Journal of Molecular Neuroscience Copyright © 2003 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/03/20:115–123/$20.00
ORIGINAL ARTICLE
Conserved Cholecystokinin Receptor Transmembrane Domain IV Amino Acids Confer Peptide Affinity Yong Ren, Michael Bläker,1 Lakshmi Seshadri, Edward W. McBride, Martin Beinborn, and Alan S. Kopin* Department of Medicine and Molecular Pharmacology Research Center, Tufts-New England Medical Center, Boston, MA 02111 Received November 10, 2002; Accepted December 8, 2002
Abstract The Cholecystokinin type 1 and type 2 receptors (CCK-1R and CCK-2R) share >50% amino acid identity, as well as subnanomolar affinity for the endogenous peptide cholecystokinin octapeptide (CCK-8). Although it is likely that these two receptor subtypes share amino acids that confer CCK-8 affinity, it has been difficult to identify such residues. We have examined the role of several transmembrane domain (TMD) IV residues that are common to both CCK receptor subtypes. In both the CCK-1R and CCK-2R, we demonstrate that alanine substitution of two TMD IV residues, which are highly conserved among all known CCK receptor subtypes and species homologs, significantly decrease CCK-8 affinity. Despite the observed decrease in peptide binding, the mutant receptors maintain close to wild-type affinity for the respective subtype selective nonpeptide ligands, 3 H-labeled L-364,714 (CCK-1R) and 3H-labeled L-365,260 (CCK-2R), suggesting conserved tertiary structure of these mutants. Assessment of CCK-8-induced inositol phosphate production at each of the mutant CCK receptors revealed normal peptide efficacy. In contrast, peptide potencies are reduced in parallel with the observed decreases in affinity. Taken together, these findings suggest that important peptide affinity determinants are localized on TMD IV, a region that has not previously been considered a major contributor to ligand affinity in either CCK receptors or other G protein-coupled peptide receptors. Index Entries: Cholecystokinin; cholecystokinin receptor; peptide affinity; transmembrane domain; G protein-coupled receptor; affinity determinant.
Introduction The mammalian cholecystokinin (CCK) receptor family is comprised of two subtypes, CCK-1R (also known as the CCK-A receptor) and CCK-2R (also known as the CCK-B/gastrin receptor). The CCK-1R is predominantly expressed in peripheral tissues, where this protein has been demonstrated to regulate gallbladder contraction, pancreatic exocrine secretion, gastric emptying, and gut motility. The CCK-1R is also expressed in limited regions
of the central nervous system (CNS) (Saito et al., 1980; Noble et al., 1999), where it has been shown to modulate food intake (Gibbs et al., 1973; Corp et al., 1997; Kopin et al., 1999). The CCK-2R is expressed more broadly in the CNS and is also present in a number of peripheral tissues including the pancreas and stomach. In addition to its well-established role in regulating gastric secretory function, it has been suggested that the CCK-2R modulates anxiety, panic attacks, and the perception of pain (Crawley and Corwin, 1994; Noble et al., 1999).
1 Present address: Medizinische Kernklinik und Poliklinik, Universitäts-Krankenhaus Eppendorf, 20246 Hamburg, Germany. *Author to whom all correspondence and reprint requests should be addressed. E-mail:
[email protected]
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116 Each of the vertebrate CCK receptors that has been cloned (Kopin et al., 1992; Nakata et al., 1992; Wank et al., 1992; de Weerth et al., 1993; Lee et al., 1993; Ulrich et al., 1993; Blandizzi et al., 1994; Reuben et al., 1994; Dufresne et al., 1996; Schmitz et al., 1996; Takata et al., 1997) recognizes amidated cholecystokinin octapeptide (CCK-8, sulfated form) with affinity in the nanomolar range. In addition to the two mammalian CCK receptor subtypes, the known CCK receptors include a Xenopus laevis homolog (CCK-XR) which, based on amino acid sequence homology, is a likely precursor of the mammalian receptors. Paired comparison among the CCK-1R, CCK-2R, and CCK-XR reveals ~50% amino acid identity. The high degree of sequence homology among these receptors, together with their similar dissociation constant (nanomolar range) for CCK-8, suggests that key determinants of CCK-8 affinity are likely conserved among all mammalian/reptilian receptor proteins. Despite the expectation that conserved amino acids among different CCK receptor subtypes should provide a common structural basis for CCK-8 affinity, only four such residues have been experimentally determined to fulfill this role in both known mammalian subtypes. Two of these putative CCK-8 affinity determinants, L103/116 and F107/120, are located in the first extracellular loops of the CCK-1R/CCK-2R, respectively (SilventePoirot et al., 1998). In addition to the extracellular loop amino acids, two conserved transmembrane domain (TMD) residues (Y48/61 located in TMD I and N333/353 located in TMD VI) have so far been defined as sites that contribute to CCK affinity (Anders et al., 1999; Gigoux et al., 1999; Bläker et al., 2000; Gouldson et al., 2000; Giragossian and Mierke, 2001). We now report the identification of additional TMD affinity determinants. These amino acids are conserved in both the CCK-1R and CCK-2R. It is of note that these CCK-1R/CCK-2R residues, M186/173 and Y189/176, are found in TMD IV of respective receptors, a domain that has not previously been recognized as being important in conferring peptide affinity.
