©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of the Cholecystokinin-B/Gastrin Receptor Transmembrane Domains in Determining Affinity for Subtype-selective Ligands (*)

(Received for publication, October 24, 1994)

Alan S. Kopin (1)(§) Edward W. McBride (1) Suzanne M. Quinn (1) Lee F. Kolakowski Jr. (2) Martin Beinborn (1)

From the  (1)Division of Gastroenterology and GRASP Digestive Disease Center, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111 and the (2)Ina Sue Perlmutter Laboratory, Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES

ABSTRACT

We have examined the role of transmembrane domain amino acids in conferring subtype-selective ligand affinity to the human cholecystokinin-B (CCK-B)/gastrin receptor. Fifty-eight residues were sequentially replaced by the corresponding amino acids from the pharmacologically distinct CCK-A receptor subtype. I-CCK-8 competition binding experiments were performed to compare all mutant CCK-B/gastrin receptor constructs with the wild type control. Affinities for the nonselective agonist, CCK-8, as well as the subtype-selective peptide (gastrin), peptide-derived (PD135,158), and nonpeptide (L365,260 and L364,718) ligands were assessed. All of the mutants retained relatively high affinity for CCK-8, suggesting that the tertiary structure of these receptors was well maintained. Only eight of the amino acid substitutions had a significant effect on subtype selective binding. When compared with the wild type, single point mutations in the CCK-B/gastrin receptor decreased affinity for gastrin, L365,260, and PD135,158 up to 17-, 23-, and 61-fold, respectively. In contrast, the affinity for L364,718 increased up to 63-fold. None of the single amino acid substitutions, however, was sufficient to fully account for the subtype selectivity of any tested compound. Rather, CCK-B/gastrin receptor affinity appears to be influenced by multiple residues acting in concert. The 8 pharmacologically important amino acids cluster in the portion of the transmembrane domains adjacent to the cell surface. The spatial orientation of these residues was analyzed with a rhodopsin-based three-dimensional model of G-protein coupled receptor structure (Baldwin, J. M.(1993) EMBO J. 12, 1693-1703). This model [Abstract] predicts that the 8 crucial residues project into a putative ligand pocket, similar to the one which is well established for biogenic amine receptors (Caron, M. G., and Lefkowitz, R. J.(1993) Recent Prog. Horm. Res. 48, 277-290; Strader, C. D., Sigal, I. S., and Dixon, R. A.(1989) Trends Pharmacol. Sci. 10, Dec. Suppl., 26-30).


INTRODUCTION

Cholecystokinin octapeptide (CCK-8) (^1)and gastrin are structurally related regulatory peptides which share high affinity for the CCK-B/gastrin receptor. This receptor is widely expressed in the central nervous system, as well as in a number of peripheral tissues including smooth muscle, pancreas, and stomach. In the central nervous system, the CCK-B/gastrin receptor has been implicated in anxiety, panic attacks, and the perception of pain (4, 5, 6, 7, 8) , making it a potentially important clinical target for receptor-specific antagonists. The CCK-B/gastrin receptor in the stomach has also been extensively studied and has been shown to modulate acid secretion as well as the proliferation of enterochromaffin-like cells(9) .

The CCK-B/gastrin receptor is a member of the seven-transmembrane domain beta-adrenergic receptor superfamily. Within this broad group, it is well established that the transmembrane domain residues of biogenic amine receptors are the primary determinants of ligand affinity(2, 3) . In contrast, relatively little is known about the molecular basis for ligand affinity to peptide receptors. We have previously shown that differences in the affinities for nonpeptide antagonists between the human and canine CCK-B/gastrin receptors result from interspecies variation in a single transmembrane domain amino acid(10) . In addition, the potential importance of the peptide receptor transmembrane domains in determining ligand affinity has been further illustrated by mutational analysis of the neurokinin(11, 12, 13, 14, 15, 16) and angiotensin (17) receptors.

