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Research ArticleArticle

Binding Sites and Transduction Process of the CholecystokininB Receptor: Involvement of Highly Conserved Aromatic Residues of the Transmembrane Domains Evidenced by Site-Directed Mutagenesis

Alexandre Jagerschmidt, Nathalie Guillaume, Bernard P. Roques and Florence Noble
Molecular Pharmacology May 1998, 53 (5) 878-885;

Abstract

The functional significance of the extracellular amino-terminal region and of three highly conserved aromatic residues present in the fifth (TM-V) and sixth (TM-VI) transmembrane domains of the rat cholecystokinin (CCK)B receptor, transfected in Cos-7 cells, was investigated by site-directed mutagenesis. The amino-terminal region of the CCKB receptor seemed to be weakly involved in CCK binding in that the affinities of CCK8 and selective agonists and antagonists were not modified by truncation of this region. Substitution of Phe347 in TM-VI with alanine produced a mutant receptor that displays the same affinity and selectivity as the wild-type receptor for agonists, but a slightly increased affinity for the selective CCKB antagonist L-365,260. However, the addition of saturating CCK8concentrations to cells expressing this mutant did not result in the production of inositol phosphates, demonstrating the critical role of Phe347 in CCKB receptor to G protein coupling. Substitution of Phe227 with alanine was without effect on the affinities of CCKB ligands and on phosphoinositide turnover but modified the affinity of the CCKA antagonist L-364,718. Residue Trp351 located within the CCKB receptor TM-VI is involved in the binding of CCK8 and CCK4 and of the CCK4-based antagonist PD-134,308, as illustrated by the decreased affinities of these ligands in W351A mutant. The lower affinity for CCK8 observed with this mutated CCKB receptor accounts for the higher EC50value for phosphotidylinositol hydrolysis. This study suggests that at least part of the binding site for the agonist is located inside the transmembrane domain of the CCKB receptor, partially overlapping that of antagonists, and gives new insights into the regions involved in the transduction process.

The effects of CCK8, the carboxyl-terminal octapeptide fragment of cholecystokinin, are mediated by two distinct receptors: the CCKA receptors, mainly found in the gastrointestinal system, and the CCKBreceptors, predominant in the central nervous system (see review inWank, 1995). Both receptors, cloned from various species, belong to the GPCR superfamily, characterized by seven membrane-spanning segments and a extracellular NH2-terminal domain. On binding of CCK8, the CCKB receptor activates PLC, triggering an increase in IP production (Jagerschmidtet al., 1995; Wank, 1995). The brain CCKB receptor is involved in several important adaptational processes. Thus, selective CCKBantagonists were shown capable to block the panic attacks induced by administration of CCK4 (Bradwejn et al., 1992) and to be endowed with antidepressant-like properties in rodents (Derrien et al., 1994). Furthermore, due to the negative feedback control achieved by CCK8 via CCKB receptor activation on the opioid system, the CCKB antagonists were shown to produce a strong potentiation of the antinociceptive effects induced by opioids (Maldonado et al., 1993). On the other hand, potent and selective CCKB receptor agonists such as BC 264 (Charpentier et al., 1988) were shown to improve vigilance and memory processes (see review in Daugé and Roques, 1995). All these data account for the great interest devoted to the design of selective CCKB ligands.

Although numerous results support the localization of binding sites for small amine ligands (e.g., histamine, 5-hydroxytryptamine, dopamine) within the TM domains of GPCRs (Schwartz and Rosenkilde, 1996), several peptide ligands were reported to interact with both TM and extracellular domains of these receptors. Thus, for instance, it has been shown by point mutagenesis and chimeric studies that opioid receptors interact with their ligands at multiple sites, both extracellular and intramembranous (Befort et al., 1996;Pepin et al., 1997). Likewise, numerous studies have provided evidence for an important role of residues in the amino-terminal domain, the extracellular loops, and the transmembrane helices for the ability of several receptors to bind their ligands, such as neurokinin receptor (Fong et al., 1992), V1a vasopressin receptor (Chini et al., 1995), oxytocin receptor (Chini et al., 1996), somatostatin receptor (Kaupman et al., 1995), and vasoactive intestinal peptide receptor (Du et al., 1997).

