1 Eight types and subtypes of the mouse prostanoid receptor, the prostaglandin D (DP) receptor, the prostaglandin F (FP) receptor, the prostaglandin I (IP) receptor, the thromboxane A (TP) receptor and the EP 1 , EP 2 , EP 3 and EP 4 subtypes of the prostaglandin E receptor, were stably expressed in Chinese hamster ovary cells. Their ligand binding characteristics were examined with thirty two prostanoids and their analogues by determining the K i values from the displacement curves of radioligand binding to the respective receptors. 2 The DP, IP and TP receptors showed high ligand binding speci®city and only bound their own putative ligands with high anity such as PGD 2 , BW245C and BW868C for DP, cicaprost, iloprost and isocabacyclin for IP, and S-145, I-BOP and GR 32191 for TP. 3 The FP receptor bound PGF 2a and¯uprostenol with K i values of 3 ± 4 nM. In addition, PGD 2 , 17-phenyl-PGE 2 , STA 2 , I-BOP, PGE 2 and M&B Á -28767 bound to this receptor with K i values less than 100 nM. 4 The EP 1 receptor bound 17-phenyl-PGE 2 , sulprostone and iloprost in addition to PGE 2 and PGE 1 , with K i values of 14 ± 36 nM. 16,16-dimethyl-PGE 2 and two putative EP 1 antagonists, AH6809 and SC-19220, did not show any signi®cant binding to this receptor. M&B-28767, a putative EP 3 agonist, and misoprostol, a putative EP 2 /EP 3 agonist, also bound to this receptor with K i values of 120 nM. 5 The EP 2 and EP 4 receptors showed similar binding pro®les. They bound 16,16-dimethyl PGE 2 and 11-deoxy-PGE 1 in addition to PGE 2 and PGE 1 . The two receptors were discriminated by butaprost, AH-13205 and AH-6809 that bound to the EP 2 receptor but not to the EP 4 receptor, and by 1-OH-PGE 1 that bound to the EP 4 but not to the EP 2 receptor. 6 The EP 3 receptor showed the broadest binding pro®le, and bound sulprostone, M&B-28767, GR63799X, 11-deoxy-PGE 1 , 16,16-dimethyl-PGE 2 and 17-phenyl-PGE 2 , in addition to PGE 2 and PGE 1 , with K i values of 0.6 ± 3.7 nM. In addition, three IP ligands, iloprost, carbacyclin and isocarbacyclin, and one TP ligand, STA 2 , bound to this receptor with K i values comparable to the K i values of these compounds for the IP and TP receptors, respectively. 7 8-Epi-PGF 2a showed only weak binding to the IP, TP, FP, EP 2 and EP 3 receptor at 10 mM concentration.

To identify domains conferring ligand binding specificity to prostanoid receptors, we constructed a series of chimeric receptors by successively replacing the regions from the carboxyl-terminal tail of mouse prostacyclin (prostaglandin I (PGI)) receptor (mIP) with the corresponding regions of the mouse PGD receptor (mDP showed no affinity for the mIP, mDP, and all the chimeric receptors. These results suggest that the sixth to seventh transmembrane domain of the mIP receptor confers the specificity of this receptor to bind selectively to PGE 1 and not to PGE 2 and that the third transmembrane domain of the mDP receptor confers the selective binding of PGD 2 to this receptor. Prostaglandins (PGs)1 contain prostanoic acid as a central structural element. PGs have two structural features in the prostanoic acid framework. First, they have functional groups on the cyclopentane ring, which classifies them into four types, D, E, F, and I. Second, they are classified into three series, 1, 2, and 3, by the number of double bonds in the side chains. Additionally, another cyclooxygenase product, thromboxane A 2 has an oxane ring instead of the cyclopentane ring. These prostanoids act on eight types and subtypes of the receptors. They are the PGD receptor (DP), the EP 1 , EP 2 , EP 3 , and EP 4 subtypes of the PGE receptor, the PGF receptor (FP), the PGI receptor (IP), and the thromboxane A 2 receptor (TP) (1-4). These receptors can recognize the structural differences of prostanoid molecules. The binding affinities of these receptors to prostanoid molecules are determined primarily by the cyclopentane ring structures of ligands. For example, the DP receptor shows the highest affinities to PGD 2 and PGD 1 , but affinities to other prostanoids are at least 2 orders of magnitude less. One exception is the IP receptor, which shows the affinity to PGE 1 almost comparable to PGI analogs such as iloprost. This receptor, however, can bind PGE 2 with much lower affinity, suggesting that the IP receptor can discriminate a difference in the side chains.We have cloned cDNAs for all of these types and subtypes of the mouse prostanoid receptors (5-13). These studies revealed that the prostanoid receptors belong to the G protein-coupled rhodopsin type receptor superfamily. They have several regions conserved specifically among them. These conserved regions may participate in the construction of binding domains for structures common to prostanoid molecules, whereas the other regions may confer specificity for ligand binding. For example, the arginine in the seventh transmembrane domain, which is conserved in all of the prostanoid receptors, was proposed to be the binding site for the carboxyl group of prostanoid molecules (5, 14, 15). In fact, Funk et al. (16) have shown that a point mutation at this arginine residue in the human TP receptor results in loss of ligand binding activity. However, structural domains of the prostanoid receptors conferring specificity for ligand binding are as yet unknown.

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