Identification of domains conferring ligand binding specificity to the prostanoid receptor - Studies on chimeric prostacyclin/prostaglandin D receptors

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), The mIP receptor expressed in COS 7 cells bound [H-3]iloprost, a PGI(2) analog and [H-3]PGE(1) with K-d values of 13 and 27 nM, respectively, This receptor did not bind [H-3]PGD(2), [H-3]PGE(2), and [H-3]PGF(2 alpha). The mDP receptor bound only [H-3]PGD(2) with a K-d value of 43 nM, The chimeric IPN-VII/DPC receptor with replacement of the carboxyl tail of the mIP receptor with that of the mDP receptor showed 12-16-fold higher affinities for [H-3]iloprost and [H-3]PGE(1) than the mIP receptor, The region extending from the sixth transmembrane domain to the carboxyl terminus of the mIP receptor was next replaced with the corresponding region of the mDP receptor, This chimeric IPN-V/DPVI-C receptor acquired the ability to bind [H-3]PGD(2) and [H-3]PGE(2) without decreasing the affinities of the mIP receptor to [H-3]iloprost and [H-3]PGE(1). These binding characteristics did not change when the fourth and fifth transmembrane domains of the mIP receptor were further replaced with She corresponding regions of the mDP receptor, However, when the first extracellular to second intracellular loop of the mIP receptor containing the third transmembrane domain was further replaced with those of the mDP receptor, the affinities for [H-3]PGE(1), [H-3]PGE(2), and [3H]iloprost were markedly decreased, whereas that far [H-3]PGD(2) was increased by about 2-fold, [H-3]PGF(2 alpha) showed no affinity for the mIP, mDP, and all the chimeric receptors. These results suggest that She 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.