From the INSERM U151, Institut Louis Bugnard, CHU de
Rangueil, Bat. L3, 31403 Toulouse Cedex, France,
§ Laboratoire de chimie théorique, Université de
Nancy, 54506 Vandoeuvre les Nancy, France, ¶ CNRS URA 1845, Faculté de Pharmacie, 34060 Montpellier, France,
Sanofi-Recherche, 195 route d'Espagne, 31036 Toulouse Cedex,
France, and ** Max Planck Institut für biochemie,
82143 Martinsried, Germany
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ABSTRACT |
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Sulfation of the tyrosine at the seventh position from the C terminus of cholecystokinin (CCK) is crucial for CCK binding to the CCK-A receptor. Using three-dimensional modeling, we identified methionine 195 of the CCK-A receptor as a putative amino acid in interaction with the aromatic ring of the sulfated tyrosine of CCK. We analyzed the role played by the two partners of this interaction. The exchange of Met-195 for a leucine caused a minor decrease (2.8-fold) on the affinity of the high affinity sites for sulfated CCK-9, a strong drop (73%) of their number, and a 30-fold decrease on the affinity of the low and very low affinity sites for sulfated CCK-9, with no change in their number. The mutation also caused a 54-fold decrease of the potency of the receptor to induce inositol phosphates production. The high affinity sites of the wild-type CCK-A receptor were highly selective (800-fold) toward sulfated versus nonsulfated CCK, whereas low and very low affinity sites were poorly selective (10- and 18-fold). In addition, the M195L mutant bound, and responded to, sulfated CCK analogues with decreased affinities and potencies, whereas it bound and responded to nonsulfated CCK identically to the wild-type receptor. Thus, Met-195 interacts with the aromatic ring of the sulfated tyrosine to correctly position the sulfated group of CCK in the binding site of the receptor. This interaction is essential for CCK-dependent transition of the CCK-A receptor to a high affinity state. Our data should represent an important step toward the identification of the residue(s) of the receptor in interaction with the sulfate moiety of CCK and the understanding of the molecular mechanisms that govern CCK-A receptor activation.
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INTRODUCTION |
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The peptide cholecystokinin
(CCK)1 is found throughout
the gastrointestinal system and the central nervous system where it acts both as a hormone and a neurotransmitter (1). Post-translational processing of CCK involves sulfation of the tyrosine at position seven
from the C-terminal and -amidation of the C-terminal phenylalanine residue (1). Studies using chemically synthesized fragments have shown
that the C-terminal sulfated and amidated octapeptide Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2 (Fig.
1) exhibits the full spectrum of biological activity. However,
fragments as small as the C-terminal tetrapeptide
Trp-Met-Asp-Phe-NH2, which CCK has in common with the
related peptide gastrin, retain biological activity (2).
The actions of CCK are mediated by membrane receptors that are divided
into two subtypes, the CCK-A and the CCK-B/gastrin (3). The cloning of
the cDNA coding for these receptors has shown that they belong to
the superfamily of G protein-coupled receptors which are characterized
by seven transmembrane domains connected by intracellular and
extracellular loops with an extracellular N-terminal and intracellular
C-terminal (4, 5). Both receptor subtypes can exist in several affinity
states for sulfated CCK and have in common the functional coupling to
phospholipase-C, presumably via binding to a Gq/11
heterotrimeric GTP-binding protein (6-9).
The CCK-B/gastrin receptor binds CCK and gastrin with the same high
affinity and discriminates poorly between sulfated and nonsulfated
forms of the peptidic ligands (10, 11). In contrast, the CCK-A receptor
has a 300- to 1000-fold higher affinity for CCK than for gastrin and a
500-fold higher affinity for sulfated than for nonsulfated CCK (2,
3). These pharmacological properties indicate that the location of the
tyrosine at the seventh position from the C terminus of CCK and the
sulfation of this tyrosine are crucial for full binding and biological
activity of CCK at the CCK-A receptor. Accordingly, the CCK-A receptor
should contain amino acids within its agonist binding site that
specifically interact with the sulfated tyrosine moiety of CCK and are
responsible for the binding and biological selectivities of this
receptor for sulfated and nonsulfated CCK. Delineation of the agonist
binding site of the CCK-A receptor requires identification of such
amino acids.