Few studies have been devoted to the CCKBreceptor. Using site-directed mutagenesis, eight residues located in the TM helices of the human CCKB receptor (Kopinet al., 1995) and His381 located in the TM-VII of the rat CCKB receptor (Jagerschmidt et al., 1996) have been suggested to be involved in CCKBversus CCKA antagonist selectivity. In addition, a segment of five amino acids in the second extracellular loop of the CCKB receptor was shown to be essential for the high affinity of the natural peptide agonist gastrin, suggesting that determinants of the binding site of the CCKBreceptor are situated, at least partially, within the extracellular domains (Silvente-Poirot and Wank, 1996). This result has been confirmed in the case of the CCKA receptor, in which two amino acids in the amino-terminal region have been identified as critical components of the agonist binding site (Kennedy et al., 1997). Moreover, we recently reported that the Asp100 residue, located within the second TM segment of the rat CCKB receptor and highly conserved among the GPCR superfamily, was involved in agonist-induced IP production (Jagerschmidt et al., 1995). All these findings were in good agreement with docking experiments performed with a three-dimensional model of the CCKB receptor (Jagerschmidt et al., 1995, 1996).

On the other hand, several aromatic residues were shown to be highly conserved within the transmembrane domains of GPCRs, in particular, in helices IV, V, and VI (Probst et al., 1992; Underwoodet al., 1994; Befort et al., 1996) (Table1). These conserved aromatic residues were proposed to play an important role in the spatial organization of the binding site (Underwood et al., 1994). Moreover, these amino acids could be involved in the signal transduction mechanism occurring after agonist-induced receptor activation as described in the case of the neurokinin type 1 receptor (Huang et al., 1994), the 5-hydroxytryptamine2 receptor (Choudharyet al., 1993), and the angiotensin II type AT1 receptor (Marie et al., 1994).

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Table 1

Sequence comparison of TM-V and TM-VI helices within various GPCRs

To gain further insights into the mechanism of ligand recognition and transduction process for the CCKB receptor, we investigated the role of the amino-terminal region and of three conserved aromatic residues located on TM-V (Phe227) and TM-VI (Phe347 and Trp351) helices of the rat CCKB receptor (Table 1 and Fig 1). Using site-directed mutagenesis, the aromatic residues were replaced by alanine, and a amino-terminally truncated (Δ1,53) CCKBreceptor was constructed. The WT, the truncated, and the F227A, F347A, and W351A mutated rat CCKB receptors were transiently expressed in Cos-7 cells, and radioligand binding experiments and IP assays were performed. This enabled us to demonstrate that Trp351 interacts with the CCK agonists and antagonists, whereas Phe227 and Phe347 are more important for CCK antagonists binding. Moreover, the amino-terminal region of the CCKB receptor seems not to be involved in CCK ligands recognition. On the other hand, at least one residue (Phe347) localized in the sixth TM domain was identified as essential for the signal transduction process.

Figure 1
Figure 1

Schematic representation of the rat CCKB receptor showing the postulated transmembrane topology. The phenylalanine (F227, F347) and tryptophan (W351) residues are highlighted; these residues were individually converted to alanine, as described in Experimental Procedures. Boxed, seven TM domains.

Materials and Methods

Reagents.

CCK4 was purchased from Bachem (Buhendrof, Switzerland). CCK8, pBC 264, L-365,260, L-364,718, and PD-134,308 (Fig.2) were synthesized in the laboratory according to reported procedures (Evans et al., 1986;Charpentier et al., 1988; Lotti and Chang, 1989; Hugheset al., 1990). Radiolabeled compounds, [α-33P]dATP (specific activity, 1000–3000 Ci/mmol), [3H]pCCK8(specific activity, 60–90 Ci/mmol), andmyo-[2-3H]inositol (specific activity, 60–90 Ci/mmol) were purchased from Amersham (Les Ulis, France). [3H]pBC 264 (specific activity, 20–30 Ci/mmol) was from NEN (Les Ulis, France). Cell culture reagents were from GIBCO-BRL (Cergy, France), and the AG1-X8 Dowex resin was from BioRad (Ivry/Seine, France).

Figure 2
Figure 2

Structures of CCK agonists and antagonists.

Site-directed mutagenesis.