The aim of this study was to analyze the role of CCK-B/gastrin receptor transmembrane domain amino acids in determining high affinity binding of subtype-selective agonists and antagonists. The strategy we utilized to identify potentially important residues was to compare the human CCK-B/gastrin receptor with the other known CCK receptor subtype, CCK-A. The two subtypes share 47% amino acid identity, as well as high affinity for CCK-8. Pharmacologically, the CCK-B/gastrin receptor can be distinguished from the CCK-A subtype by its unique affinity profile for peptide as well as nonpeptide ligands(9) . Comparison between the transmembrane segment amino acid sequences of the CCK-B/gastrin and the CCK-A receptors revealed 58 differences, residues which could potentially confer affinities characteristic of a CCK-B/gastrin receptor. These divergent amino acids were sequentially substituted in the CCK-B/gastrin receptor with their CCK-A homologs. The resultant mutant receptors were assessed for alteration in their agonist and antagonist binding profiles. We report here that 8 of the 58 residue substitutions significantly influence ligand affinity for the CCK-B/gastrin receptor.


EXPERIMENTAL PROCEDURES

Generation of Mutant Receptors

Putative transmembrane regions within the human CCK-B/gastrin and CCK-A receptors were predicted by hydropathy analysis(1) . Each of the 58 amino acid differences between the two subtypes was considered a candidate for influencing the pharmacologic profile of the CCK-B/gastrin receptor. To evaluate this hypothesis, each of the 58 CCK-B/gastrin receptor transmembrane domain residues was sequentially replaced by its corresponding CCK-A receptor homolog. These substitutions were introduced as units of 1-4 amino acids/mutant (underlined in Fig. 1) using oligonucleotide-directed mutagenesis(10) . All mutant receptor cDNA sequences were confirmed by the chain termination method (18) .


Figure 1: Amino acid substitutions which alter ligand affinity cluster in the outer third of the transmembrane domains adjacent to the extracellular space. The inset shows a schematic representation of the CCK-B/gastrin receptor; transmembrane domains are numbered as in the main figure. The upper line shows the amino acid sequence corresponding to transmembrane domains of the human CCK-B/gastrin receptor(32, 33, 34) . The middle line shows residues in the rat CCK-A receptor sequence (22) which differ from the human CCK-B/gastrin receptor. The lower line shows human CCK-A receptor sequence (23, 24) which diverges from the rat CCK-A receptor. Divergent CCK-A receptor residues were sequentially substituted into the CCK-B/gastrin receptor as units of 1-4 amino acids/mutant (underlined). Analysis of mutants was done by I-CCK-8 binding experiments, using as competitors two agonists, sulfated CCK-8 and gastrin (B-specific), and two benzodiazepine-based subtype-selective antagonists, L365,260 (B-specific) and L364,718 (A-specific), n geq 2. The wild type receptor was tested in parallel for every experiment, and mutant IC values were always expressed relative to wild type. Data shown for each mutant represent only the largest affinity shift which was observed for any of the four ligands tested. Units on the y axis correspond to either mutant/wild type IC ratios for affinity decreases or to wild type/mutant IC ratios for affinity increases. Table 1shows the complete binding profile of mutants with an IC ratio greater than 2.5 for any ligand. For 3 of the amino acids in the CCK-B/gastrin receptor, two different mutants were tested, one corresponding to rat and the other to human CCK-A sequence. In these cases, a large dot denotes IC ratios of mutants which include human CCK-A receptor sequence. Amino acid numbering corresponds to the position in the human CCK-B/gastrin receptor. N, amino terminus; C, carboxyl terminus; EC, extracellular loop; IC, intracellular space; TM, transmembrane domain; aa, amino acids.