Previously, using site-directed mutagenesis and molecular modeling, we had shown that two amino acids, Trp-39 and Gln-40 interact with the N-terminal region of CCK octa- and nonapeptides (12). We present in this work an optimized three-dimensional model of the CCK agonist·CCK-A receptor complex, which enabled us to propose that the residue methionine 195, located in the second extracellular loop of the CCK-A receptor, may interact with the tyrosyl residue of CCK. Such interactions between an aromatic ring and neighboring functional groups have been previously found in several proteins and shown to contribute to the overall stability of biological structures (13). Here we demonstrate that this interaction is required for agonist-dependent transition of the CCK-A receptor to a high affinity state, and for CCK's full binding and biological activity to the CCK-A receptor.
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EXPERIMENTAL PROCEDURES |
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Materials-- The C-terminal nonapeptide of CCK, [Thr28,Ahx31]CCK-25-33 ([Thr,Nle]CCK-9, named sulfated CCK-9), was synthesized by Luis Moroder (Max Planck Institut für Biochimie, Munchen, Germany). The sulfated and nonsulfated C-terminal octapeptides, named sulfated CCK-8 and nonsulfated CCK-8, were from Neosystem, Strasbourg, France. The other analogues of CCK, namely (Phe)-CCK-8, (Ala)-CCK-8, 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) (14) (see Fig. 1). 125INa was from Amersham, Les Ulis, France. [Thr,Nle]CCK-9 was conjugated with Bolton-Hunter reagent, purified, and radioiodinated as described previously (15). The specific activity of radioiodinated peptide was 1600-2000 Ci/mmol. Oligonucleotide primers were from Bioprobe Systems (Montreuil, France). All other chemicals were obtained from commercial sources.
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Computer Modeling of the CCK-A Receptor and CCK Agonist·CCK-A Receptor Complex-- The molecular model of the human CCK-A receptor was constructed as described previously (12). The docking of CCK into the receptor was achieved using manual preliminary positioning inside the receptor grove guided by the inspection of the molecular electrostatic potential representations of the ligand and receptor. Then, the docking was improved by intensive annealing calculations. The resulting structure obtained for the ligand-receptor complex was further refined using molecular dynamics and energy minimization. All the annealing refinement and docking calculations were produced using the Biosym/MSI molecular modeling package.
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 (16). Mutation was introduced into the human CCK-A receptor cDNA cloned into pRFENeo vector (17) using mutagenic primer based on the published human CCK-A receptor cDNA sequence (4, 5). Selection primers mutated a unique SmaI restriction site to a unique NruI site and vice versa. The mutation was confirmed by sequencing on an automated sequencer (Applied Biosystem). Two receptor mutants were constructed: in the first the methionine 195 was substituted by a leucine, in the second, the methionine 195 was subtituted by a glutamine, which is the equivalent amino acid in the human CCK-B/gastrin receptor.
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 an 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 post-transfection, 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 phosphates assay, the cells were resuspended in complete medium in the presence of 2 µCi/ml myo-2-[3H]inositol (Amersham Pharmacia Biotech) and incubated overnight in 24-well dishes.
Receptor Binding Assay on Cells or Membranes-- 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% BSA and then incubated for 60 min at 37 °C in 0.5 ml Dulbecco's modified Eagle's medium, 0.1% BSA with either 71 pM (wild-type receptor) or 350 pM (mutated receptors) 125I-BH-(Thr,Nle)CCK-9 or 1.83 nM [3H]SR-27,897 and in the presence or absence of competing agonists or antagonists. The cells were washed twice with phosphate-buffered saline, pH 6.95, containing 2% BSA, and cell-associated 125I-BH-(Thr,Nle)CCK-9 or [3H]SR-27,897 was collected with NaOH 0.1 N added to each well.
Plasma membrane from COS-7 cells were prepared as described previously in detail. Aliquots of 10-20-µg protein (18) were incubated in binding buffer containing 0.5 mg/ml BSA for 90 min at 25 °C (steady-state conditions) with 350 pM 125I-BH-(Thr,Nle)CCK-9 in the presence or absence of competing agonist. The binding reaction was stopped by the addition of 500 µl of ice-cold binding buffer, and bound radioligand was separated from the free fraction by centrifugation at 10,000 × g for 10 min at 4 °C. Pellets were washed two times more and the radioactivity was directly counted for radioiodinated ligands. Binding data were analyzed using the EBDA LIGAND program (19) or GraphPad Prism program (Software).Inositol Phosphate Assay-- Approximately 24 h after the transfer to 24-well 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 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 at pH 7.45 (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% BSA. 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 MeOH/HCl to each well, and the content was transferred to a column (Dowex AG 1-X8 formate form, Bio-Rad) to release 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 4 ml of 1 M ammonium formate, 100 mM formic acid.