The cDNA of the rat CCKB receptor was obtained as described previously (Jagerschmidt et al., 1995). For construction of the amino-terminally truncated (1–53) CCKBreceptor, the PvuII site at residue 53 was used for restriction endonuclease cleavage. Three oligonucleotides were designed to replace the codon for phenylalanine (TTC) located at amino acid positions 227 and 347 with a codon for alanine (5′-CTGCTTTTGGCCTTCATCCCG-3′ and 5′-GTTTTGCTTGCCTTCCTGTGT-3′, respectively) and to replace the codon for tryptophan (TGG) located at amino acid position 351 with a codon for alanine (5′-TTCCTGTGTGCGCTGCCAGTG-3′). Double-strand mutagenesis was carried out as described previously (Jagerschmidt et al., 1995). Authenticity of the mutations was confirmed by sequencing the constructions over the entire protein-coding region with the Sequenase Version 2.0 DNA sequencing kit (United States Biochemical, Cleveland, OH) and [α-33P]dATP.

Radioligand binding assays.

Cos-7 cells, which do not express CCKB receptor, were grown onto 24-well plates and transfected, using the calcium phosphate precipitation method, with 0.5 μg of plasmid DNA/105 cells as described previously (Jagerschmidt et al., 1996). Then, 8 μg of plasmid containing either the WT or truncated receptor cDNA also were transfected into Cos-7 (105 cells) using the calcium phosphate precipitation method as described by Graham and van der Erb (1973). At 48 hr after the transfections, the binding assays were performed directly on cells in Dulbecco’s modified Eagle’s medium containing 5 mm MgCl2and 0.2 mg/ml bacitracin. Each assay (90 min, 25°) was performed in a final volume of 0.5 ml. For saturation binding experiments, the concentration of the [3H]pCCK8 or [3H]pBC 264 varied from 50 to 6000 pm. For competition experiments, a fixed concentration of 500 pm of the radioligand [3H]pCCK8 or [3H]pBC 264 were used in the presence of various concentrations of the competitor. Nonspecific binding was determined by using 1 μm CCK8. Incubations were stopped by removing the media. The cells were harvested and the radioactivity was counted as described previously (Jagerschmidt et al., 1995). Parameters describing [3H]pCCK8 saturation binding (i.e., Kd andBmax) were determined using the computer program EBDA. Ki values were determined by using the Cheng-Prussof equation:Ki = IC50/[1 + (radioligand concentration/Kd value of the radioligand)].

IP assays.

Cos-7 cells transiently expressing WT and mutated CCKB receptors were assayed for CCK8-stimulated PI hydrolysis, as described previously (Jagerschmidt et al., 1995). Briefly, transfected cells were grown in the presence of 1 μCi/mlmyo-[2-3H]inositol for 16 hr at 37°. Cells then were treated with 10 mm LiCl for 30 min at 37° and various concentrations of CCK8 were added to the cells. After 45 min at 37°, the incubation medium was removed, and the cells were washed twice with 1 ml of PBS. The reaction was stopped by adding 400 μl of ice-cold 75% methanol and 300 μl of 0.12% Triton. The cells were scraped, and the suspension was subjected to chloroform extraction. Then, 0.5 ml of the aqueous phase was added to 4.5 ml of H2O. The solution was loaded onto a 0.5-ml column containing AG1-X8 Dowex anion exchange resin. The column was washed with 1 ml of distilled water followed by 5 ml of 5 mm sodium borate/60 mm sodium formate. Total [3H]IP then were eluted into scintillation vials with 5 ml of 1 m ammonium formate/0.1m formic acid. After the addition of scintillation mixture, radioactivity was counted.

Statistical analysis.

The results presented are the mean ± standard error. Data were analyzed using one-way analysis of variance and paired Student’s t tests. Differences were considered statistically significant at p < 0.05.

Results

Identification of aromatic residues of the rat CCKBreceptor important for the binding of CCK ligands.

