Evidence of the residues involved in ligand recognition in the second extracellular loop of the prostacyclin receptor characterized by high resolution 2D NMR techniques.
In previous studies, we have determined the solution structure of the second extracellular loop (eLP(2)) of the human thromboxane A(2) receptor (TP) and identified the residues in the eLP(2) domain involved in ligand recognition, by using a combination of approaches including a constrained synthetic peptide, 2D NMR spectroscopy, and recombinant proteins. These findings led us to hypothesize that the specific ligand recognition sites may be localized in the eLP(2) for all the prostanoid receptors. To test this hypothesis, we have investigated the ligand recognition site for another prostanoid receptor, the prostacyclin receptor (IP), which mediates an opposite biological function compared to that of the TP receptor. The identification of the interaction between the IP receptor and its agonist, iloprost, was achieved with a constrained synthetic peptide mimicking the eLP(2) region of the receptor. The IP eLP(2) segment was designed and synthesized to form a constrained loop, using a homocysteine disulfide bond connecting the ends of the peptide, based on the distance predicted from the IP receptor model created by homology modeling using the crystal structure of bovine rhodopsin as a template. The evidence of the constrained IP eLP(2) interaction with iloprost was found by the identification of the conformational changes of the eLP(2) induced by iloprost using fluorescence spectroscopy, and was further confirmed by 1D and 2D 1H NMR experiments. In addition, the IP eLP(2)-induced structure of iloprost in solution was elucidated through a complete assignment of the 2D 1H NMR spectra for iloprost in the presence of the IP eLP(2) segment. In contrast, no ordered structure was observed in the 2D 1H NMR experiments for iloprost alone in solution. These studies not only identified that the eLP(2) segment of the IP receptor is involved in ligand recognition, but also solved the 3D solution structure of the bound-form of iloprost, which could be used to study the receptor-ligand interaction in structural terms.
D001665:Binding Sites; D006801:Humans; D016285:Iloprost / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000737:chemistry* / Q000378:metabolism
D008024:Ligands; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D016285:Iloprost