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RESULTS |
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Identification of the Putative Amino Acid of the CCK-A Receptor in
Interaction with the Tyrosine of CCK--
A view of the
three-dimensional model of the CCK-A receptor is presented in Fig.
2. Besides the interactions that have
already been described and involved the Trp-39 and Gln-40 residues of the CCK-A receptor and the N-terminal part of CCK octa- and
nonapeptides (12), this three-dimensional model enabled us to identify
another interaction, which may explain the high selectivity of the
CCK-A receptor for sulfated versus nonsulfated CCK. The key
amino acid of the receptor involved in this additional interaction is
the methionine 195 located in the second extracellular loop of the CCK-A receptor. The sulfur atom of Met-195 side chain is shown on the
model to interact with the aromatic ring of the sulfated tyrosine of
the CCK agonist. In this interaction, the sulfur atom of Met-195 is
positioned toward the center of the aromatic ring of the tyrosine at a
distance of about 6 Å, and the methyl group attached to the sulfur
atom is pointed away from the aromatic plane. Such positioning is
typical of quadrupole/quadrupole interactions between a sulfur atom and
the electron cloud of an aromatic ring. Moreover, ab
initio calculations performed on a model built from toluene and
dimethylsulfur have clearly demonstrated that positioning of the
aromatic ring and sulfur atom shown in the model of the CCK-A
receptor·CCK complex is a favorable one (13).
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Analysis of the Contribution of Methionine 195 of the CCK-A Receptor to CCK Binding and CCK-A Receptor Function-- To evaluate the role of the putative interaction between Met-195 of the CCK-A receptor and the tyrosine of CCK, we first exchanged Met-195 for a leucine in the CCK-A receptor. Such a substitution, which essentially consists of the removal of the sulfur from the side chain of the amino acid, should eliminate the quadrupole/quadrupole interaction and therefore hinder the correct positioning of the sulfated group in the receptor binding site. In a second mutant receptor, the methionine 195 was subtituted by a glutamine that is the equivalent positioned amino acid in the human CCK-B/gastrin receptor. Since the human CCK-A receptor exists in three different affinity states for sulfated CCK, we evaluated the contribution Met-195 to these different affinity states (8, 9). We used the sulfated CCK-9 agonist, 125I-BH-(Thr,Nle)CCK-9, and the non-peptide antagonist, [3H]SR-27,897 as radioligands because they have the capability to detect the different affinity states of the receptor.
Scatchard analysis of competition binding between 125I-BH-(Thr,Nle)CCK-9 and sulfated CCK-9 to the wild-type receptor demonstrated two classes of binding sites, a high affinity site with a Kd of 0.56 ± 0.19 nM and a maximal binding capacity (Bmax) of 0.086 ± 0.020 pmol/106 cells, and a low affinity site with a Kd of 44 ± 16 nM and a maximal binding capacity of 2.0 ± 0.4 pmol/106 cells (Table I). On the other hand, the M195L mutant demonstrated a very low binding of 125I-BH-(Thr,Nle)CCK-9 when the standard radioligand concentration (71 pM) was used. By contrast, significant binding was observed in the presence of 350 pM of radioligand, allowing Scatchard analysis of this binding. In fact, the M195L mutant demonstrated two classes of binding sites similar to the wild-type receptor. The binding parameters were modified, the high affinity sites had a Kd of 1.59 ± 0.42 nM and a maximal binding capacity of 0.023 ± 0.005 pmol/106 cells, and the low affinity sites had a Kd of 1,310 ± 362 nM and a maximal binding capacity of 2.35 ± 0.80 pmol/106 cells (Table I).
