The Seventh Transmembrane Domain of Gastrin/CCK Receptors Contributes to Nonpeptide Antagonist Binding

doi:10.1006/bbrc.1994.1856

Biochemical and Biophysical Research Communications

Volume 201, Issue 3, 30 June 1994, Pages 1382-1389

https://doi.org/10.1006/bbrc.1994.1856 Get rights and content

Abstract

The related rat cholecystokinin (CCK)-A and gastrin/CCK-B receptors can be selectively blocked by the antagonists L364718 and L365260, respectively. In order to determine receptor domains which are important in conferring specificity for L365260 and L364718 we constructed by overlap-PCR a rat gastrin/CCK-B receptor chimaera which contained the seventh transmembrane domain of the rat CCK-A receptor. Receptor binding assays on transiently transfected COS cells demonstrated a selective reduction in the affinity of the chimaeric receptor for L364718, so that the L365260 and L364718 affinities were of a similar order. Since the chimaera differs from the wildtype gastrin/CCK-B receptor by only six amino acids we conclude that one or more of these six amino acids contributes to L364718 binding and that the affinity determinants of L365260 and L364718 must, at least in part, be different. Furthermore, the affinity of the chimaera for gastrin is essentially the same as the gastrin/CCK-B receptor, indicating that the six different amino acids probably do not contribute to peptide agonist binding.

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Cited by (32)