Materials and Methods Materials CCK-8 was obtained from Peninsula Laboratories (Belmont, CA). L-365,260 (3R[+] - N - {2,3-dihydro - 1 -methyl-2-oxo-5-phenyl-1H - 1,4 - benzodiazepine3-yl}-N′-{3-methyl-phenyl}urea) and L-364,718 Journal of Molecular Neuroscience
Ren et al. (3S[–]-N-{2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H1,4-benzodiazepoine-3-yl}-1H-indole-2-carboxamide) were provided by Merck, Sharp, and Dohme Research Laboratories (Harlow, UK). 125I-Labeled Asp1-Bolton-Hunter-conjugated CCK-8 (125I-labeled CCK-8), (3-methylphenyl-2,4,6-3H)-labeled L-365, 260 ( 3 H-labeled L-365,260), (±)-(N-methyl- 3 H)labeled L364,718 (3H-labeled L-364,718), and myo(1,2- 3 H[N])-inositol were obtained from New England Nuclear (Boston, MA). Other chemicals were purchased from Sigma (St. Louis, MO).
Generation of Mutant Receptors Human CCK-1R and CCK-2R mutants were generated using oligonucleotide-directed, site-specific mutagenesis, as described previously (Beinborn et al., 1993). The mutations were designed to substitute respective wild-type amino acids in each receptor with an alanine. The alteration in each cDNAwas confirmed by DNA sequencing using the chain termination method. Radioligand-Binding Experiments COS-7 cells (106/10-cm dish) were transiently transfected with 5 µg of either wild-type or mutant human CCK receptor cDNA subcloned into pcDNA1.1 (Invitrogen, Carlsbad, CA). The following day, cells were trypsinized and seeded into 24well plates (5,000–50,000 cells/well). Forty-eight hours after transfection, competition binding experiments were performed on intact cells expressing the recombinant receptors. The assays were carried out in Hank’s balanced salt solution supplemented with 25 mM HEPES (pH 7.3) 0.2% BSA, and 0.15 mM PMSF, as described previously (Bläker et al., 2000). Affinity for CCK-8, L-365,260, or L-364,718 was determined by competition assays using either 125I-labeled CCK-8 (20 pM), 3H-labeled L-365,260 (2 nM), or 3 H-labeled L-364,718 (2 nM) as the respective radioligand. The half maximal inhibitory concentration of 50% (IC50) values were calculated by computerized nonlinear curve fitting (GraphPad, Prizm v. 2.0, San Diego, CA). Binding capacities were calculated using the Ligand computer program (Kell v. 6 for Windows; Biosoft, Cambridge, UK). Measurement of Inositol Phosphate Accumulation COS-7 cells were transiently transfected as described for binding assays. The following day, cells were seeded into 12-well plates (2 × 105 cells/well). After an additional 6 h, the cells were prelabeled overnight with 3 µCi/mL 3H-labeled myo-inositol Volume 20, 2003
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Fig. 1. Schematic representations of human CCK receptor TMDs. (A) The cartoon represents a cross-sectional view of the seven TMDs of the CCK receptor. Large open circles represent TMD helices I–VII; shaded circles illustrate selected amino acids that are conserved in all known vertebrate CCK receptor subtype/species homologs and are postulated to comprise part of a conserved CCK-8 ligand pocket. The small open circle represents a residue that differs between the CCK-1R and the CCK-2R. TMD IV residues M173, Y176, and S180 (CCK-1R) correspond to M186, Y189, and T193 in the CCK-2R. TMD I residue Y48 (CCK-1R) corresponds to Y61 in the CCK-2R. TMD VI residue N333 (CCK-1R) corresponds to N353 in CCK-2R. (B) Two-dimensional view of the proposed topology of the CCK-2R seven TMDs. Membrane boundaries are outlined by horizontal lines. The positions of residues Y61, M186, Y189, T193, and N353 adjacent to the extracellular space are indicated. Abbreviations: TMD, transmembrane domain; NH2, amino terminus; COOH, carboxyl terminus.
