Insights into the molecular basis of ligand binding by the cholecystokinin receptor

IAP/APA Meeting Pancreatology 2001;1:336–342

Insights into the Molecular Basis of Ligand Binding by the Cholecystokinin Receptor Laurence J. Miller Xi-Qin Ding Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minn., USA

Key Words Cholecystokinin W G-protein-coupled receptors W Photoaffinity labeling W Mutagenesis

Abstract The receptor for the peptide hormone, cholecystokinin, is a G-protein-coupled receptor in the rhodopsin/ß-adrenergic receptor family. A number of methodological approaches have been utilized to gain insights into the molecular basis for natural peptide ligand binding and activation of this physiologically important receptor. Insights into this have come from sequence analysis, ligand and receptor structure-activity data, receptor mutagenesis, conformational analysis of ligand and receptor fragments, and photoaffinity labeling. In this work, we review the contributions of each of these complementary approaches and provide a current integrated view of the active complex of cholecystokinin bound to its receptor. Copyright © 2001 S. Karger AG, Basel and IAP

Cholecystokinin (CCK) represents one of the classical gastrointestinal hormones, with its earliest recognized activities on gallbladder and pancreas described in 1928 [1] and 1943 [2], and with its sequence solved in 1971 [3]. Its spectrum of biological actions is now known to be quite

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broad, and to be mediated by two distinct types of receptors, the type A receptor that is the focus of this report and the type B receptor, representing the predominant CCK receptor in the brain. Most of the type A actions of CCK are relevant to nutrient assimilation, with this hormone acting as a physiological stimulant of post-cibal gallbladder contraction and pancreatic enzyme secretion, affecting gastric emptying and bowel transit, and inducing satiety [4]. Due to this broad spectrum of important actions on a variety of cell types, CCK receptors represent potentially important drug targets. Therefore, there has been much interest in the nature of the hormone-binding domain, with the hope that such structural data will facilitate the rational design and improvement of highly selective and potent drugs. The type A CCK receptor was first identified by photoaffinity labeling in 1987 as a Mr = 85,000–95,000 glycoprotein in rat pancreas [5]. It was subsequently purified and had its cDNA cloned [6], demonstrating sequence typical of a member of the rhodopsin/ß-adrenergic receptor family of guanine nucleotide-binding protein (G-protein)-coupled receptors. This superfamily is remarkable for the diversity of ligands and themes of binding that have been proposed. The ligands range from extremely small photons, odorants, and biogenic amines to larger peptides, proteins, and even intact viruses [7]. Of these, those receptors having the smallest ligands have the best understood themes for

Laurence J. Miller, MD Center for Basic Research in Digestive Diseases Guggenheim 17, Mayo Clinic Rochester, MN 55905 (USA) Tel. +1 507 284 0680, Fax +1 507 284 0762, E-Mail [email protected]

Fig. 1. Shown is a diagram of the different molecular forms of human CCK, along with the current numbering convention that is based on the form of CCK-33 that was initially purified and characterized. All forms of this hormone share their carboxyl-terminal domain. The pharmacophoric domain key for type A CCK receptor binding and action is shown in the shaded area.

binding, with ligand-binding domains believed to be within the confluence of seven transmembrane helices within the lipid bilayer [7, 8]. As the ligands increase in size, they no longer are accomodated in this receptor domain, and binding sites are believed to migrate toward extracellular loop and amino-terminal tail domains [9]. However, we know far less about the structure of these more flexible receptor domains, and we understand little about the molecular basis for their interactions with ligands. CCK is a peptide hormone that occurs as a series of linear peptides with lengths of 72, 58, 39, 33, 22, 8, 5 and 4 residues, all sharing their carboxyl-terminal domain (fig. 1) [4]. Each of the form’s eight residues or longer are potent and high-affinity agonists of the type A CCK recep-

tor. Primary structure-activity data demonstrate that the pharmacophoric domain represents the carboxyl-terminal heptapeptide amide of this hormone [10]. By first principles, one would expect this region of CCK to be quite flexible. However, solution structures have suggested that it may achieve preferred folded conformations [11–13]. Since the environment of CCK when bound to its receptor is likely quite distinct from conditions used in these studies, it is hard to know how to use such solution conformational data in the determination of ligand docking to the receptor. The best current understanding of the conformation of the CCK receptor comes from expected homology with the very recently described crystal structure of rhodopsin

