Abstract
Using chimeras of the mouse prostaglandin (PG) I receptor (mIP) and the mouse PGD receptor (mDP), we previously revealed that the cyclopentane ring recognition by these receptors is specified by a region from the first to third transmembrane domain of each receptor; recognition by this region of mIP is broad, accommodating the D, E, and I types of cyclopentane rings, whereas that of mDP binds the D type of PGs alone (Kobayashi, T., Kiriyama, M., Hirata, T., Hirata, M., Ushikubi, F., and Narumiya, S. (1997) J. Biol. Chem. 272, 15154-15160). In the present study, we performed a more detailed chimera analysis, and narrowed the domain for the ring recognition to a region from the first transmembrane domain to the first extracellular loop. One chimera with the replacement of the second transmembrane domain and the first extracellular loop of mDP with that of mIP bound only iloprost. The amino acid substitutions in this chimera suggest that Ser(50) in the first transmembrane domain of mIP confers the broad ligand recognition of mIP and that Lys(75) and Leu(83) in the second transmembrane domain of mDP confer the high affinity to PGD(2) and the strict specificity of ligand binding of mDP, respectively.
Highlights
Using chimeras of the mouse prostaglandin (PG) I receptor and the mouse PGD receptor, we previously revealed that the cyclopentane ring recognition by these receptors is specified by a region from the first to third transmembrane domain of each receptor; recognition by this region of mIP is broad, accommodating the D, E, and I types of cyclopentane rings, whereas that of mDP binds the D type of PGs alone (Kobayashi, T., Kiriyama, M., Hirata, T., Hirata, M., Ushikubi, F., and Narumiya, S. (1997) J
We found that further replacement of the region containing the COOH-terminal end of the second transmembrane domain (Phe102) to the second intracellular loop of mIP with that of mDP resulted in loss of the broad recognition of mIP and gain of the strict specificity of mDP
This IPN-II(Val101)/ DPII(Leu83)-C receptor, formerly designated as IPN-II/DPIII-C (15), markedly decreased the affinities to PGE1, PGE2, and iloprost, and bound PGD2 alone. These results suggest that this region of mDP has amino acid residue(s) determining the strict specificity of ligand binding of mDP
Summary
Materials—PGD2, PGE1, PGE2, and PGF2␣ were generous gifts from Ono Pharmaceuticals Co. The Chimeric IPN-Ex1/DPIII-C Receptor—Fragments N-1 and C-1 were amplified by PCR with primer pairs and the template shown in Table I (Fig. 3B). The Chimeric IPN-I/DPII-C Receptor—Fragments N-2 and C-2 were amplified by PCR with primer pairs and the template shown in Table I (Fig. 3B). The Chimeric IPN/DPI-C Receptor—Fragments N-3 and C-3 were amplified by PCR with primer pairs and the template shown in Table I (Fig. 3B). The Chimeric DPN-I/IPII-Ex1/DPIII-C Receptor—Fragments N-4 and C-4 were amplified by PCR with primer pairs and the template shown in Table I (Fig. 3B). Fragment N-4 was digested with Asp[718] and Eco47III, and fragment C-4 was digested with Eco47III and BamHI Both digested fragments were ligated into the Asp[718] and BamHI sites of pCMX-mIP. Four mutants were constructed by site-directed mutagenesis of this chimeric DPN-I/IPII-Ex1/DPIII-C receptor
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