in serum-free Dulbecco’s modified Eagle medium. Cells were then stimulated for 1 h at 37°C with increasing concentrations of CCK-8 in Dulbecco′s phosphate-buffered saline (GIBCO/BRL, Grand Island, NY) containing 10 mM LiCl. After stimulation, the cells were lysed and extracted with methanol/chloroform. The upper phase was analyzed for inositol phosphates (IPs) by strong anion exchange chromatography. The ligand-induced production of tritiated IPs was expressed as a fraction of the total tritium content incorporated by the cells during overnight exposure to 3H-labeled myoinositol. Concentration-response curves were generated using GraphPAD Prizm software.
Statistical Analysis IC50 values were converted to pIC50 (–logIC50) before statistical analysis, which was performed by one-way ANOVA. Post-tests were done by Tukey-Kramer multiple comparison analysis (Instat; GraphPad Software). Journal of Molecular Neuroscience
Results This study explored the role of selected TMD IV residues in conferring affinity for CCK-8. Our focus was on CCK-2R residues M186, Y189, and T193, as well as the respective residues in the homologous positions of the CCK-1R (Fig. 1). In the CCK-2R, alanine substitution of M186 or T193 resulted in a significant decrease in CCK-8 affinity. The loss of CCK-8 affinity with these TMD IV mutants was comparable or greater than that observed with an alanine substitution of the established TMD I affinity determinant Y61 (Anders et al., 1999; Bläker et al., 2000). Alanine substitution of another residue in TMD IV, Y189, led to a complete loss of detectable 125 I-labeled CCK-8 binding (Table 1). To explore whether these alanine substitutions induced a selective loss of peptide affinity, each mutant receptor was also assessed using the tritiated nonpeptide ligand, 3H-labeled L-365,260. In
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Ren et al. Table 1 CCK-2R TMD Mutations Selectively Decrease CCK-8 Affinitya CCK-8
Receptor WT Y61A M186A Y189A T193A
L-365,260
Expression
IC50 (nM)b
IC50 ratio (vs WT)
IC50 (nM)b
IC50 ratio (vs WT)
Bmax (fmol/104 cells)
Bmax ratio (vs WT)
0.1 ± 0.0c** 1.0 ± 0.1**c 0.9 ± 0.2**c NDB 4.0 ± 0.6**c
11.0 11.6 10.2
18.1± 0.9**b 14.4± 4.0**b 15.8± 4.4**b 24.1± 4.1**b 19.9± 4.6**b
1.0 1.8 2.0 3.0 1.3
59 ± 51 45 ± 21 50 ± 61 33 ± 11 64 ± 15
1.0 0.8 0.8 0.6 1.1
44.4
a CCK-8 affinities (IC50 values) were calculated from 125I-labeled CCK-8 competition binding experiments. Respective data for L-365,260, as well as Bmax values, were determined from homologous competition experiments using 3H-labeled L-365,260 as the radioligand. Data represent the means ± S.E.M. of four to six independent experiments, each performed with triplicate samples. Mutation-induced changes exceeding twofold were examined for significance vs wild-type controls. b ** p < 0.01. c The CCK-8 affinities for Y61A, M186A, and T193A have been previously reported (Bläker et al., 1998); respective values are included for comparison. Abbreviations: WT, wild type; NDB, no detectable binding.