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[8]. That structure, achieving a 2.8 Å resolution, confirmed the prevailing prediction of the number and organization of transmembrane helices, but provided a number of surprises as well. At the end of the seventh helix was another ·-helical structure that ran parallel with the membrane. Several of the loops ‘plugged’ the end of the helical bundle, even attempting to insert portions into the bilayer. It is not yet clear how generally applicable this structure might be for other members of the superfamily, particularly for those that bind larger ligands. Rhodopsin is unique, with abundant natural sources and inherent stability making large-scale purification possible. There are still considerable methodological challenges to overcome in the areas of overexpression and purification of other G-protein-coupled receptors to be able to study them in an analogous way. There is not currently any good template to use as a model for the loop and tail domains of the CCK receptor that are believed to be most relevant for peptide binding. Since the receptor-binding region of CCK represents a flexible peptide, docking two flexible structures with a high degree of confidence is not yet possible. Efforts to achieve meaningful peptide hormone docking to the CCK receptor have been limited to three types of studies. These include receptor mutagenesis [14–21], structural analysis of fragments of receptor incubated with peptides [22], and photoaffinity labeling [23–25]. Each of these has unique strengths and weaknesses. Together, however, they provide important complementary insights. Receptor mutagenesis studies can be divided into three general types: loss-of-function, gain-of-function, and twodimensional mutagenesis with complementary changes in both receptor and ligand [26]. Loss-of-function mutations are the most common and the most difficult to interpret, reflecting the reality that loss of function can result from a long series of possible mechanisms. These can range from interference in the biosynthesis and transport of the receptor construct to the cell surface, to global disruption of conformation, more subtle allosteric effects, and direct effects on ligand binding. In gain-of-function mutations, a receptor domain believed to be responsible for a specific activity is inserted into a functionally deficient receptor construct, leading to the positive addition of this functionality to that construct. When successful, this approach is more useful than the loss-of-function approach. Studies involving structurally-related but functionally-distinct receptors that are used to form chimeric constructs, are the most common way to achieve this result. However, this can also be affected by allosterism. The two-dimensional

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mutagenesis studies are the most useful in this series of mutagenesis studies for determining interactions between residues. In these studies, mutations are made in receptor and ligand that result in loss of function of each, while these constructs exhibit normal function when interacting together. Unfortunately, this has only been successfully achieved for small molecule ligands at the ß2-adrenergic receptor [26]. Several mutants of the CCK receptor have been reported. Deletion of the amino-terminal 37 residues of the receptor, without having negative impact on CCK binding, was particularly useful [27]. This demonstrated that the distal amino-terminal tail region did not play an important role in binding of natural hormone. Additional truncation of only five residues caused loss of function, supporting an important role for this region [27]. A series of CCK receptor residues have been mutated to alanine residues [15, 18, 21]. While some of these mutations (mutation of Lys37, Asn102, Lys105, Phe109, Lys187, Phe198, His210, Arg345, or Arg346) have had no effect, other mutations (mutation of Trp39, Gln40, Leu103, Phe107, Lys115, Met195, Arg197, Asn334, and Arg337) have had substantial negative impact on CCK binding or biological activity (fig. 2). Mutations with quantitatively significant negative impact were those at Leu103, Phe107, Lys115, Arg197 and Arg337. The basic residues in this group have particular interest as potential partners for chargecharge interaction with Tyr-sulfate-27 and Asp-32, key acidic residues within the pharmacophoric domain of CCK. Chimeric receptor studies have been performed with type A and type B CCK receptors [28–30]. Major differences in these receptors include the role of the tyrosinesulfate in position 27 for the type A receptor and the highaffinity binding and potency of the carboxyl-terminal tetrapeptide at the type B receptor. Unfortunately, these studies have not been able to clearly dissociate the domains contributing to this selectivity. This may reflect differences in the way the peptide docks at each of these receptors. Indeed, conformationally-constrained CCK analogues have been reported that can distinguish between these two receptors [31, 32]. As noted above, two-dimensional mutagenesis has not yet been successfully applied to the CCK-receptor complex. An attempt was made to interpret results for CCK receptor binding using such a scheme, but the receptor construct never even achieved an affinity for CCK that was high enough to measure, and required the use of a pharmacological ligand having only millimolar affinity for the receptor [18]. With such substantial loss in binding

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Fig. 2. Shown is a schematic representation of the rat type A CCK receptor with its proposed membrane topology.

The highlighted residues have been shown to have substantial functional impact in mutagenesis studies in this or other species.

energy, it is quite likely that the normal positioning of the ligand had also been lost. The conformation of the amino-terminal 47 residues of the CCK receptor has been determined by nuclear magnetic resonance (NMR) [22]. For this, the peptide was studied in an environment of H2O/dodecylphosphocholine-d38 (DPC) to mimic a lipid environment. This region of the CCK receptor is normally highly glycosylated, while the segment studied was not, and this work utilized nonsulfated carboxyl-terminal octapeptide of CCK, a lowaffinity and low-potency ligand [10]. NMR also requires

very high (millimolar) concentrations of reagents, making it impossible to determine whether the interaction between peptide and receptor fragment has an affinity appropriate for their biological interaction. While such work may provide useful insights into the conformation of this flexible region of the CCK receptor, its relevance to ligand interaction in the absence of the rest of the receptor is less clear. Photoaffinity labeling takes advantage of the direct spatial approximation necessary between a photolabile residue sited within the pharmacophoric domain of the

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Table 1. Photoaffinity labeling of the CCK receptor Position of crosslinking

Fragment or site labeled in the CCK receptor

References

Within CCK pharmacophore, position 33

Trp39 in the amino-terminal tail, just above TM1 of the receptor

23

125I-D-Tyr-Gly-[(Nle28,31,Bpa33)CCK-26–33]

