Differential Spatial Approximation between Secretin and Its Receptor Residues in Active and Inactive Conformations Demonstrated by Photoaffinity Labeling

Abstract

Understanding of the conformational changes in G protein-coupled receptors associated with activation and inactivation is of great interest. We previously used photoaffinity labeling to elucidate spatial approximations between photolabile residues situated throughout the pharmacophore of secretin agonist probes and this receptor. The aim of the current work was to develop analogous photolabile secretin antagonist probes and to explore their spatial approximations. The most potent secretin antagonist reported is a pseudopeptide ([psi(4, 5)]secretin) in which the peptide bond between residues 4 and 5 was replaced by a psi(CH(2)-NH) peptide bond isostere. We have developed a series of [psi(4, 5)]secretin analogs incorporating photolabile benzoyl phenylalanine residues in positions 6, 22, and 26. Each bound to the secretin receptor saturably and specifically, with affinity similar to their parental peptide. At concentrations with no measurable agonist activity, each probe covalently labeled the secretin receptor. Peptide mapping using proteolytic cleavage, immunoprecipitation, and radiochemical sequencing identified that each of these three probes labeled the amino terminus of the secretin receptor. Whereas the position 22 probe labeled the same residue as its analogous agonist probe and the position 6 probe labeled a residue within two residues of that labeled by its analogous agonist probe, the position 26 probe labeled a site 16 residues away from that labeled by its analogous agonist probe. Thus, whereas structurally related agonist and antagonist probes dock in the same general region of this receptor, conformational differences in active and inactive states result in substantial differences in spatial approximation at the carboxyl-terminal end of secretin analogs.

Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites

“…We hypothesized that the receptor conformation stabilized by a U-II agonist might differ from a conformation stabilized by a weak partial agonist and that these changes may be detected by photoaffinity labelling. Such an approach has recently been exploited for the cholecystokinin and secretin receptors [31,32].…”

Effects of pH and temperature on photoaffinity labeling of Family B G protein-coupled receptors

“…This approach has been particularly useful for the study of Family B G protein-coupled receptors, where the natural ligands are moderately large peptides with extended pharmacophoric domains. The prototypic secretin receptor has been extensively studied using this technique to establish spatial approximation constraints potentially useful in molecular modeling [12–18, 22–25]. We have previously used secretin probes incorporating a photolabile benzophenone, Bpa or (BzBz)Lys, in various positions throughout the length of the 27-residue peptide [12–18, 22–25].…”

“…The prototypic secretin receptor has been extensively studied using this technique to establish spatial approximation constraints potentially useful in molecular modeling [12–18, 22–25]. We have previously used secretin probes incorporating a photolabile benzophenone, Bpa or (BzBz)Lys, in various positions throughout the length of the 27-residue peptide [12–18, 22–25]. These spatial constraints have been key in building a working model of the ligand-bound secretin receptor complex [24].…”

Class B GPCRs: A Hidden Agonist Within?: Fig. 1.

“…Current evidence suggests that such antagonists primarily act by competing with conventional agonists for binding to the receptor's amino terminus (Lopez de Maturana et al, 2003). If so, reported antagonists at the secretin receptor (Dong et al, 2006a) should be ineffective in blocking stimulation induced by oligopeptides derived from built-in agonist sequence.…”