]
Fig. 3.
Ligands used to map the CCK-AR
binding site.
Summary of the binding properties of the wild-type and mutated CCK-AR
100,000 nM),
binding of (Arg-8)-CCK to the (R336D)-CCK-AR mutant occurred with an
apparent affinity that was close to that of unmodified CCK for the very
low affinity state of (WT)-CCK-AR (2683 ± 330 nM,
n = 3 versus 1873 ± 331 nM, n = 6). Unmodified CCK and (Arg-8)-CCK bound with intermediate affinities to (R336M)-CCK-AR
(Ki 
220,000 nM and 23,300 nM, respectively, data not shown). These results, showing
that interchange of partner amino acids Arg-336/Asp-8 produced a gain
of affinity, are consistent with the view that Arg-336 is the residue
of CCK-AR in interaction with Asp-8 of CCK.
View larger version (30K):
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Fig. 4.
Effect of mutations Arg-336 
Asp in CCK-AR
and Asp-8 Arg in CCK on CCK-AR·CCK complex formation. COS-7
cells expressing (WT)-CCK-AR or (R336D)-CCK-AR were incubated in
presence of the nonpeptide antagonist radioligand
[3H]SR-27,897 alone or in the presence of increasing
concentrations of unmodified CCK (A) or (Arg-8)-CCK
(B) as described under "Experimental Procedures."
Binding is expressed as percentage of specific binding in the absence
of competitor. Results are mean of at least three individual
determinations. The data indicate that simultaneous double-mutation in
CCK-AR and in CCK yielded a complex ((R336D)-CCK-AR·(Arg-8)-CCK), the
affinity of which is identical to that resulting from the binding of
CCK to the very low affinity state of the wild-type CCK-AR
((WT)-CCK-AR·CCK complex). In the bottom graphs
(C and D), effects of the single mutations
Arg-336 Asp in CCK-AR and Asp-8 Arg in CCK on inositol
phosphate production are shown. D50 values were
as follows: (WT)-CCK-AR·CCK, 0.47 ± 0.15 nM;
(R336D)-CCK-AR·CCK, 6315 ± 425 nM;
(WT)-CCK-AR·(Arg-8)-CCK, 6060 ± 670 nM
(n = 3). The (R336D)-CCK-AR·(Arg-8)-CCK complex did
not produce detectable inositol phosphates (data not shown).
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Fig. 5.
Effect of the Arg-336 Asp mutation on
CCK-AR affinity for the nonpeptide antagonists SR-27,897 and
L-364,718. COS-7 cells expressing (WT)-CCK-AR or (R336D)-CCK-AR
were incubated in presence of nonpeptide antagonist radioligand
[3H]SR-27,897 alone or in the presence of increasing
concentrations of SR-27,897 (A) or L-364,718 (B)
as described under "Experimental Procedures." Binding is expressed
as a percentage of specific binding in the absence of competitor.
Results are the mean of at least three individual determinations.
View larger version (16K):
[in a new window]
Fig. 6.
Effects of the Asn-333 Ala mutation in
CCK-AR and amide elimination in CCK on inositol phosphate
production. The experiments were conducted as described under
"Experimental Procedures," and results are expressed as indicated
in Fig. 2. The D50 values were as follows:
(WT)-CCK-AR·CCK, 0.47 ± 0.15 nM;
(WT)-CCK-AR·(PheCH3)CCK, 3300 ± 1227 nM; (N333A)-CCK-AR·CCK, 635 ± 171 nM;
(N333A)-CCK-AR·(PheCH3)CCK, 7250 ± 488 nM (n = 3-6).
View larger version (13K):
[in a new window]
Fig. 7.
Effect of the Asn-333 Ala mutation on
inhibition of CCK-induced inositol phosphate production by CCK-AR
antagonists and peptide partial agonist JMV 180. COS-7 cells
expressing (WT)-CCK-AR or (N333A)-CCK-AR were incubated in presence of
unmodified CCK alone (0.5 nM for (WT)-CCK-AR and 500 nM for (N333A)-CCK-AR) or in the presence of increasing
concentrations of SR-27,897 (A), L-364,718 (B),
JMV 179 (C), or JMV 180 (D). Inositol phosphate
synthesis was measured as described under "Experimental Procedures"
and is expressed as a percentage of inositol phosphates in the absence
of competitor. Results are the mean of three individual determinations.
IC50 values were as follows: (WT)-CCK-AR·SR-27,897,
5.1 ± 1.2 nM (N333A)-CCK-AR·SR-27,897, 579 ± 154 nM; (WT)-CCK-AR·L-364,718, 1.6 ± 0.7 nM; (N333A)-CCK-AR·L-364,718, 49.3 ± 5.4 nM; (WT)-CCK-AR·JMV 179, 21.0 ± 2.5 nM;
(N333A)-CCK-AR·JMV 179, 24.7 ± 4.5 nM;
(WT)-CCK-AR·JMV 180, 17.1 ± 6.5 nM;
(N333A)-CCK-AR·JMV 180, 10.9 ± 2.0 nM
(n = 3).
DISCUSSION
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Asp in CCK-AR and Asp-8
Arg in CCK yielded a CCK-AR·CCK complex
((R336D)-CCK-AR·(Arg-8)-CCK), the affinity of which was identical to
that resulting from CCK binding to the very low affinity state of the
wild-type CCK-AR ((WT)-CCK-AR·CCK). The specific and direct roles of
Arg-336 in the CCK-AR binding site was further supported by the
differential effect of its mutation on antagonist binding. Indeed,
Arg-336 mutation caused stronger changes in receptor affinity for
CCK-related peptides and SR-27897 than for L-364,718. Structural
differences between these compounds account for such data because only
CCK-related peptides and SR-27,897 contain a carboxylate residue (Fig.