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Analysis of the Contribution of the Sulfated Tyrosine to CCK Binding to the Wild-type Human CCK-A Receptor-- The effects of the mutation of Met-195 on the decrease of the number of high affinity sites for sulfated CCK-9 suggested a role of Met-195 in the transition of the CCK-A receptor to a high affinity state. Moreover, the three-dimensional model suggested that the interaction between the aromatic ring of the sulfated tyrosine of CCK and Met-195 of the CCK-A receptor serves to correctly position the sulfated moiety of the ligand within the receptor binding site. We therefore analyzed the contribution of the different chemical functions of the sulfated tyrosine to CCK binding to the three affinity states of the wild-type CCK-A receptor. For this, we tested new CCK analogues modified at the seventh position from the C terminus for their ability to interact with the wild-type CCK-A receptor.
Competition binding using 125I-BH-(Thr,Nle)CCK-9 showed that in contrast to sulfated CCK-9 or CCK-8, nonsulfated CCK-8 bound to the CCK-A receptor with a single affinity (Ki, 447 ± 105 nM) that was 800-fold lower than the affinity of sulfated CCK-9 for the high affinity sites and 10-fold lower than the affinity of sulfated CCK-9 for the low affinity sites (Fig. 4A). Competition binding using [3H]SR-27,897 showed that nonsulfated CCK-8 bound to the very low affinity sites of the CCK-A receptor with an affinity only 18-fold lower than the affinity of sulfated CCK-9 to these sites (Fig. 4B, Ki, 22,842 ± 4,776 nM. Thus, the sulfated moiety of sulfated CCK is highly important for binding to the high affinity sites of the human CCK-A receptor, whereas it is much less important for binding to the low and very low affinity sites.
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Demonstration of the Interaction between the Methionine 195 of the CCK-A Receptor and the Sulfated Tyrosine of CCK-- We attempted to experimentally verify that Met-195 of the CCK-A receptor was indeed in interaction with the sulfated tyrosine of CCK. We tested CCK-related peptides modified at the seventh position from the C terminus of CCK as well as JMV 179, JMV 180, (Phe)-CCK-8, and (Ala)-CCK-8 for their ability to bind to the M195L mutant. The two peptides, JMV 179 and JMV 180, possess the sulfated tyrosine but are antagonists of the human CCK-A receptor. We used [3H]SR-27,897 because this ligand bound identically to the M195L mutant and the wild-type receptor and could easily reveal the effect of the mutation on the affinity of the receptor for CCK. The binding and biological results are summarized in Table II.
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DISCUSSION |
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The aim of this work was to identify amino acids of the human CCK-A receptor that are in interaction with the sulfated tyrosine moiety of the natural agonist CCK. We hypothetized the presence of such amino acids in the CCK-A receptor on the basis of the main pharmacological characteristic of this receptor; namely its ability to bind sulfated CCK with a 500- to 1,000-fold higher affinity than nonsulfated CCK (2). To identify such putative amino acid(s), we used the three-dimensional model of the CCK agonist·CCK-A receptor complex (12). In this three-dimensional model, we focused our interest on the residue methionine 195, which represents a highly probable point of interaction with the sulfated tyrosine moiety of CCK, although it could not be involved in ionic interactions with the negatively charged sulfated group of CCK.
We first showed that the exchange of the methionine 195 for a leucine caused a minor decrease (2.8-fold) on the affinity of the high affinity sites for sulfated CCK-9 and a strong drop (73%) of their number, and a 30-fold decrease on the affinity of the low and very low affinity sites for sulfated CCK-9, with no change in their number. By contrast, the mutation did not affect binding of the nonpeptide antagonist SR-27,897. Finally, the mutation also caused a 54-fold decrease of the potency with which the CCK-A receptor coupled to phospholipase-C after stimulation by sulfated CCK-9.
Since the effects of the mutation of Met-195 on the decrease of the number of high affinity sites for sulfated CCK-9 suggested a role of Met-195 in the transition of the CCK-A receptor to a high affinity state, we then analyzed the contribution of the sulfated tyrosine to the CCK binding to the three affinity states of the wild-type human CCK-A receptor. We confirmed the data obtained with the biological models naturally expressing this receptor that nonsulfated CCK-8 has a 800-fold lower affinity than sulfated CCK for the high affinity sites of the CCK-A receptor and demonstrated that the phenyl moiety does not strongly contribute to binding of nonsulfated CCK. In addition, we showed that nonsulfated CCK-8 has only 10- to 18-fold lower affinity than sulfated CCK-9 for the low and very low affinity sites of the CCK-A receptor. Thus, we demonstrated for the first time that the low and very low affinity sites of the human CCK-A receptor exhibit a similar binding selectivity for sulfated and nonsulfated CCK as the native or cloned CCK-B/gastrin receptors (10, 11, 20, 21). This suggested a role of the sulfated tyrosine of CCK in the induction or stabilization of the high affinity state of the CCK-A receptor.