Radioligand Binding Experiments

Full-length wild type and mutant CCK-B/gastrin receptor cDNAs subcloned into pcDNAI were transiently expressed in COS-7 cells(19) . Forty-eight hours after transfection, binding experiments were performed in 24-well plates (2,500-50,000 cells/well), using 20 pMI CCK-8 (DuPont NEN) as the radioligand(10) . Binding affinities for two agonists, sulfated CCK-8 and gastrin (Peninsula Laboratories, Inc.), and three subtype-selective antagonists, L365,260, L364,718 (Merck Sharp and Dohme Research Laboratories) and PD135,158 (Parke-Davis Neuroscience Research Center) were determined by radioligand competition studies with increasing concentrations of unlabeled ligand. The respective IC values were calculated by computerized nonlinear curve fitting (Inplot 4.0, GraphPad). In all experiments, the wild type CCK-B/gastrin receptor was included as a control. All IC values were expressed as ratios relative to the wild type value. The characterization of each mutant receptor included two or more (n geq 2) independent competition binding experiments for each of the ligands tested.

Receptor Modeling

The spatial orientation of transmembrane domain amino acids in the CCK-B/gastrin receptor (Fig. 3) was predicted from a model of G-protein-coupled receptor structure(1) . In this model, the relative positions of the transmembrane domains were predicted from a projection map of rhodopsin derived from electron crystallographic analysis. The orientation of the helices in the model was proposed based on structural comparison of the sequences of 204 G-protein-coupled receptors and was corroborated by mutagenesis data on rhodopsin and the biogenic amine receptors. Corresponding positions in the CCK-B/gastrin, neurokinin-1, endothelin-B, and thyrotropin-releasing hormone receptors were identified by multiple sequence alignments(1, 20, 21) .


Figure 3: Overlapping cross-sectional views of the seven transmembrane domains of the CCK-B/gastrin receptor. This diagram is based on a model of G-protein coupled receptors (1) derived from the two-dimensional crystalline structure of rhodopsin(25) . Large circles represent alpha-helices 1-7. The inward faces of the seven helices define the borders of a putative ligand pocket. The small numbered circles correspond to amino acids in the human CCK-B/gastrin receptor. Gray shading indicates amino acid identity between the human CCK-A and CCK-B/gastrin receptors. Open circles represent pharmacologically silent differences, whereas black solid circles mark amino acids in the CCK-B/gastrin receptor which result in affinity shifts when substituted with their CCK-A counterparts. CCK-B/gastrin receptor residues are shown in single letter amino acid code, followed by homologous CCK-A residues in parentheses. A, the outer third of the transmembrane domains adjacent to the extracellular space defines a putative ``ligand binding pocket.'' Pharmacologically significant amino acid substitutions




RESULTS AND DISCUSSION

The transmembrane domain amino acid sequences of the human CCK-B/gastrin and CCK-A receptors differ in 58 positions. For each residue where the sequence of the two subtypes is not conserved, CCK-B/gastrin receptor amino acids were substituted with the corresponding CCK-A homolog. In total, 38 mutant receptors (1-4 amino acid substitutions/mutant) were constructed. Initially, the amino acid substitutions were based on the rat CCK-A receptor sequence(22) . When the human CCK-A receptor sequence became available(23, 24) , several additional mutants were constructed to include all human CCK-A residues which differ from the rat (Fig. 1). Receptors were expressed in COS-7 cells, and the affinities for CCK-8, gastrin, L365,260, and L364,718 were determined by I-CCK-8 competition binding experiments. Only 8 of the 58 divergent amino acids, Arg, Thr, Ser, Ser, Val, Tyr, Thr, and His, shifted affinity for any of the tested competitors by more than 2.5-fold relative to wild type (p < 0.05) (Table 1).

All mutant receptors except for H376L had IC values for CCK-8 in the range between the wild type CCK-B/gastrin and CCK-A receptors (7.6-fold difference), suggesting that the tertiary structure of all receptors was relatively well conserved. While the substitution mutant H376L had a slight decrease in affinity for CCK-8 (to approximately 4-fold lower than observed for the CCK-A receptor), it also demonstrated a 63-fold increase in affinity for the CCK-A receptor specific antagonist, L364,718, in comparison with the wild type CCK-B/gastrin receptor (Table 1). This suggests that the overall conformation of the H376L mutant remained intact.

