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 prostanoi d receptors, we constructed a series of chimeric receptors by successi vely replacing the regions from the carboxyl-terminal tail of mouse pr ostacyclin (prostaglandin I (PGI)) receptor (mIP) with the correspondi ng 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) w ith K-d values of 13 and 27 nM, respectively, This receptor did not bi nd [H-3]PGD(2), [H-3]PGE(2), and [H-3]PGF(2 alpha). The mDP receptor b ound 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 exten ding from the sixth transmembrane domain to the carboxyl terminus of t he mIP receptor was next replaced with the corresponding region of the mDP receptor, This chimeric IPN-V/DPVI-C receptor acquired the abilit y to bind [H-3]PGD(2) and [H-3]PGE(2) without decreasing the affinitie s 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 correspond ing 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 recep tor, the affinities for [H-3]PGE(1), [H-3]PGE(2), and [3H]iloprost wer e markedly decreased, whereas that far [H-3]PGD(2) was increased by ab out 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 s eventh transmembrane domain of the mIP receptor confers the specificit y 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.