[Determination of ligand binding domains of the prostanoid receptors]

Prostanoid receptors are the G-protein-coupled, rhodopsin-type receptors with seven transmembrane domains and consist of eight types and subtypes. Although the overall homology is not high, there are several regions specifically conserved among them. These regions are considered to form the ligand binding pocket for the structures common to prostanoid molecules, and the other regions to confer specificity for ligand binding. The PGI and PGD receptors have relatively high homology (40%) at the amino acid level and share the same signalling pathway. To determine which structural domains of these receptors confer ligand binding specificity, we constructed a series of chimeric receptors from the mouse PGI and PGD receptors. These chimeric receptors were expressed in COS-7 cells, and their abilities to bind prostaglandins and their analogues were examined. The region from the sixth transmembrane domain to the carboxyl terminus of the PGI receptor was first replaced by the corresponding region of the PGD receptor. This chimeric receptor binds both PGD2 and PGE2, though the ability to bind iloprost, a PGI receptor agonist, and PGE1 does not change. This result indicates that the sixth and seventh transmembrane domains of the PGI receptor play an important role in distinction of structural difference between PGE1 and PGE2 in the alpha-side chain. These binding characteristics did not change when the region up to the third transmembrane domain of the PGI receptor was replaced with the corresponding region of the PGD receptor. However, when the first extracellular loop including a portion of the second transmembrane domain was further replaced, the abilities to bind PGE1, PGE2 and iloprost were eliminated. This result indicates that this domain of the PGD receptor is responsible for distinction of structural differences between PGD2 and PGE2 on the cyclopentane ring.