Structurally Related Peptide Agonist, Partial Agonist, and Antagonist Occupy a Similar Binding Pocket within the Cholecystokinin Receptor

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103

Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa 488 , nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodide and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.Cell surface receptors present on essentially every excitable cell of the body are important targets for pharmacotherapy. A detailed understanding of the structure of these molecules and the molecular basis of their activation should contribute to the rational design and refinement of ligands for these receptors. Receptor-bound, environmentally sensitive fluorescent reporters can provide information regarding ligand-binding domains (1). In this work, we utilize this approach to gain insight into agonist-and antagonist-binding domains of the type A cholecystokinin (CCK) 1 receptor, a physiologically important member of the rhodopsin/␤-adrenergic receptor family of guanine nucleotide-binding protein (G protein)-coupled receptors.The superfamily of G protein-coupled receptors represents the largest group of membrane receptors. They are remarkable for the diversity in structure of the natural agonist ligands that can activate them and initiate intracellular signaling cascades. These range in size from small photons and odorants to peptides, proteins, and even large viral particles. Tertiary structure determination by x-ray crystallography has provided the most incisive insight into the structure of superfamily members, which bind small ligands in the intramembranous helical bundle domain ...

Section: Lifetime Measurements For Fluorescent Ligandsmentioning

confidence: 62%

Section: Lifetime Measurements For Fluorescent Ligandsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Environment and Mobility of a Series of Fluorescent Reporters at the Amino Terminus of Structurally Related Peptide Agonists and Antagonists Bound to the Cholecystokinin Receptor

Harikumar

Pinon

Wessels

et al. 2002

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103

“…In the angiotensin AT 1 and the endothelin ET A receptors, minimal overlapping binding sites between peptide agonists and antagonists have been demonstrated, respectively (40,41). Very recently, it has been observed that structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the rat cholecystokinin CCK-A receptor (42). Similarly, we describe in the present study major transmembrane overlapping binding sites in the V 1a vasopressin receptor for both peptide agonist and two linear peptide antagonists.…”

Section: Receptor Interactions As Observed In the Modelmentioning

confidence: 99%

Docking of Linear Peptide Antagonists into the Human V1a Vasopressin Receptor

Phalipou¹,

Seyer²,

Cotte³

et al. 1999

Phpa-LVA (covalent attachment to transmembrane domain VII), threedimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V 1a receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.Over the past few years, interest in locating ligand-binding sites in G protein-coupled receptors has increased exponentially. Indeed, identification of these binding sites is of prime importance both for a better understanding of the structure and the function of the G protein-coupled receptor superfamily and for facilitating rational design of potential therapeutic agents. Extensive mutational analysis and receptor three-dimensional molecular modeling have led to valuable information concerning "small ligand" and peptide/protein ligand receptor-binding sites (for review, see Refs. 1-6).In 1995, we published the mapping of arginine-vasopressin (AVP) 1 -binding site in the V 1a receptor subtype and described a major localization within transmembrane regions (TMR) in a position equivalent to that defined for the cationic neurotransmitters (7). Because all receptor residues potentially interacting with AVP are conserved in the different members of the AVP/oxytocin (OT) receptor family, we proposed that the binding pocket identified in the V 1a might be common to V 2 , V 1b , and OT receptor subtypes. Extracellular residues responsible for receptor-selective and species-selective binding have also been identified (8 -10). Unfortunately, these first analyses of AVP receptor structure/function relationships did not provide much information on AVP receptor antagonist-binding domains (Refs. 11 and 12, and for review see Ref. 13). The photoaffinity labeling technique is an essential complement to modeling and mutagenesis approaches and allows direct unambiguous identification of the contact regions between a receptor and its specific photoactivatable ligands (for review see Ref. 14). At the present time, very few photoaffinity labeling studies have led to the direct determination of labeled amino acid residues in peptide G protein-coupled receptor; remarkable results with bovine V 2 receptor (15), human NK1 tachykinin receptor (16), and rat type A cholecystokinin receptor (17) allowed identification of covalently labeled residues with photoactivatable agonist analogues of AVP, substance P, and cholecystokinin, respectively.Very recently, a first radioiodinated photoreactive linear peptide antagonist has been used in our laboratory to photolabel the human and rat V 1a receptors (12,18,19). Our results have clearly indicated that covalent attachment of the [ 125 I]3N 3 Phpa-LVA occurs in a restricted domain of the human receptor including TMR VII. Based both on this photolabeling result and on th...

“…These probes have similar primary structures, sharing the critical amino acid functional groups that have been shown to be responsible for the specificity of CCK receptor binding, pointing toward similarity in the position of their docking to this receptor. Additional support for similar docking comes from photoaffinity labeling studies in which the same segment of the CCK receptor was covalently labeled by each of three analogous photolabile probes, with sites of covalent attachment through a benzoyl phenylalanine at position 24, the same position as that of the fluorescence indicator in the probes used in the current work (16). For still further validation of the similarity of the docking of these probes and for purposes of registration, in the current work all three of these structurally similar ligands resulted in a fixed distance measured between the fluorophore at the amino terminus of the probes at position 24 and receptor residue 94 within the intramembranous region of helix two.…”

Section: Development and Characterization Of Receptor Probes-the Fluomentioning

confidence: 99%

Fluorescence Resonance Energy Transfer Analysis of the Antagonist- and Partial Agonist-occupied States of the Cholecystokinin Receptor

Harikumar

Miller

2005

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Fluorescence resonance energy transfer (FRET)1 is a powerful biophysical tool to determine the distances between a donor and an acceptor (2). This methodology can be applied to distinct positions within a ligand and receptor as they exist within a molecular complex to elucidate spatial constraints. By utilizing ligands with distinct biological properties, stabilizing the receptor into fully active, partially active, and inactive conformations, this approach can be useful for the elucidation of the conformational changes that are associated with receptor activation.Recently, we utilized this technique to measure a series of intermolecular distances between a fixed point at the aminoterminal end of a CCK analogue that represents a full agonist and distinct points on the exposed surfaces of the CCK receptor (1). This was accomplished by the individual introduction of reactive Cys residues at positions thought likely to accommodate this modification without having negative functional implications based on structure-activity relationships among structurally related receptors. This was fully validated (3). Subsequently, cells expressing these receptor constructs were allowed to react with a cell-impermeant, fluorescent, thiolreactive methanethiosulfonate (MTS) reagent that was specially prepared (1, 3). The fluorescent donor was provided to the system in a highly specific way, taking advantage of the structural specificity of interaction between hormone and receptor, utilizing a fluorescent analogue of the natural peptide ligand of this receptor. A series of controls established that transfer only occurred between the receptor-bound ligand and the specific sites within the receptor (1).The data from the current approach utilizing FRET between ligand and receptor complements other experimentally derived constraints that have come from structure-activity studies of ligand and receptor, receptor mutagenesis, photoaffinity labeling studies, and studies of the fluorescence properties of ligand probes (1, 4 -7). These have provided a workable model of the CCK-ligand-occupied type A CCK receptor as well as insights into the types of conformational changes that might occur upon receptor activation. The data in the current work provide the first quantitative measurements to be applied to these working models to help describe the details of the conformational changes that are associated with agonist-stimulated receptor activation and initiation of signaling. EXPERIMENTAL PROCEDURESMaterials-Synthetic CCK octapeptide (CCK-26 -33, using the numbering convention based on the 33-residue CCK peptide first isolated from porcine duodenum) was purchased from Peninsula Laboratories (Belmont, CA