To discuss our results, we assumed that the CCK-A receptor behaves
according to the prevailing model of the ternary complex (TCM) for
activation of G protein-coupled receptors. This model, in its initial
version, describes the active form of a receptor as a ternary complex
(R*GL) between ligand (L), receptor
(R), and G protein (G), which is believed to
result from sequential binding of ligand and G protein, in either
order, to the receptor (22). This initial model was further refined and
now integrates the fact that the unliganded receptor naturally exists
as an equilibrium between receptors in an inactive
(R) and an active (R*) state (23).
In this revised TCM so-called "allosteric TCM" the ligand serves to
either select or induce the active state(s) of the receptor. A
simplified representation of this model accounting for our findings is
shown in Fig. 5. The three affinity
states of the receptor called R
,
R', and R* can lead to the formation of
corresponding complexes R
L(G),
R'L(G) and R*LG.
Results from this study indicate that the sulfated tyrosine plays a
crucial role in inducing or stabilizing the high affinity state of the
receptor, whereas appropriate mutation of Met-195, which is in
interaction with the sulfated tyrosine, caused a strong drop in the
number of high affinity sites for sulfated CCK. These concordant
results support the view that interaction between the sulfated tyrosine
and Met-195 plays a role in the receptor's transition to a high
affinity state (R*GL, route 1). On the other
hand, the receptor mutant M195L was still able to bind some sulfated
CCK-9 with a high affinity, and this residual binding was sensitive to
GTP
S suggesting that the receptor mutant remained capable of
exhibiting a high affinity G protein-coupled state. This result fits
well with the allosteric TCM, the population of high affinity receptors
identified despite the mutation of Met-195 as likely to represent
receptors that spontaneously oscillate between inactive and active
states independently of agonist binding (species
R*G and R*GL after CCK
binding, route 2). The existence of such spontaneously active receptors
has been demonstrated for several G protein-coupled receptors,
including the CCK-B/gastrin receptor (24). Therefore, the
pharmacological study of M195L mutant allowed us to demonstrate the
existence of two populations (agonist-dependent and
agonist-independent) of high affinity sites of the CCK-A receptor. The
fact that these two populations of high affinity sites bound sulfated
CCK with almost equal affinities suggests that the receptor molecules
that account for these respective sites have the same conformation at
their binding site level. However, the question of whether
agonist-dependent and agonist-independent receptor high
affinity sites are functionally identical remains to be clarified. As a
second feature, the M195L CCK-A receptor mutant displayed low and very
low affinity sites (R
and R'
species), which bound sulfated CCK-9 with a
30-fold reduced affinity
relative to the wild-type CCK-A receptor. This value is in the same
range as the difference between the affinities of sulfated and
nonsulfated CCK for these sites on the wild-type receptor.
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Finally, the M195L CCK-A receptor mutant coupled to phospholipase-C after stimulation by sulfated CCK-9 with the same efficacy as the wild-type receptor but with a 54-fold lower potency. This loss of biological potency could result from the decrease of the number of high affinity sites or from the decrease of the affinity of the low affinity sites or both. The hypothesis that would explain the loss of biological potency of the mutant by the decrease of the affinity of the low affinity sites for sulfated CCK-9 is supported by the similarity between the 30-fold decrease in the affinity of the low affinity sites for sulfated CCK and the 54-fold decrease in biological potency. This view agrees with data suggesting the functional coupling of the low affinity sites to the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase-C in pancreatic acini and their involvement in enzyme exocytosis (3, 25). On the other hand, in the present study, the potency of the wild-type CCK-A receptor to induce inositol phosphate production was in perfect agreement with the affinity of the high affinity sites for sulfated CCK-9 suggesting that these sites also contribute to the overall biological response.