We have previously shown that V349I increased affinity for L364,718 by 6-fold relative to the wild type CCK-B/gastrin receptor(10) . A similar 5-fold increase in affinity was observed with Y350F (Table 1). These affinity shifts were additive; the double mutant V349I/Y350F increased affinity for L364,718 thirty-eight fold in comparison with wild type (Fig. 2). This combination mutant illustrates how multiple residues may interact to determine overall affinity for a given ligand. In addition, the V349I/Y350F substitution demonstrates how agonist and antagonist affinities can be independently affected, since this mutation did not alter affinity for either CCK-8 or gastrin.


Figure 2: Multiple amino acids act in concert to determine L364,718 affinity. Comparison of I-CCK-8 binding to wild type and mutant CCK-B/gastrin receptors in the presence of increasing concentrations of L364,718. Respective IC values (nM ± S.E.) for L364,718 were as follows: wild type (WT), 204 ± 10 (n = 11); V349I, 28.1 ± 3.7 (n = 3); Y350F, 31.9 ± 4.1 (n = 7); V349I/Y350F, 5.6 ± 0.5 (n = 5).



Four amino acid substitutions, R57Q, S131T, S219H, and H376L, decreased affinity for gastrin, an agonist with high specificity for the CCK-B/gastrin receptor subtype. In each of these mutant receptors, the loss of affinity for gastrin was mirrored by an increase in affinity for the CCK-A receptor specific antagonist, L364,718, by 6.7-, 3.6-, 4.8-, and 62.5-fold, respectively (Table 1). The increases in affinity for L364,718 suggest that these substituted receptors are not merely ``loss of function'' variants. Rather, these mutations in the CCK-B/gastrin receptor shift its ligand specificity toward that of the CCK-A subtype.

Four amino acids substitutions, T111N, S219H or Q, and H376L, reduced affinity for the CCK-B/gastrin receptor specific antagonist, L365,260 (Table 1). The substitution, T111N, decreased L365,260 affinity with little effect on agonist binding, as did substitution of Ser with the corresponding residue (glutamine) from the rat CCK-A receptor. In contrast, S219H, the human homolog of the same mutant, and H376L not only decreased L365,260 binding, but also reduced affinities for CCK-8 and gastrin closer to the values expected for the CCK-A receptor.

Taken together, our data indicate that no single residue substitution is sufficient to completely reverse subtype selectivity for gastrin, L365,260, or L364,718. It therefore appears that ligand specificity is determined by multiple amino acids working in concert.

Hydropathy analysis of the CCK-B/gastrin receptor sequence suggests that the 8 amino acids which affect ligand affinity cluster toward the outer third of the transmembrane domains, adjacent to the extracellular space (Fig. 1). To further examine the potential spatial orientation of these CCK-B/gastrin receptor amino acids, we utilized a rhodopsin-based model of G-protein coupled receptor transmembrane domain structure(1, 25) . As shown in Fig. 3, the model depicts three overlapping transmembrane domain cross-sectional views, partitioned into an outer (A), middle (B), and inner third (C). When the CCK-B/gastrin receptor is examined using this model, all 8 amino acids which alter subtype selective binding are positioned on the inward pointing faces of the helices, outlining a putative ligand pocket within the CCK-B/gastrin receptor (Fig. 3A).

Neither the middle nor the inner thirds of the transmembrane segments appear to be important in determining subtype selective ligand affinity. In the middle third of the transmembrane domains, amino acids which project into the putative ``binding pocket'' tend to be identical when the CCK-B/gastrin and CCK-A receptors are compared (Fig. 3B). This suggests that the central portion of the transmembrane domain helices provides a conserved structural requirement for both CCK receptor subtypes. As in Fig. 3A, functionally silent amino acid substitutions tend to project away from the putative pocket. In the inner third of the transmembrane domains, none of the residue substitutions altered the affinity for subtype-selective compounds (Fig. 3C).