Within CCK pharmacophore, position 33

Trp39 in the amino-terminal tail, just above TM1 of the receptor

40

125I-D-Tyr-Gly-[(Nle28,31,6-NO -Trp30) 2

Within CCK pharmacophore, position 30

Not yet localized

41

Within CCK pharmacophore, position 29

His347 and Leu348 in the third extracellular loop, above TM7 of the receptor

24

Outside CCK pharmacophore, position 24

Glu345 in the third extracellular loop and a segment between Asn10 and Lys37 in the amino-terminal tail of the receptor

25

Photoreactive CCK analogues 125I-D-Tyr-Gly-[(Nle28,31,pNO -Phe33) 2

125I-D-Tyr-Gly-[(Nle28,31,Bpa29)

CCK-26–33]

CCK-26–33]

CCK-26–33]

125I-D-Tyr-Gly-Bpa-Gly-[(Nle28,31) CCK-26–33]

ligand and the receptor residue that is covalently labeled. This provides great power for establishing useful constraints for molecular modeling. However, this approach requires specialized synthetic photochemical capabilities and structure-activity considerations must accomodate modification of the ligand in a given site. In the earliest affinity labeling studies designed to identify the CCK receptor, radioiodinated CCK-33 was used in conjunction with ultraviolet light or bifunctional chemical crosslinking [33–35]. Labeling in these studies identified a glycoprotein migrating on an SDS-polyacrylamide gel in the region between Mr = 76,000 and 85,000, having a protein core of Mr = 65,000. This glycoprotein turned out to represent a molecule that was a near neighbor to the true type A CCK receptor. The receptor itself was first identified using affinity labeling with shorter probes having sites of covalent attachment closer to and within the carboxyl-terminal octapeptide [5, 36–38]. The spatial relationship between these two proteins was further established using a series of unique topographical mapping probes that incorporated flexible polyethylene glycol spacers between the receptor-binding domain and the site of covalent attachment [39]. Several affinity labeling studies have utilized analogues of CCK with photolabile residues intrinsic to the pharmacophore (table 1) [23–25]. In these, the radiolabeled photolabile analogues of CCK were shown to bind with high affinity in a specific manner to the CCK receptor and to represent full agonists, helping to ensure the relevance of the data to the normal position of CCK bound to the receptor. Position 33 photoprobes have covalently labeled receptor residue Trp39 just above the first transmembrane segment [23]. The position 29 photo-

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probe established covalent bonds with receptor residues His347 and Leu348 just above the seventh transmembrane segment [24]. Each of these sets of labeling data represented focused contacts with a single receptor domain, as might be expected from the high-affinity tight interaction between native ligand and the ligand-binding domain of the receptor. We recently prepared a CCK analogue probe with a photolabile residue sited at the amino-terminal end of the CCK pharmacophore (table 1). This has given us the opportunity to explore the spatial approximations between this residue and specific residues within the CCK receptor. The constraints provided by these data have been the key for the refinement of our molecular model of the agonist-occupied CCK receptor [23, 25, 40]. In contrast to the studies with sites of covalent attachment within the pharmacophoric domain, studies with the probe having its photolabile residue sited outside this domain at the amino-terminus of the peptide established covalent bonds with two distinct regions of the CCK receptor [25]. This probe labeled both the third extracellular loop of the receptor and a portion of the distal aminoterminal tail of the receptor. Labeling of the latter domain was eliminated by truncation of the receptor amino-terminus, without having any negative effects on ligand binding or signaling [27]. While the labeled third extracellular loop of the receptor is known to be important for function, that contact was also eliminated when the amino-terminus of the receptor was truncated [25]. The photoaffinity labeling data for the ligand aminoterminus, combined with crosslinking data for two positions within the peptide pharmacophore, have enabled us to propose a distinct topological model for the peptide

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ligand-type A CCK receptor complex. Only a counterclockwise helical bundle arrangement for the CCK receptor can accomodate the full set of covalent crosslinks we observed in the photoaffinity labeling experiments. Our model now supports spatial approximation between tyrosine-sulfate in position 27 of CCK and Arg197 in the second extracellular loop of the receptor. These residues represent prominent potential partners for charge-charge interaction. Arg197 is one of only three residues in the extracellular domain of the CCK receptor reported to have strong negative impact on CCK binding when mutated to Ala [21]. More definitive biophysical data will be needed to ultimately verify this prediciton. Of note, another distinct molecular model has placed the amino-terminus of CCK much closer to the membrane and directed toward the other side of the helical bundle [14, 18]. This is inconsistent with the residue-residue approximations directly established by photoaffinity labeling studies. It is interesting that, despite being so dif-

ferent, this model also places the tyrosine-sulfate in the ligand close to Arg197. Our understanding of the molecular basis of the binding of CCK to its receptor will require continued refinement as additional data are generated. These data will likely add new specific constraints that can improve the model. All data generated from the types of studies described above should be accommodated and explained by such a model, taking into consideration the strengths and weaknesses of each of these complementary experimental approaches. Ultimately, a high-quality model will emerge that will make substantial contribution to the rational development and refinement of receptor-active drugs.

Acknowledgments Supported by grants from the National Institutes of Health (DK32878) and the Fiterman Foundation.

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