3).
-Ala decreases biological
potency of CCK on gall bladder and pancreatic acinar cells by several
hundred fold (17, 37, 38). Moreover, the importance of the C-terminal
amide for biological activity of CCK has been demonstrated in
structure-function studies with a series of CCK peptides having
modified C termini. In fact, CCK peptides in which the C-terminal
phenylalanine was replaced by a phenylethylamide
(Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-NH-CH2CH2-C6H5) stimulated amylase release from rat pancreatic acini with a 1000-fold lower potency than amidated CCK, whereas it had only a 50-fold lower
affinity for CCK-AR (11). JMV 180 is the most commonly used peptide of
this series of CCK analogues. JMV 180 has its C-terminal phenylalanine
substituted by a phenylethyl ester (Fig. 3). It was shown that JMV 180 binds to native rat pancreatic CCK-AR with a 10-fold lower affinity
than amidated CCK and that it behaves as a partial agonist on amylase
release (11, 15, 39). Although it is well recognized that JMV 180 induces distinct pharmacological effects and intracellular signals from
CCK, the underlying molecular mechanisms remain to be precisely determined.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed. Tel.:
33-5-61-32-24-05; Fax: 33-5-61-32-24-03; E-mail:
Daniel.Fourmy@rangueil.inserm.fr.
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 M. Dufresne, C. Seva, and D. Fourmy Cholecystokinin and gastrin receptors. Physiol Rev, July 1, 2006; 86(3): 805 - 847. [Abstract] [Full Text] [PDF]
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I. Langer, I. G. Tikhonova, M.-A. Travers, E. Archer-Lahlou, C. Escrieut, B. Maigret, and D. Fourmy Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin J. Biol. Chem., June 10, 2005; 280(23): 22198 - 22204. [Abstract] [Full Text] [PDF]
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E. Archer-Lahlou, C. Escrieut, P. Clerc, J. Martinez, L. Moroder, C. Logsdon, A. Kopin, C. Seva, M. Dufresne, L. Pradayrol, et al. Molecular Mechanism Underlying Partial and Full Agonism Mediated by the Human Cholecystokinin-1 Receptor J. Biol. Chem., March 18, 2005; 280(11): 10664 - 10674. [Abstract] [Full Text] [PDF]
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 C. Gales, M. Poirot, J. Taillefer, B. Maigret, J. Martinez, L. Moroder, C. Escrieut, L. Pradayrol, D. Fourmy, and S. Silvente-Poirot Identification of Tyrosine 189 and Asparagine 358 of the Cholecystokinin 2 Receptor in Direct Interaction with the Crucial C-Terminal Amide of Cholecystokinin by Molecular Modeling, Site-Directed Mutagenesis, and Structure/Affinity Studies Mol. Pharmacol., May 1, 2003; 63(5): 973 - 982. [Abstract] [Full Text] [PDF]
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K. G. Harikumar, D. I. Pinon, W. S. Wessels, F. G. Prendergast, and L. J. Miller Environment and Mobility of a Series of Fluorescent Reporters at the Amino Terminus of Structurally Related Peptide Agonists and Antagonists Bound to the Cholecystokinin Receptor J. Biol. Chem., May 17, 2002; 277(21): 18552 - 18560. [Abstract] [Full Text] [PDF]
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X.-Q. Ding, D. I. Pinon, K. E. Furse, T. P. Lybrand, and L. J. Miller Refinement of the Conformation of a Critical Region of Charge-Charge Interaction between Cholecystokinin and Its Receptor Mol. Pharmacol., May 1, 2002; 61(5): 1041 - 1052. [Abstract] [Full Text] [PDF]
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C. Escrieut, V. Gigoux, E. Archer, S. Verrier, B. Maigret, R. Behrendt, L. Moroder, E. Bignon, S. Silvente-Poirot, L. Pradayrol, et al. The Biologically Crucial C Terminus of Cholecystokinin and the Non-peptide Agonist SR-146,131 Share a Common Binding Site in the Human CCK1 Receptor. EVIDENCE FOR A CRUCIAL ROLE OF MET-121 IN THE ACTIVATION PROCESS J. Biol. Chem., February 22, 2002; 277(9): 7546 - 7555. [Abstract] [Full Text] [PDF]
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 M. C. Gershengorn and R. Osman Minireview: Insights into G Protein-Coupled Receptor Function Using Molecular Models Endocrinology, January 1, 2001; 142(1): 2 - 10. [Abstract] [Full Text] [PDF]
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X.-Q. Ding, V. Dolu, E. M. Hadac, E. L. Holicky, D. I. Pinon, T. P. Lybrand, and L. J. Miller Refinement of the Structure of the Ligand-occupied Cholecystokinin Receptor Using a Photolabile Amino-terminal Probe J. Biol. Chem., February 2, 2001; 276(6): 4236 - 4244. [Abstract] [Full Text] [PDF]
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K. Tokita, S. J. Hocart, T. Katsuno, S. A. Mantey, D. H. Coy, and R. T. Jensen Tyrosine 220 in the 5th Transmembrane Domain of the Neuromedin B Receptor Is Critical for the High Selectivity of the Peptoid Antagonist PD168368 J. Biol. Chem., January 5, 2001; 276(1): 495 - 504. [Abstract] [Full Text] [PDF]
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K. Tokita, T. Katsuno, S. J. Hocart, D. H. Coy, M. Llinares, J. Martinez, and R. T. Jensen Molecular Basis for Selectivity of High Affinity Peptide Antagonists for the Gastrin-releasing Peptide Receptor J. Biol. Chem., September 21, 2001; 276(39): 36652 - 36663. [Abstract] [Full Text] [PDF]
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