homologous competition binding experiments, the respective IC50 values for L-365,260 at the Y61A, M186A, Y189A, and T193A mutants were 14.4, 15.8, 24.1, and 9.9 nM, each within threefold of the wildtype value (8.1 nM). Expression levels (B max) of mutant CCK-2Rs were also within twofold of the wild-type receptor. It thus appeared that alanine substitution of these four residues was relatively selective in decreasing endogenous peptide affinity. Among the CCK-2R mutants tested, only the Y189A receptor totally lacked displaceable binding of 125I-labeled CCK-8 (Table 1). Despite this marked decrease in peptide radioligand binding, it is of note that the maximal level of CCK-8-induced second messenger signaling at the Y189A mutant was comparable to that of the wild-type protein (see Fig. 2, legend). However, the potency of CCK-8 at the Y189A mutant was reduced by ~100-fold compared to the respective wild-type value (EC50 [50% effective concentration] = 78.9 vs 0.7 nM). The marked reduction in CCK-8 potency at the Y189A mutant suggests that the inability to detect 125I-labeled CCK-8 binding (assessed at 20 pM radioligand concentration) is explained by markedly reduced CCK-8 affinity. According to our previously proposed model of CCK-2R TMD structure, the side chains of residues Y61, M186, Y189, and T193 are predicted to project into the aqueous cavity of a putative ligand pocket (Kopin et al., 1995). These residues are positioned at the upper portion of either TMD I or TMD IV
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Fig. 2. The CCK-2R mutant Y189A shows a marked reduction in potency for CCK-8. Wild-type CCK-2R and mutant Y189A were each transiently expressed in COS-7 cells; CCK-8-stimulated IP production was assessed. Wildtype and Y189A CCK-2Rs triggered comparable maximal levels of CCK-8-induced IP production ([ 3 H]IP/total incorporated 3H-labeled myo-inositol = 0.20 ± 0.00 and 0.21 ± 0.02, respectively). EC50 values for the wild-type and Y189A receptor were 0.7 and 79 nM, respectively. Data represent the mean ± S.E.M. of three independent experiments, each performed with duplicate samples. Abbreviations: IP, inositol phosphate; WT, wild type.
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119 (Fig. 1). In an attempt to further examine the role of these residues in conferring CCK-8 affinity, combination mutants were generated. These constructs included alanine replacement of Y61 (TMD I), together with substitution of either M186 or T193 (TMD IV). In contrast to three of the single alaninesubstituted receptors that showed decreased yet detectable 125I-labeled CCK-8 binding (Table 1), each of the double mutants, Y61A/M186A and Y61A/T193A, lacked detectable binding of this radioligand (Fig. 3A). At the same time however, homologous competition experiments utilizing 3H-labeled L-365,260, revealed IC50 values of 5.9, 9.9, and 5.0 nM for the wild-type, Y61A/M186A, and Y61A/T193A proteins, respectively (Fig. 3B). The conserved L-365,260 affinity of the double mutants suggests that the overall tertiary structure of the respective proteins was maintained despite the introduction of two alanine substitutions. When the double mutants Y61A/M186A and Y61A/T193A were stimulated with saturating concentration of CCK-8 (1 µM), the maximal level of signaling by each receptor was comparable to that of the wild-type protein (see Fig. 3C, legend). In contrast, CCK-8 potency was reduced 27-fold (Y61A/M186A) and 137-fold (Y61A/T193A), respectively, relative to wild type. Based on the decreased potency it may be presumed that the lack of detectable 125I-labeledCCK-8 binding (Fig. 3A) in each of the double mutants reflects a marked reduction in CCK-8 affinity. To explore the functional significance of CCK-1R homologs corresponding to CCK-2R residues M186, Y189, and T193, amino acids M173, Y175, and S180
Fig. 3. CCK-2R double mutants show decreased CCK-8 affinity and potency. (A) CCK-2R double mutants lack detectable binding of 125I-labeled CCK-8. Homologous competition experiments were performed using 125I-labeled CCK-8 as the radioligand and unlabeled CCK-8 as the competitor. Displaceable binding for each receptor was expressed as fmol/10,000 transfected cells. Data represent the means ± S.E.M. of three or four independent experiments, each performed in triplicate. (B) CCK-2R double mutants maintain L-365,260 affinity comparable to the wild-type receptor. Homologous competition experiments were performed using 3 H-labeled L-365,260 as the radioligand and unlabeled
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L-365,260 as the competitor. IC50 values for wild-type, Y61A/M186A, and Y61A / T193A CCK-2Rs were 5.9, 9.9, and 5.0 nM, respectively. Data represent the means ± S.E.M. of four independent experiments, each performed in triplicate. (C) CCK-2R double mutants have reduced CCK-8 potency. Wild-type and mutant CCK-2Rs were transiently expressed in COS-7 cells, and CCK-8-stimulated IP production was measured. EC50 values were 0.7, 19, and 96 nM for wild-type, Y61A / M186A, and Y61A / T193A receptors, respectively. Wild-type and double mutants had comparable maximal levels of CCK-8-induced IP production ([3H]IP/total incorporated 3H-labeled myo-inositol ) = 0.26 ± 0.02 (wild type), 0.23 ± 0.02 (Y61A/M186A), and 0.22 ± 0.04 (Y61A/T193A), respectively. Data represent the means ± S.E.M. of three independent experiments, each performed in duplicate. Abbreviations: IP, inositol phosphate; WT, wild type.