The WT and mutant CCKB receptors were expressed transiently in Cos-7 cells. To determine whether the mutated receptors were expressed correctly by the cells and displayed CCK affinities comparable to that of the WT receptor, saturation studies were first performed. Except for cells expressing the W351A mutated receptor, the binding of [3H]pCCK8 to transfected cells was specific and saturable. In the case of the W351A mutant receptor, the binding experiments performed with [3H]pCCK8 were difficult to interpret due to high nonspecific binding. Moreover, the absence of saturation at a concentration of 6 nm of this radioligand suggested a decrease in its affinity (data not shown). To overcome this problem, we used the CCK8-derived peptidomimetic [3H]pBC264, a selective CCKB receptor agonist endowed with both a higher affinity and a lower nonspecific binding than [3H]pCCK8 (Charpentieret al., 1988; Durieux et al., 1992). With this radioligand, specific and saturable binding was observed for both WT and W351A receptors. [3H]pBC264 was then chosen for further characterization of the W351A mutant receptor. On the other hand, in the experimental conditions used (0.5 μg of DNA transfected), the Bmax value for the amino-terminal truncated CCKB receptor (Δ1,53) was strongly decreased. Thus, to facilitate the determination of the binding characteristics of this mutant, the amount of DNA used in the transfection was increased (8 μg).

Table 2 shows theKd andBmax values obtained for the WT and mutant CCKB receptors. [3H]pCCK8 and [3H]pBC264 binding to the WT receptor yieldedKd values of 1.03 ± 0.30 and 0.78 ± 0.16 nm, respectively, andBmax values of 37 ± 10 and 35 ± 8 fmol/106 cells, respectively. No binding of both radiolabeled probes was detected for nontransfected cells (data not shown), demonstrating the absence of CCK receptor on native cells. The mutation of Phe227 to alanine did not modify the expression of CCKB receptors in Cos-7 cells. The mutation of Phe347 to alanine gave only 33% expression, whereas the W351A mutation strongly decreased the expression of receptor, yielding 12% of the WT level. The affinity constants (Kd values) of the F227A, F347A, and W351A mutants showed no significant change as compared with the WT value. The truncation of the amino-terminal domain in Δ1,53 yielded also a large reduction in amounts of expressed receptors, with only 10% of that of the WT receptor. Again for this mutant, changes occurred in the level of binding sites rather than in the affinities for CCK agonists (Table 2).

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Table 2

Binding parameters of WT and mutated CCKB receptors expressed in Cos-7 cells

The effects of mutations on the pharmacological properties of the mutant receptors were studied by competition binding experiments using [3H]pCCK8 or [3H]pBC264 as radioligands. In these conditions, both Kd (1.03 nm or 0.78) andKi (0.58 or 1.40 nm) values of pBC264 or CCK8, respectively, were found to be almost identical, as expected (Tables 2-4). Moreover, the four mutations, F227A, W351A, F347A, and Δ1,53, have no effect on the binding of the highly selective CCKB agonist pBC264 (Tables 3 and 4). However, the exchange of Trp351 for an alanine caused a 6-fold decrease in the affinity of the natural ligand CCK8 as determined in the competition experiments using [3H]pBC264 as radioligand (Table 4). Interestingly, the carboxyl-terminal tetrapeptide CCK4 showed also a reducedKi value (10-fold) for this mutant receptor compared with the WT receptor (Table 4, Fig.3).

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Table 3

Compared pharmacological properties of WT and mutated CCKBreceptors expressed in Cos-7 cells

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Table 4

Compared pharmacological properties of WT and mutated CCKBreceptors expressed in Cos-7 cells

Figure 3
Figure 3

Competition of CCK ligands toward the binding of [3H]pBC264 to WT and W351A rat CCKBreceptors. Cos-7 cells transiently expressing WT (•) or W351A mutant (▴) receptors were tested for competition experiments using pBC 264, CCK4 and PD-134,308 as described in Experimental Procedures. Each point is the average of triplicate determinations. Data, which are representative of three or four separate experiments, are depicted with standard error values in Table 4.

The Ki values of several antagonists that belong to different chemical classes were determined. In this study, the CCK4-derived peptoid PD-134,308 and the benzodiazepine-derived compound L-365,260, which are selective CCKB receptor antagonists, and the CCKA receptor-selective antagonist L-364,718, a benzodiazepine-derived compound, have been used. As shown in Table 3, the exchange of Phe227 for an alanine caused a 3-fold decrease in affinity of L-364,718 (Ki = 68 and 207 nm for WT and F227A, respectively) without affecting the affinity of the CCKB antagonists L-365,260 and PD-134,308 (Table 3). The mutant F347A demonstrated only increased binding for L-365,260 (Table 3). The exchange of Trp351 for an alanine produced a 3- and 10-fold decrease in the affinity of the benzodiazepine-derived CCKA antagonist L-364,718 and of the CCK4-derived peptoid PD-134,308 (Table4, Fig. 3), respectively, without affecting the affinity of the benzodiazepine CCKB antagonist L-365,260 (Table4). The deletion of the amino-terminal region of the CCKB receptor had no effect on the binding of the CCKB and CCKA receptor antagonists (Table 3).