Molecular basis for binding and subtype selectivity of 1,4-benzodiazepine antagonist ligands of the cholecystokinin receptor
2012, Journal of Biological Chemistry
Citation Excerpt :
Those studies, however, were more indirect, utilizing the orthosteric radioligand based on the natural peptide that is known to bind to extracellular loop and tail regions rather than a radioligand that binds directly to the intramembranous docking site of these small molecules. Two of the residues identified as important in the current work (residues 6.51 and 7.39) have also been identified as important for the binding of the benzodiazepine antagonist L364,718 to the CCK1R (25, 46, 47). This is helpful in confirming the more general relevance of these residues for small molecule ligand binding to this receptor.
Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2R-selective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs.
Structure-activity function for binding and signaling in CHO-K1 and COS-7 cells expressing the cholecystokinin A receptor
2004, Biochemical and Biophysical Research Communications
Citation Excerpt :
Replacement of His381 to Leu or Phe of the CCK-BR 7TM improved the affinity of CCK-AR antagonists, L-364,718 and SR-27,897B [25]. Furthermore, one or more six amino acids on the CCK-AR 7TM may contribute to L-364,718 binding, but not CCK binding [26]. Thus, CCK-AR antagonists may bind to either TM6 or TM7, independent of the agonist binding.
Key amino acids of the cholecystokinin (CCK) peptide for receptor binding are sulfated Y27, W30, D32, and F33-NH₂. Three-dimensional modeling showed that the CCK-A receptor (CCK-AR) antagonist devazepide penetrated into the transmembrane (TM) domains, whereas CCK was placed on the surface of the CCK-AR. Four types of rat CCK-AR cDNAs were transfected into CHO-K1 and COS-7 cells: normal CCK-AR cDNA transfected cells (wild type, WT); K120 substituted with V; K130V; and R352V. Binding of [³H]CCK-8 was observed in WT and K130V, but not in K120V and R352V. CCK caused Ca²⁺ spiking in WT and K130V, whereas K120V and R352V had no effect. Three chimeras including the CCK-AR/3iβ2 adrenergic receptor (β2AR), 3Niβ2AR, and 3Ciβ2AR were constructed. Two groups of point mutations in the CCK-AR3i were also made: Y252V, S274V, S281V, and S289V (non-phospho-acceptor Y or S); S260V, S264V, S271V, and S275V (phospho-acceptor S). WT and CCK-AR/3Ciβ2AR increased [Ca²⁺]_i in response to CCK; 3Niβ2AR was vice versa. CCK failed to increase [IP₃] in phospho-acceptor S to V without affecting binding. Non-phospho-acceptor S or Y to V showed normal response. Thus, Lys120 outside the TM2 and Arg352 outside the TM6 of the CCK-AR are amino acids interacting with Tyr[SO₃H]27 and Asp32 of the CCK peptide for binding. Phospho-acceptor Ser groups in the CCK-AR 3Ni are amino acids for initiating cell signaling.
Conformational and molecular modeling studies of sulfated cholecystokinin-15
2002, Biochemical and Biophysical Research Communications
Conformational features of the C-terminal carboxyamidated pentadecapeptide of CCK (S¹⁹HRISDRD[SO₄]–YMGWMDF³³–NH₂) were determined by NMR spectroscopy in a zwitterionic membrane-mimetic solvent system, composed of DPC micelles. The C-terminal octapeptide consisted of a well-defined pseudohelix that was nearly identical to the structure previously reported for nonsulfated CCK-8 in the same solvent system. N-terminal amino acids of CCK-15 were highly disordered, with no clear conformational preference. Extensive NOE-restrained molecular dynamics simulations of the CCK-15/CCK₁–R complex suggested that almost all the experimentally determined intermolecular contact points provided by NMR, site-directed mutagenesis, and photoaffinity labeling could be simultaneously satisfied, when the N-terminus of the ligand is placed in close spatial proximity to the N-terminus of the receptor.
Biologically Active Recombinant Human Progastrin<inf>6-80</inf> Contains a Tightly Bound Calcium Ion
2001, Journal of Biological Chemistry
Citation Excerpt :
After three further rinses the coverslips were mounted on slides with cytifluor and observed on a fluorescence microscope. COS7 cells were transiently transfected by the DEAE-dextran method as described previously (21). One day before transfection, 0.7–1.0 × 106 COS cells were seeded in 10-cm plates in Dulbecco's modified Eagle's medium and grown in 5% CO2 such that on the day of transfection the cells were 60% confluent.
Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin_6–80 and to investigate its structure and biological activities in vitro. Human progastrin_6–80 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin_6–80 was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin_6–80 at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin_6–80 did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin_6–80suggested that progastrin_6–80 stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin_6–80 is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.
Characterization of the type A cholecystokinin receptor hormone-binding domain: Use of contrasting and complementary methodologies
2001, Peptides
Insights into the molecular basis of binding of the peptide hormone, cholecystokinin, to its G protein-coupled receptor is of substantial interest and may contribute to the successful production and refinement of receptor-active drugs. A number of methodological approaches provide complementary data to contribute to these insights. These include receptor mutagenesis, ligand structure-activity data, conformational analysis of ligand and receptor fragments, and photoaffinity labeling. In this work, we compare and contrast each of these methods and provide our current view of the cumulative impact of the current data on molecular conformational models of the agonist-occupied type A cholecystokinin receptor. These support the key roles played by extracellular loop and tail regions of this receptor for binding its natural peptide ligand.
Insights into the molecular basis of ligand binding by the cholecystokinin receptor
2001, Pancreatology
The receptor for the peptide hormone, cholecystokinin, is a G-protein-coupled receptor in the rhodopsin/β-ad-renergic 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.

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Biochemical and Biophysical Research Communications

Regular ArticleThe Seventh Transmembrane Domain of Gastrin/CCK Receptors Contributes to Nonpeptide Antagonist Binding

Abstract

Molecular basis for binding and subtype selectivity of 1,4-benzodiazepine antagonist ligands of the cholecystokinin receptor

Structure-activity function for binding and signaling in CHO-K1 and COS-7 cells expressing the cholecystokinin A receptor

Conformational and molecular modeling studies of sulfated cholecystokinin-15

Biologically Active Recombinant Human Progastrin<inf>6-80</inf> Contains a Tightly Bound Calcium Ion

Characterization of the type A cholecystokinin receptor hormone-binding domain: Use of contrasting and complementary methodologies

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

Regular Article
The Seventh Transmembrane Domain of Gastrin/CCK Receptors Contributes to Nonpeptide Antagonist Binding