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Ren et al. Table 2 CCK-1R TMD Mutations Selectively Decrease CCK-8 Affinitya CCK-8
Receptor WT M173A Y176A S180A
L-364,718
Expression
IC50 (nM)b
IC50 ratio (vs WT)
IC50 (nM)b
IC50 ratio (vs WT)
Bmax (fmol/104 cells)
Bmax ratio (vs WT)
0.5 ± 0.1**b 2.7 ± 1.0**b NDB 0.5 ± 0.3**b
1.0 5.4
3.9 ± 0.6 3.6 ± 0.5 3.8 ± 0.2
1.0 0.9 1.0
27 ± 5 29 ± 2 24 ± 7
1.0 1.1 0.9
1.0
c
c
a CCK-8 affinities (IC50 values) were calculated from 125I-labeled CCK-8 competition binding experiments. Respective data for L-364,718 and Bmax values were determined from homologous competition experiments using 3H-labeled L-364,718 as the radioligand. Data represent the means ± S.E.M. of three independent experiments, each performed with triplicate samples. Mutation-induced changes exceeding twofold were examined for significance vs wild-type controls. b **p < 0.01. c , Not determined. Abbreviations: WT, wild type; NDB, no detectable binding.
in the CCK-1R subtype were sequentially replaced with alanine. The affinities of these CCK-1R mutants for CCK-8 were assessed by 125I-labeled CCK-8 homologous competition experiments (Table 2). The M173A mutant exhibited a small but significant (fivefold) reduction in CCK-8 affinity, whereas no 125 I-labeled CCK-8 binding was detectable in cells expressing the Y176A substitution mutant. In contrast to these findings, alanine substitution of CCK1R residue S180 (corresponding to T193 in CCK-2R) did not alter CCK-8 affinity. Thus, only residues that were conserved among all CCK receptors (Fig. 4) appeared to be significant determinants of CCK-8 affinity at the CCK-1R. To further characterize the CCK-1R mutants, respective affinities for a selective CCK-1R nonpeptide ligand, L-364,718, were assessed. Homologous competition experiments utilizing 3 H-labeled L-364,718 as the radioligand revealed that the respective IC50 values for the M173A and Y176A mutants were comparable to the wild-type value (Table 2). In addition, the Bmax values of the mutant CCK-1Rs were similar to wild type. These findings suggest that the observed decrease at the alanine-substituted receptors was selective for CCK-8 and that respective mutants were normally expressed. Assessment of second messenger signaling in cells expressing the CCK-1R mutants revealed that the maximal level of CCK-8-induced IP production was comparable to levels observed in cells expressing the wild-type protein (see Fig. 5, legend). Whereas CCK-8 efficacy was conserved, the potency of
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CCK-8 at the mutant CCK-1Rs was decreased, consistent with the observed decrease in CCK-8 affinity (M173A) or lack of detectable 125I-labeled CCK-8 binding (Y176A as shown in Table 2).