Functionality of the mutant receptors.

Rat CCKB receptors expressed in Cos-7 cells display agonist-mediated dose-dependent increases in PI hydrolysis (Jagerschmidt et al., 1995; Wank, 1995). To determine the functionality of each of the mutant receptors, the CCK8-induced accumulation of IP in transfected Cos-7 cells was measured.

As illustrated in Fig. 4, CCK8 induced a dose-dependent stimulation of the production of IP to a 7-fold maximal increase in Cos-7 cells transfected with the WT, whereas no change in PI hydrolysis was observed in control cells transfected with pcDNA3 alone (Fig.5). Moreover, as previously shown (Table2), F347A and W351A mutants have a lower binding capacity than the WT. Therefore, preliminary studies were carried out with the WT CCKB receptor to determine the effect of receptor density on CCK8-mediated PI hydrolysis. As shown in Table 5, an increase in amounts of transfected DNA into Cos-7 cells induced a proportional increase inBmax values and led to an enhanced level of the total IP produced after CCK8 stimulation without modification of the EC50 values (Table 5, Fig. 5). On the other hand, in a previous study, we have shown that theBmax value was optimal when the WT CCKB receptor was transfected in Cos-7 cells with 8 μg of DNA/105 cells (Jagerschmidt et al., 1995). Thus, in the current study, we used this amount of DNA for transfection of the mutant receptors in Cos-7 cells. In cells expressing the F227A receptor, the accumulation of inositol phosphates induced by CCK8 was in the same range (∼7-fold) as that observed in Cos-7 cells transfected with the WT receptor, and the concentrations giving half-maximal responses were similar (EC50 = 1.41 and 1.3 nm for F227A and WT, respectively). The exchange of Trp351 for an alanine decreased the efficacy of CCK8 to stimulate PI hydrolysis (EC50 = 8.7 nm) compared with WT, with a slight decrease in potency (Table 5, Fig. 5). In contrast, cells expressing the F347A mutant receptor were almost completely unable to produce IP in response to CCK8 stimulation (Figs.4 and 5), although the binding affinity for this ligand was found to be unaffected by the mutation (Table 2). This result cannot be attributed to the decrease in expression level of the mutated receptor (Bmax = 166 ± 15 fmol/106 cells) because cells expressing similar levels of WT CCKB receptor (Bmax = 180 ± 12 fmol/106 cells) produced large amounts of IP (Table 5, Fig. 5).

Figure 4
Figure 4

CCK8 stimulation of IP formation in Cos-7 cells expressing WT (○), F227A (▪), F347A (□), and W351A (▴) CCKB receptors. Cells were transfected using 8 μg of DNA/105 cells, and assays were performed as described in Experimental Procedures. Data points represent mean values of triplicate determinations in three experiments. There was no CCK8-stimulated IP formation in nontransfected Cos-7 cells. For comparison, the maximum amount of [3H]IP accumulation induced by CCK8 activation of WT CCKB receptor, in a typical experiment, was 1700 cpm, with a basal accumulation of 250 cpm.

Figure 5
Figure 5

CCK8 stimulation of IP formation in Cos-7 cells expressing WT (▪) and mutated (□) CCKBreceptors. After transfection, Cos-7 cells were stimulated using 0.1 μm CCK8, and assays were performed as described in Experimental Procedures. Histograms, mean values of triplicate determinations in three experiments for cells expressing 100–200 (A) and 700–800 (B) fmol receptor/106cells. Hatched bars, CCK8 stimulation of IP formation in nontransfected Cos-7 cells.

