Abstract
Fluorescence techniques can provide insight into the environment of fluorescence indicators situated at distinct sites within a ligand as it is bound to its receptor. Here, we have developed a series of analogues of the 27-amino acid hormone, secretin, that incorporate a fluorescent Alexa Fluor 488 into the amino terminus, the carboxyl terminus, and positions 13 and 22. Each probe bound with high affinity and was biologically active, stimulating full cAMP responses in receptor-bearing Chinese hamster ovary-SecR cells. Treatment with 10 mum guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp) shifted the agonist-bound receptor into a G protein-uncoupled low affinity state. Fluorescence spectra for the probes in solution and bound to the receptor demonstrated maximal emission at 521 nm after excitation at 481 nm. Collisional quenching of fluorescence with potassium iodide revealed that Alexa at the amino terminus of secretin was more accessible than at the other three positions within the probes. Of note, quenching constants for each probe were higher when bound in the active state than in the G protein-uncoupled, low affinity state of the receptor, with the most marked changes occurring for the two midregion probes. Anisotropy values and fluorescence lifetimes confirmed this, with higher anisotropy and longer lifetimes observed for position 13 and 22 probes bound to the receptor in its uncoupled state than in its active state. These observations suggest that the amino terminus of secretin as docked to the receptor is most exposed to the hydrophilic aqueous milieu, and that the major changes in conformation and exposure to the medium occur in the midregion of secretin. Photoaffinity labeling studies have demonstrated approximation of each of these ligand residues with distinct receptor residues. Combining the fluorescence data with photoaffinity labeling data provides insights into the conformation and dynamics of a natural peptide ligand docked to a Family B G protein-coupled receptor.
Highlights
Insights for the largest family of G protein-coupled receptors, the A Family, which includes rhodopsin and the -adrenergic receptor [1, 2]
Competition binding studies demonstrated that these fluorescent peptide probes were able to bind to the secretin receptor saturably, and with high affinity, the probe with Alexa at the amino terminus had its affinity reduced by approximately 1 order of magnitude (Fig. 2A and Table 1)
Molecular basis of ligand binding, and mechanism of receptor activation can provide useful leads to help in the development of receptor-active drugs and to the refinement of drug candidates
Summary
Incorporated into the amino terminus, carboxyl terminus, and two positions within the midregion of the peptide (positions 13 and 22). We measured the fluorescence emission spectra, potassium iodide quenching, anisotropy, and lifetimes of these probes bound to the secretin receptor in both active and G protein-uncoupled conformations, manipulated using GppNHp, a non-hydrolysable analogue of GTP. The two probes in which Alexa was incorporated into positions Lys and Lys of secretin exhibited lower anisotropy and shorter average lifetimes with the receptor in its active conformation than in its G protein-uncoupled conformation. Consistent with these observations, the Alexa fluorescence of these probes was more quenched by potassium iodide with the receptor in the active conformation. The environments of both the amino-terminal and carboxyl-terminal probes were less affected by shifting the conformation of the receptor, the trends were similar to those of the midregion probes
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