Discussion Prior work has established the existence of a CCK2R ligand pocket comprised of TMD amino acids, where small molecule drugs interact with this receptor (Beinborn et al., 1993; Kopin et al., 1995; Bläker et al., 1998; Bläker et al., 2000). These studies have shown that alteration of the amino acid side chain of residues projecting into the putative pocket alters the affinity (Beinborn et al., 1993; Kopin et al., 1995) and/or functional activity of synthetic CCK-2R ligands (Bläker et al., 1998; Bläker et al., 2000). Extending from this concept, there is now emerging evidence by our group and by others that a similar TMD pocket may also be involved in CCK-2R recognition of its endogenous peptide ligand, CCK-8. The current study initially focused on the role of CCK-2R TMD IV residues M186, Y189, and T193 as candidate peptide affinity determinants. According to a well-established model of G protein-coupled receptor (GPCR) TMD structure (Baldwin, 1993; Baldwin et al., 1997; Unger et al., 1997), which is now further supported by the crystal structure of rhodopsin (Palczewski et al., 2000), these residues are localized on the outermost third of the TMD, with the corresponding amino acid side chains projecting into the aqueous cavity of the putative ligand pocket. In each
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Fig. 4. Two CCK-2R TMD IV residues, M186 and Y189, are conserved among all known vertebrate CCK receptors. The amino acid sequences corresponding to TMD IV from all known CCK receptors are aligned. In the CCK-2R, TMD IV residues M186, Y189, and T193 were shown to be significant affinity determinants. The residues corresponding to M186 and Y189 (arrows) are fully conserved among all CCK receptor species homologs and subtypes. In contrast, the threonine residue in position 193 of the human CCK-2R (asterisk) is conserved only among CCK-2R species homologs. Abbreviations: EC, extracellular; IC, intracellular; TMD, transmembrane domain.
case, alanine substitution results in a significant loss of CCK-8 binding affinity and signaling potency. However, the conserved affinity of each of these mutants for a nonpeptide ligand, L-365,260, together with normal cellular expression and the ability to trigger IP production to a level comparable with wild type, suggests that the overall tertiary structure of the corresponding receptors is intact despite disruption of the peptide affinity determinants. The additive nature of the observed decreases in CCK-8 affinity with introduction of either of two alanine substitutions, Y61A/M186A or Y61A/T193A, further supports the potential importance of these residues as affinity determinants. The marked loss of CCK-8 binding with the double mutants suggests that the outer portion of TMD I and TMD IV may represent two distinct yet interacting regions for ligand-receptor interaction between CCK-8 and CCK-2R. In this scenario, mutation of Y61, M186, or
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Fig. 5. CCK-1R mutants show decreases in CCK-8 potency that parallel the observed changes in CCK-8 affinity. Wildtype, M173A, andY176A CCK-1Rs were transiently expressed in COS-7 cells. CCK-8-stimulated IP production was assessed. Respective EC50 values were 0.2, 0.8, and 19 nM for wildtype, M173A, and Y176A receptors, respectively. Wild-type and all mutant receptors had comparable maximal levels of CCK-8-induced IP production. [3H]IP/total incorporated 3 H-labeled myo-inositol was 0.19 ± 0.05 (WT), 0.25 ± 0.05 (M173A), and 0.24 ± 0.03 (Y176A), respectively. Data represent the means ± S.E.M. of three independent experiments, each performed with duplicate samples. Abbreviations: IP, inositol phosphate; WT, wild type.
T193 may either disrupt direct binding of CCK-8 to these residues or indirectly reduce peptide affinity (e.g., by an allosteric mechanism). Our findings are consistent with current models suggesting that peptides interact at multiple sites with their corresponding receptors (Ji et al., 1998; Gether, 2000). One hallmark feature of CCK receptors, as reflected by their nomenclature, is nanomolar affinity for CCK-8. Teleologically, one might therefore expect that during evolution, critical determinants of CCK-8 affinity were, at least to some extent, conserved among the structurally distinct members of this receptor family. Alignment of all known mammalian and Xenopus CCK receptors reveals that the residues corresponding to human CCK-2R amino acids M186 and Y189 are fully conserved. (Fig. 4). This high degree of conservation, despite more limited overall structural homology among CCK-1Rs, CCK-2Rs, and the CCK-XR (~50% amino acid identity), suggests the possibility that these shared amino acids have a similar function in each of the CCK receptor homologs (i.e., they serve as affinity