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Table 5

Expression levels and functional properties of WT and mutant CCKB receptors expressed in Cos-7 cells

Discussion

Numerous studies have examined peptide ligand binding domains of GPCRs showing a considerable diversity in both the number and location of ligand interacting determinants. Several aromatic residues located in the TM helices are conserved among most of the GPCRs (Probstet al., 1992). This is the case for the Ar-X2-Pro-X7-Ar motif (where Ar is aromatic and X is any residue), which is very often found in the TM-V domain, and for Ar-X3-Ar-X-Pro, which is found in the TM-VI helix of most of the GPCRs (Underwood et al., 1994). These motifs also were found in the CCKB receptor, and the high conservation of these constituting amino acids (Table 1) suggests that they could have a critical role in the structural organization and/or in the functioning of GPCRs. Furthermore, the importance of the external amino-terminal region of several GPCRs for the binding of peptide ligands has been reported (Hjorth et al., 1994; Hong et al., 1994). Thus, Miyake (1995) has shown that a splice variant of the CCKB receptor, in which the amino-terminal extracellular domain and almost all the residues of the first transmembrane helix were absent, displayed altered binding and functional properties. Therefore, in the current study, we evaluated the involvement in ligand binding and signal transduction of three aromatic residues belonging to the highly conserved domains (Phe227 in TM-V, Phe347 and Trp351 in TM-VI) and of the external amino-terminal region of the CCKB receptor (Fig. 1).

Exchange of Phe227 for alanine produced only a slight decrease in the affinity of the selective CCKA antagonist L-364,718, without effects on CCK8 and CCKB antagonists affinities and second messenger production. A recent study on the human NK1receptor demonstrated that the exchange of a tyrosine localized in the same TM-V domain (Tyr205) (Table 1) for alanine resulted in a decrease of substance P affinity, associated with a similar loss in the biological potency illustrated by a parallel reduction in IP production (Huang et al., 1994). Together, these results emphasize the diversity of GPCRs binding sites.

The mutation of Phe347 for alanine in the rat CCKB receptor did not alter the binding of CCK agonists. This result suggests that in the rat CCKB receptor, the removal of the aromatic ring of residue 347 located in the TM-VI does not affect the binding site for agonists. Furthermore, the mutant rat CCKBreceptor (F347A) exhibits a slightly improved affinity for the CCKB antagonist L-365,260, whereas affinity for the benzodiazepine-derived antagonist L-364,718 remains unchanged (Table 3). This could be explained by differences in the spatial orientation of the two antagonists within the receptor binding site. Accordingly, in a previous study, we have shown that the two structurally close benzodiazepine-derived compounds L-365,260 and L-364,718 recognize some specific residues into the ligand binding pocket of the CCKB receptor (Jagerschmidtet al., 1996). Moreover, mutation F347A of the CCKB receptor, while slightly affecting the binding of CCK antagonists, did not affect the binding of CCK agonists, suggesting that both classes of molecules do not interact with the same region of the receptor. Differences between chemical determinants for agonists and antagonists binding has already been shown for peptide receptors, including the CCKB receptor (Beinbornet al., 1993; Mantamadiotis and Baldwin, 1994).

Studies on the functionality of the CCKB receptor showed a different pattern of responses in the case of F347A and F227A mutants. Thus, mutation of Phe347 disrupts PI signaling, indicating a complete loss of the transduction process associated with agonists binding. In contrast, at comparable expression levels, both efficacy and potency values were found to be very close for the WT and F227A receptors, indicating that the Phe227 residue of the rat CCKB receptor is not involved in the signal transduction mechanism. The loss in pharmacological activity of CCK8 with the F347A mutant receptor was not accompanied by a similar loss in affinity for CCK8. Moreover, the observed lack of functional activity cannot be attributed to differences in receptor expression level; for similar amounts of F347A and WT receptors, only the latter induced phosphatidylinositol hydrolysis (Fig. 4). This observation suggests that mutation of Phe347 affects a region involved in the agonist-induced second messenger production without affecting the overall integrity of the receptor. It is interesting to observe that the amino acids involved in G protein activation and located in TM VI and VII domains of GPCRs are very often tyrosine (Wess et al., 1992; Marie et al., 1994), threonine (Wesset al., 1992), or proline (Wess et al., 1993) residues. Nevertheless in the 5-hydroxytryptamine2 receptor, a phenylalanine residue also has been shown to be implicated in second messenger production (Choudhary et al., 1993). Recently, we have shown that the Asp100 residue of the rat CCKB receptor is involved in signal transduction. It was hypothesized that Asp100 points in a direction of a cluster of basic amino acids (Lys333/Lys334/Arg335) located in the third intracellular loop of the receptor at the bottom of the TM-VI domain (Jagerschmidt et al., 1995). This was confirmed by results reported by Wang (1997)showing that these three basic amino acids play a critical role in CCKB receptor activation of Gq proteins. Thus, although not directly involved in the binding of CCKB ligands as shown by the lack of change in binding affinities after its replacement by alanine, Phe347, which belongs to the TM-VI domain, could be a residue implicated in transduction processes by playing a key role in agonist-induced changes in receptor conformation triggering G protein stimulation. The same explanation could be proposed if we take into account the allosteric model of receptor. In both cases, the exchange of Phe347 by alanine could produce a conformational change in the sequence containing the basic triplet Lys333/Lys334/Arg335, located just beneath TM VI, in the third intracellular loop, known to be involved in G protein coupling (Schwartz and Rosenkilde, 1996).