The large number (50) of pharmacologically silent amino acid substitutions makes it unlikely that the observed affinity shifts result from random disruption of transmembrane domain helical packing. The specificity of the eight mutation-induced functional changes is also supported by the predictable configuration of these residues in the extracellular third of the membrane. The significance of this clustering is further highlighted by the relative absence of residues which determine affinity in the middle and intracellular cross-sections of the transmembrane domain helices (Fig. 3, B and C).

To further support the hypothesis that the amino acids projecting into the ligand pocket affect subtype selectivity, we examined the binding of PD135,158 (CAM-1028), which exemplifies an additional class of CCK-B/gastrin receptor antagonists(26) . This compound binds with much higher affinity to the wild type CCK-B/gastrin than to the CCK-A receptor (IC ratio = 250.5 ± 35.8; mean ± S.E. from three independent experiments). Substitutions in three of the eight positions previously shown to influence ligand binding, T111N, S219H, and T354A, significantly decreased PD135,158 affinity toward the value expected for a CCK-A receptor: 3.7 ± 0.4, 60.9 ± 20.1, and 5.3 ± 0.7-fold, respectively (mean IC ratio ± S.E., n geq 2, p < 0.01).

Further studies are required to determine whether the CCK-B/gastrin receptor residues influencing affinity form a true ligand pocket or indirectly affect agonist and antagonist binding. Localization of these amino acids to the transmembrane domains is based on hydropathy algorithms and comparison with other G-protein-coupled receptors(1) . The distinction between extracellular loop amino acids which are close to the membrane, and residues which are within the outer portion of the transmembrane domains is a best approximation. Thus, some of the amino acids shown in Table 1may actually influence ligand affinity as part of an extracellular loop. The contribution of the extracellular loops to peptide receptor affinity is well supported by work on the neurokinin-1 (12, 27, 28) and thrombin (29) receptors. It is therefore likely that the complete CCK-B/gastrin receptor binding pocket is defined by both transmembrane domain and extracellular loop amino acids.

To investigate whether the proposed transmembrane domain binding pocket is applicable to other peptide receptors, the human CCK-B/gastrin and the neurokinin-1 (NK-1) receptors were compared. Ten transmembrane domain residues in the human NK-1 receptor, Asn, Asn, Val, Gln, Ser, His, His, Tyr, Ile, and Met, have been implicated in ligand affinity by mutational analysis(11, 12, 13, 14, 15, 16) . When projected into the CCK-B/gastrin receptor model, all 10 NK-1 residues are found on the inward face of the transmembrane domain helices and project toward the putative binding pocket (Fig. 3A). Four of these 10 residues map to the same positions as amino acids which influence CCK-B/gastrin receptor subtype selectivity, T111N, S219H, Y350F, and H376L. These observations suggest that the binding pocket of the NK-1 and the CCK-B/gastrin receptors are structurally similar.

The extent to which these findings can be generalized remains to be further investigated. Two established binding determinants in other peptide receptors, tyrosine 106 of the thyrotropin-releasing hormone receptor (30) and lysine 181 of the endothelin B receptor(31) , appear to project into the putative ligand pocket (Fig. 3A). In contrast, a number of affinity determinants in the angiotensin AT-1 receptor are not consistent with the rhodopsin-based model. Some of the crucial AT-1 receptor transmembrane domain amino acids project away from the binding pocket and others are located in close proximity to the cytoplasm(17) .

In summary, our analysis suggests that the specific ligand affinity profile of the CCK-B/gastrin receptor is, in part, determined by residues which occupy the outer third of the transmembrane domains adjacent to the extracellular space and project into a putative ligand pocket in the membrane. An analogous model is well established for the biogenic amine receptors (2, 3) , suggesting that the organization of the transmembrane domain binding determinants has been conserved, at least to some degree, across the broad family of G-protein-coupled receptors. These findings should enhance the understanding of high affinity ligand binding to the CCK-B/gastrin receptor, which may expedite the design and development of subtype selective receptor antagonists.


©1995 by The American Society for Biochemistry and Molecular Biology, Inc.