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122 determinants for the common endogenous ligand CCK-8 ). Consistent with this speculation, followup study of mutant CCK-1Rs confirmed that alanine substitutions of respective conserved TMD IV amino acids (M173 and Y176) resulted in a selective decrease in CCK-8 affinity without altering the binding of the CCK-1R nonpeptide ligand L-364,718. In contrast to human CCK-2R residues M186A and Y189A, amino acid T193 is less conserved among members of the CCK receptor family. Although present in all known CCK-2R species homologs, the corresponding residues in CCK-1Rs and in the CCKXR are serine and asparagine, respectively. The lack of conservation of this amino acid between CCK receptor subtypes suggests less generalized functional relevance of this residue. Consistent with this speculation, we found that alanine replacement of S180, which occupies the corresponding position in the human CCK-1R, did not significantly decrease CCK-8 affinity. Taken together, our findings further support the emerging model that high-affinity binding of CCK8 is conferred in part by TMD amino acids, including ones, as suggested by this study, that reside in TMD IV. The TMD IV amino acids that have now been defined as CCK-8 affinity determinants likely act in concert with two other well-established TMD residues, Y48/61 (TMD I) and N333/353 (TMD VI) (Fig. 1), which appear to play a similar role in conferring peptide affinity. Furthermore, CCK-8, a relatively large peptide ligand, also requires additional contributions from extracellular loop residues to attain subnanomolar affinity, as demonstrated previously for both the CCK-1R and the CCK-2R (Silvente-Poirot et al., 1998). Cholecystokinin thereby extends a growing list of peptide hormones, in which affinity determinants include both extracellular and TMD residues (Gether, 2000). It appears increasingly likely that peptide ligands have a considerably more complex interaction with the receptor than corresponding nonpeptide small molecules. Understanding the similarities and differences between peptide and nonpeptide ligandreceptor interactions should help expedite the development of therapeutically useful synthetic drugs, which mimic the function of endogenous hormones.
Acknowledgments This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grants
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Ren et al. DK46767 (A.K.), T32 DK07701 (Y.R.), and P30 DK3928 (GRASP Center). M. Beinborn was supported by an AGA Industry Research Scholar Award. A. Kopin is a Tufts-New England Medical Center MCRI investigator. M. Bläker was supported by the Deutsche Forschungsgemeinschaft.
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CCK Receptor TMD Confer Peptide Affinity Gibbs J., Young R. C., and Smith G. P. (1973) Cholecystokinin decreases food intake in rats. J. Comp. Physiol. Psychol. 84, 488–495. Gigoux V., Escrieut C., Fehrentz J. A., Poirot S., Maigret B., Moroder L., et al. (1999) 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. J. Biol. Chem. 274, 20457–20464. Giragossian C. and Mierke D. F. (2001) Intermolecular interactions between cholecystokinin-8 and the third extracellular loop of the cholecystokinin A receptor. Biochemistry 40, 3804–3809. Gouldson P., Legoux P., Carillon C., Delpech B., Le Fur G., Ferrara P., and Shire D. (2000) The agonist SR 146131 and the antagonist SR 27897 occupy different sites on the human CCK(1) receptor. Eur. J. Pharmacol. 400, 185–194. Ji T. H., Grossmann M., and Ji I. (1998) G protein-coupled receptors. I. Diversity of receptor-ligand interactions. J. Biol. Chem. 273, 17299–17302. Kopin A. S., Lee Y. M., McBride E. W., Miller L. J., Lu M., Lin H. Y., et al. (1992) Expression cloning and characterization of the canine parietal cell gastrin receptor. Proc. Natl. Acad. Sci. USA 89, 3605–3609. Kopin A. S., Mathes W. F., McBride E. W., Nguyen M., Al-Haider W., Schmitz F., et al. (1999) The cholecystokinin-A receptor mediates inhibition of food intake yet is not essential for the maintenance of body weight. J. Clin. Invest. 103, 383–391. Kopin A. S., McBride E. W., Quinn S. M., Kolakowski L. F. Jr., and Beinborn M. (1995) The role of the cholecystokinin-B/gastrin receptor transmembrane domains in determining affinity for subtype-selective ligands. J. Biol. Chem. 270, 5019–5023. Lee Y. M., Beinborn M., McBride E. W., Lu M., Kolakowski L. F. Jr., and Kopin A. S. (1993) The human brain cholecystokinin-B/gastrin receptor. Cloning and characterization. J. Biol. Chem. 268, 8164–8169. Nakata H., T. M., Ito M., Taniguchi T., Naribayashi Y., Arima N., Nakamura A., et al. (1992) Cloning and characterization of gastrin receptor from ECL carcinoid
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