To further explore which amino acids are critical for ligand binding, we have shown by site-directed mutagenesis that Trp351, another conserved aromatic amino acid, when mutated to alanine, leads to a loss in affinity for CCK8 and CCK4 and for the CCK4-based antagonist, PD-134,308 (6-, 10-, and 10-fold, respectively). These results indicate that the Trp351 residue seems to be involved in the agonist binding site of the rat CCKB receptor. This observation and the fact that the binding of the pseudopeptide agonist pBC264 and of nonpeptide ligands, such as benzodiazepine-derived antagonists L-365,260 and L-364,718, was unaffected by the mutation, suggest that replacement of Trp351 by alanine directly affects a region of the binding site for CCK on the receptor without affecting the overall integrity of the receptor. These results emphasize previous results obtained with the NK1 (Fong et al., 1994), CCKA (Kennedy et al., 1997), and CCKB (Jagerschmidt et al., 1996) receptors, suggesting that within the receptor binding site, the residues involved in ligand recognition are different, with molecules belonging to different chemical classes, a result also found in δ-opioid receptors (Befort et al., 1996). A Trp residue, located at an equivalent position in the TM-VI domain of M3 muscarinic receptor, also was shown to be involved in both agonist and antagonist binding (Wess et al., 1993), demonstrating the critical role of this highly conserved Trp residue. In good agreement with the current experimental data, molecular modeling studies have suggested that this Trp residue, in concert with several other aromatic amino acids, may be directly involved in ligand/receptor recognition (Hibert et al., 1991; Underwood et al., 1994). Furthermore, the mutation of the Trp351 residue in the rat CCKB receptor, while strongly affecting the binding of CCK4 and CCK4-derived ligand (PD-134, 308), affects to a lesser extent the binding of CCK8, suggesting the existence of specific interaction or interactions between the carboxyl-terminal part of CCK8[Asp-Tyr(SO3H)-Met-Gly] and the CCKB receptor, stabilizing the ligand/receptor complex. On the other hand, the reduction in affinity for CCK8 of the mutant W351A receptor resulted in a loss in the biological efficacy of CCK8 to produce IP. However, as for the WT receptor, the efficacy of CCK8 to stimulate PI hydrolysis (EC50 = 8.7 nm) was in the same range as its affinity (Ki = 8.14 nm) for the sulfated octapeptide. Moreover, in the absence of stimulation, no IP release was observed, with the W351A mutant showing that this receptor is not constitutively activated.

