Measurement of Intermolecular Distances for the Natural Agonist Peptide Docked at the Cholecystokinin Receptor Expressed in Situ Using Fluorescence Resonance Energy Transfer

Abstract

Fluorescence resonance energy transfer is a powerful biophys-ical technique used to analyze the structure of membrane proteins. Here, we used this tool to determine the distances between a distinct position within a docked agonist and a series of distinct sites within the intramembranous confluence of helices and extracellular loops of the cholecystokinin (CCK) receptor. Pseudo-wild-type CCK receptor constructs having single reactive cysteine residues inserted into each of these sites were developed. The experimental strategy included the use of the full agonist, Alexa 488 -CCK, bound to these receptors as donor, with Alexa 568 covalently bound to the specific sites within the CCK receptor as acceptor. Site-labeling was achieved by derivatization of intact cells with a novel fluorescent methanethiosulfonate reagent. A high degree of spectral overlap was observed between receptor-bound donor and receptor-derivatized acceptors, with no transfer observed for a series of controls representing saturation of the receptor binding site with nonfluorescent ligand and use of a null-reactive CCK receptor construct. The measured distances between the fluorophore within the docked agonist and the sites within the first (residue 102) and third (residue 341) extracellular loops of the receptor were shorter than those directed to the second loop (residue 204) or to intramembranous helix two (residue 94). These distances were accommodated well within a refined molecular model of the CCK-occupied receptor that is fully consistent with all existing structure-activity and photoaffinitylabeling studies. This approach provides the initial insights into the conformation of extracellular loop regions of this receptor and establishes clear differences from analogous loops in the rhodopsin crystal structure.An understanding of the molecular details of agonist ligand binding to a receptor provides powerful insights into the tertiary structure of the receptor in its active conformation, which, in turn, can afford valuable information for possible structure-based drug design. In this work, we have applied fluorescence resonance energy transfer (FRET) to the determination of distances between a fixed position in an agonist ligand and a series of defined positions within its receptor. Experimentally derived distance constraints so determined can complement insights gained from ligand structure-activity series (Ding et al., 2002) and studies using receptor mutagenesis (Kennedy et al., 1997;Gigoux et al., 1999) and photoaffinity labeling (Ji et al., 1997;Hadac et al., 1998Hadac et al., , 1999Ding et al., 2001). These experimentally determined intermolecular distances can also be extremely useful as constraints for three-dimensional model construction and refinement for receptor-ligand complexes.The type A CCK receptor, a member of class I guanine nucleotide-binding protein (G protein)-coupled receptors, is normally activated by a linear peptide hormone. CCK is important for the regulation of nutritional homeostasis, playing roles in th...

Structural basis of cholecystokinin receptor binding and regulation

“…The human CCK 1 receptor structure is shown in gray. The position of the docked CCK ligand (Tyr-GlyAsp-Tyr(SO 3 )-Nle-Gly-Trp-Nle-Asp-Phe(NH 2 ), representing amino-terminally-extended CCK-26-33 with the natural Met residues replaced by Nle residues) (shown as space-filling in white with the peptide backbone shown from amino terminus to carboxyl terminus blue-tored) reflects the model based on photoaffinity labeling data (Harikumar et al, 2004). Key affinity labeled residues within the receptor are illustrated in tan.…”

“…Application of these insights have resulted in the proposal of molecular models of natural ligand-occupied CCK receptors (Harikumar et al, 2004;Henin et al, 2006). It is remarkable that, even after the collection of so many experimentally-derived constraints of different types, two quite distinct three-dimensional models continue to be actively considered in the current literature.…”

“…Studies ranging from mutagenesis, complementary mutagenesis, and photoaffinity labeling have all placed the acidic tyrosine-sulfate moiety of CCK in spatial approximation with arginine 197 in the second extracellular loop (Gigoux et al, 1999b;Ding et al, 2002;Arlander et al, 2004). The major difference between these two molecular models is the placement of the carboxyl-terminal region of CCK (Harikumar et al, 2004;Henin et al, 2006). In the model based primarily on photoaffinity-derived constraints, this portion of the peptide is also at the surface of the lipid bilayer approximated with the aminoterminal tail, just above the first transmembrane segment (Harikumar et al, 2004).…”

“…The major difference between these two molecular models is the placement of the carboxyl-terminal region of CCK (Harikumar et al, 2004;Henin et al, 2006). In the model based primarily on photoaffinity-derived constraints, this portion of the peptide is also at the surface of the lipid bilayer approximated with the aminoterminal tail, just above the first transmembrane segment (Harikumar et al, 2004). In contrast, in the model relying more heavily on receptor mutagenesis, this part of CCK is shown to dip into the bilayer within the helical bundle (Henin et al, 2006).…”

“…In the one based predominantly on photoaffinity labeling data (Harikumar et al, 2004;Hadac et al, 2006), the proposed benzodiazepine binding site would be predicted to represent an allosteric site, while the other model based predominantly on receptor mutagenesis data (Henin et al, 2006) illustrates the binding sites for peptide and non-peptidyl ligands as overlapping. Once this important question is resolved, there may be leads for the development of allosteric modulators of these important receptors.…”