Fluorescence resonance energy transfer analysis of the antagonist- and partial agonist-occupied states of the cholecystokinin receptor

Abstract

Changes in receptor conformation are believed to be key for ligand-induced regulation of cellular signaling cascades. However, little information exists about specific conformations of a receptor. We recently applied fluorescence resonance energy transfer to determine distances from distinct points distributed over the surface and within the helical bundle of the cholecystokinin receptor to the amino terminus of a full agonist CCK analogue (Harikumar, K. G., Pinon, D. I., Wessels, W. S., Dawson, E. S., Lybrand, T. P., Prendergast, F. G., and Miller, L. J. (2004) Mol. Pharmacol. 65, 28-35). Here, we apply the same experimental strategy to determine distances from the same receptor positions to an analogous point at the amino terminus of structurally related partial agonist (Alexa488-Gly-[(Nle(28,31))CCK-26-32]phenethyl ester) and antagonist (Alexa488-Gly-[(D-Trp31, Nle(28,31))CCK-26-32]phenethyl ester) ligands. A high degree of spectral overlap and fluorescence transfer was observed for ligand-occupied fluorescent-tagged receptors with no transfer observed for the ligand-occupied pseudo-wild type null cysteine-reactive mutant receptor (C94S). For the partial agonist, calculated distances to receptor positions 94, 102, 204, and 341, representing sites within the helical confluence, and the first, second, and third loops, were 21 +/- 0.4, 18 +/- 0.4, 25 +/- 1, and 17 +/- 1 angstroms, not different from those measured previously for the analogous full agonist. For the antagonist, the analogous distances were 21 +/- 2, 28 +/- 2, 15 +/- 1 and 21 +/- 1 angstroms. Distances to the first and third loops were longer and the distance to the second loop was shorter for the antagonist relative to both the full and partial agonist probes, whereas all three probes demonstrated similar distances to the intrahelical reference point. This supports the possibilities of changes in the conformation of the probe and/or the receptor induced by structurally similar ligands having distinct intrinsic biological activities.