The importance of the amino-terminal region of the CCKB receptor was demonstrated by the discovery of a splice variant that was deleted in the amino-terminal extracellular region (Δ1,63) and the upper part of the first transmembrane domain and had different binding properties than the WT CCKB receptor (Miyake, 1995). Moreover, a segment of five amino acids in the second extracellular loop of the CCKB receptor was shown to be essential for the high affinity of this receptor for gastrin, suggesting that determinants of the binding site of the CCKBreceptor could be located within the extracellular domains (Silvente-Poirot and Wank, 1996). To further explore this hypothesis, we evaluated the affinity of several CCK ligands for a mutant receptor in which only the external amino-terminal domain has been deleted (Δ1,53). The results obtained show that this truncation did not affect the binding of the CCK ligand, which is in contrast to the 5-fold reduction in affinity observed with the splice variant (Δ1,63) (see above). Thus, it seems that the extracellular amino-terminal region of the CCKB receptor is not important for ligand/receptor complex formation. In the case of the CCKA receptor, Kennedy et al. (1997)identified two amino acids located in the amino-terminal region that play a role in agonist binding. The difference between the two CCK receptor types is reflected by the low sequence homology between their amino-terminal domains. The importance of this amino-terminal region has been investigated in several GPCRs, and the results show divergent results. Thus, ligands have been shown to bind to the external amino-terminal domain of several receptors, such as κ-opioid (Konget al., 1994) or AT1 (Hjorth et al., 1994) receptors. In contrast, deletion of the 64 amino-terminal amino acids of the μ-opioid receptor did not affect binding of agonists and antagonists, indicating that the amino-terminal domain does not contribute to ligand binding (Surratt et al., 1994). This also seems to be the case for the CCKB receptor. Nevertheless, this amino-terminal domain that possesses glycosylation sites seems to be important for the expression of the receptor at the membrane levels, as the truncated mutant shows an expression level 10-fold lower than that of the CCKB WT receptor.

In conclusion, using site-directed mutagenesis of the CCKB receptor and analysis of the binding affinity and biological potency of CCK ligands, we have shown that the amino-terminal region is not involved in the formation of the ligand/receptor complex. Moreover, we demonstrated that the highly conserved aromatic amino acid residues in GPCRs, Phe227 and Phe347, do not play an important role in the recognition of the agonists, whereas a loss in the affinity of the antagonists to the mutated receptors was observed. We also have identified, for the first time, one amino acid (Trp351) in the agonist binding site of the receptor that is involved in the binding of the carboxyl-terminal sequence of CCK8 as illustrated by the similar reduction in affinity for both CCK8 and CCK4. With regard to signal transduction, it seems that the Phe347 residue plays a key role in CCK8-stimulated IP production. The results obtained with the W351A mutant also seem to demonstrate that the Trp351 residue could be involved in receptor/G protein coupling. These findings represent an important step toward the complete delineation of the agonist and antagonist binding sites, as well as the determination of the regions involved in the specificity of receptor/G protein coupling.

Acknowledgments

We thank H. J. Weng and A. Blommaert for synthesizing the CCK ligands, Dr. A. Beaumont for critical reading of the manuscript, and C. Dupuis for her help in drafting the manuscript.

Footnotes

  • Send reprint requests to: Professor B. P. Roques, U266 INSERM, URA D1500 CNRS, 4 Avenue de l’Observatoire, 75270 Paris Cedex 06, France.

  • ↵1 Current affiliation: Département de Pharmacochimie Moléculaire-Synthélabo, 92500 Reuil-Malmaison, France.

  • Abbreviations:
    CCK
    cholecystokinin
    CCK8
    cholecystokinin octapeptide
    CCK4
    cholecystokinin tetrapeptide
    pBC 264
    propionyl-Boc-Tyr(SO3H)-gNle-mGly-Trp-(NMe)-Nle-Asp-PheNH2
    L-365
    260, 3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3yl)-N′-(3-methylphenyl)urea
    L-364
    718, 3S-(−)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3yl)-1H-indole-2-carboxamide
    PD-134
    308, 4-[2-([3-(1H-indol-3-yl)-2-methyl-1-oxo-2-{[(tricyclo[3.3.1.13,7]dec-2yloxy)carbonyl]aminopropyl}amino]-1-phenyl ethyl)amino]-4-oxo-]R-(R*,R*)]-butanoateN-methyl–glucamine
    GPCR
    G protein-coupled receptor
    PI
    phosphoinositide
    PLC
    phospholipase C
    TM
    transmembrane domain
    WT
    wild-type
    IP
    inositol phosphate
    • Received September 8, 1997.
    • Accepted January 28, 1998.

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Binding Sites and Transduction Process of the CholecystokininB Receptor: Involvement of Highly Conserved Aromatic Residues of the Transmembrane Domains Evidenced by Site-Directed Mutagenesis

Alexandre Jagerschmidt, Nathalie Guillaume, Bernard P. Roques and Florence Noble
Molecular Pharmacology May 1, 1998, 53 (5) 878-885;
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