The last part of the study concerns the validation of the hypothesis of an interaction existing between Met-195 of the CCK-A receptor and the sulfated tyrosine of CCK. Two types of experimental data validate the existence of this intermolecular interaction. First, peptides containing the sulfated tyrosine moiety bound to and activated the mutated M195L receptor with decreased affinities and potencies relative to the wild-type receptor, whereas peptides lacking this chemical moiety bound to and stimulated both receptors identically. Second, the selectivity of the biological response of the mutated receptor to sulfated versus nonsulfated CCK was 17-fold, whereas it was 447-fold for the wild-type receptor.
From a physico-chemical point of view, the current work experimentally validates the existence of an interaction between the aromatic ring of the sulfated tyrosine of CCK and the methionine 195 of the CCK-A receptor. Interaction involving aromatic and neighboring functional groups have already been underlined, albeit rarely (13). Several studies where devoted to understand the nature and the role of such interactions (26, 27). Recently, experimental probing of polar interactions involving aromatic and sulfur-containing side chains in cytochrome C suggested that such contribution to protein stability ranges from 0.3-0.7 kcal/mol (28). Such information can be related for instance to estimations indicating that, in barnase, the interaction of the methylene group of Thr-16 and of the aromatic face of Tyr-17 contributes to 1.9 kcal/mol of the protein stability. In the case of the CCK-A receptor-sulfated CCK complex, the current work illustrates how the interaction between the aromatic ring of the sulfated tyrosine of CCK and the methionine 195 of the receptor, albeit its low energy, is essential for full binding and biological activity of the receptor. Now, the importance of the contribution of sulfur/aromatic interactions to the stabilization of the high affinity state of the CCK-A receptor can be more easily understood by considering this interaction as required for the right positioning of the sulfated moiety of CCK in regard to the proper amino acid partner(s) in the receptor. Of particular interest, the methionine 195 of the CCK-A receptor, which likely represents part of the CCK binding site, is not uniquely involved in ligand binding as are residues Trp-39 and Gln-40.
From a general point of view it is worthy to note that Met-195 is located in the second extracellular loop of the CCK-A receptor. In the G protein-coupled receptor superfamily, the importance of extracellular domains of the receptor, including the second extracellular loop, for agonist binding and receptor function have been documented. In the rat CCK-B/gastrin receptor, a segment of five amino acids in the second extracellular loop including Gln-204, which is the corresponding residue of Met-195 in the human CCK-A receptor, has been demonstrated to be essential for high affinity binding of gastrin agonist (29). Mutagenesis studies of the NK1 receptor have revealed that several residues in the first and second extracellular loop are involved in the peptide binding (30, 31) in addition to different amino acids in the transmembrane domains II, V, and VII, which are crucial for high affinity binding and biological response to peptide agonists (32). However in both the CCK-B/gastrin and NK1 receptors, amino acids of the peptide agonists interacting with the determinants of the receptor binding site have not been identified. In the thrombin receptor, the second extracellular loop is also directly involved in agonist recognition and in receptor activation with residue Glu-260 interacting probably with Arg-5 of the agonist peptide (33).
To summarize, data from this study lead us to conclude that 1) Met-195 of the CCK-A receptor interacts with the aromatic ring of the sulfated tyrosine; 2) post-translational sulfation of CCK is essential for agonist-dependent transition of the CCK-A receptor to a high affinity state; 3) the interaction between Met-195 and the sulfated tyrosine, albeit its low energy, is essential for the right positioning of the sulfated group of CCK in the binding site of the receptor. Our data should represent an important step toward the complete delineation of the CCK binding site within the CCK-A receptor.
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ACKNOWLEDGEMENTS |
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We thank Michèle Bouisson for DNA sequencing and Dr. Elena Puente de las Cuevas for reading the manuscript.
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FOOTNOTES |
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* This study was supported by Grants from the Association pour la Recherche sur le Cancer (6234) and 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.
To whom correspondence should be addressed. Tel.:
33-05-61-32-24-05; Fax: 33-05-61-32-24-03; E-mail:
Daniel.Fourmy{at}rangueil.inserm.fr.
1
The abbreviations used are: CCK,
cholecystokinin; BSA, bovine serum albumin; GTPS, guanosine
5'-3-O-(thio)triphosphate; TCM, allosteric ternary complex
model; SR-27,897,
1-(2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl) acetic
acid.
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REFERENCES |
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