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. 2003 Apr 15;371(Pt 2):443–449. doi: 10.1042/BJ20021429

Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor.

Frank Neuschäfer-Rube 1, Eva Engemaier 1, Sina Koch 1, Ulrike Böer 1, Gerhard P Püschel 1
PMCID: PMC1223288  PMID: 12519077

Abstract

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF(2alpha) and PGD(2). Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF(2alpha)-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.

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Selected References

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  1. Abramovitz M., Boie Y., Nguyen T., Rushmore T. H., Bayne M. A., Metters K. M., Slipetz D. M., Grygorczyk R. Cloning and expression of a cDNA for the human prostanoid FP receptor. J Biol Chem. 1994 Jan 28;269(4):2632–2636. [PubMed] [Google Scholar]
  2. Audoly L., Breyer R. M. Substitution of charged amino acid residues in transmembrane regions 6 and 7 affect ligand binding and signal transduction of the prostaglandin EP3 receptor. Mol Pharmacol. 1997 Jan;51(1):61–68. doi: 10.1124/mol.51.1.61. [DOI] [PubMed] [Google Scholar]
  3. Audoly L., Breyer R. M. The second extracellular loop of the prostaglandin EP3 receptor is an essential determinant of ligand selectivity. J Biol Chem. 1997 May 23;272(21):13475–13478. doi: 10.1074/jbc.272.21.13475. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Breyer R. M., Bagdassarian C. K., Myers S. A., Breyer M. D. Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol. 2001;41:661–690. doi: 10.1146/annurev.pharmtox.41.1.661. [DOI] [PubMed] [Google Scholar]
  6. Böer U., Neuschäfer-Rube F., Möller U., Püschel G. P. Requirement of N-glycosylation of the prostaglandin E2 receptor EP3beta for correct sorting to the plasma membrane but not for correct folding. Biochem J. 2000 Sep 15;350(Pt 3):839–847. [PMC free article] [PubMed] [Google Scholar]
  7. Chang C. S., Negishi M., Nishigaki N., Ichikawa A. Characterization of functional interaction of carboxylic acid group of agonists and arginine of the seventh transmembrane domains of four prostaglandin E receptor subtypes. Prostaglandins. 1997 Jul;54(1):437–446. doi: 10.1016/s0090-6980(97)00064-6. [DOI] [PubMed] [Google Scholar]
  8. Chang C., Negishi M., Nishigaki N., Ichikawa A. Functional interaction of the carboxylic acid group of agonists and the arginine residue of the seventh transmembrane domain of prostaglandin E receptor EP3 subtype. Biochem J. 1997 Mar 1;322(Pt 2):597–601. doi: 10.1042/bj3220597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chiang N., Kan W. M., Tai H. H. Site-directed mutagenesis of cysteinyl and serine residues of human thromboxane A2 receptor in insect cells. Arch Biochem Biophys. 1996 Oct 1;334(1):9–17. doi: 10.1006/abbi.1996.0423. [DOI] [PubMed] [Google Scholar]
  10. Coleman R. A., Smith W. L., Narumiya S. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev. 1994 Jun;46(2):205–229. [PubMed] [Google Scholar]
  11. Funk C. D., Furci L., Moran N., Fitzgerald G. A. Point mutation in the seventh hydrophobic domain of the human thromboxane A2 receptor allows discrimination between agonist and antagonist binding sites. Mol Pharmacol. 1993 Nov;44(5):934–939. [PubMed] [Google Scholar]
  12. Huang C., Tai H. H. Expression and site-directed mutagenesis of mouse prostaglandin E2 receptor EP3 subtype in insect cells. Biochem J. 1995 Apr 15;307(Pt 2):493–498. doi: 10.1042/bj3070493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang C., Tai H. H. Ser-268 plays an important role in ligand binding of prostaglandin E2 receptor EP3alpha subtype. Arch Biochem Biophys. 1996 Mar 1;327(1):161–166. doi: 10.1006/abbi.1996.0104. [DOI] [PubMed] [Google Scholar]
  14. Kedzie K. M., Donello J. E., Krauss H. A., Regan J. W., Gil D. W. A single amino-acid substitution in the EP2 prostaglandin receptor confers responsiveness to prostacyclin analogs. Mol Pharmacol. 1998 Sep;54(3):584–590. doi: 10.1124/mol.54.3.584. [DOI] [PubMed] [Google Scholar]
  15. Kobayashi T., Kiriyama M., Hirata T., Hirata M., Ushikubi F., Narumiya S. Identification of domains conferring ligand binding specificity to the prostanoid receptor. Studies on chimeric prostacyclin/prostaglandin D receptors. J Biol Chem. 1997 Jun 13;272(24):15154–15160. doi: 10.1074/jbc.272.24.15154. [DOI] [PubMed] [Google Scholar]
  16. Kobayashi T., Ushikubi F., Narumiya S. Amino acid residues conferring ligand binding properties of prostaglandin I and prostaglandin D receptors. Identification by site-directed mutagenesis. J Biol Chem. 2000 Aug 11;275(32):24294–24303. doi: 10.1074/jbc.M002437200. [DOI] [PubMed] [Google Scholar]
  17. Kunapuli P., Lawson J. A., Rokach J., FitzGerald G. A. Functional characterization of the ocular prostaglandin f2alpha (PGF2alpha) receptor. Activation by the isoprostane, 12-iso-PGF2alpha. J Biol Chem. 1997 Oct 24;272(43):27147–27154. doi: 10.1074/jbc.272.43.27147. [DOI] [PubMed] [Google Scholar]
  18. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  19. Narumiya S., Sugimoto Y., Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev. 1999 Oct;79(4):1193–1226. doi: 10.1152/physrev.1999.79.4.1193. [DOI] [PubMed] [Google Scholar]
  20. Pierce K. L., Regan J. W. Prostanoid receptor heterogeneity through alternative mRNA splicing. Life Sci. 1998;62(17-18):1479–1483. doi: 10.1016/s0024-3205(98)00093-9. [DOI] [PubMed] [Google Scholar]
  21. Rehwald M., Neuschäfer-Rube F., de Vries C., Püschel G. P. Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2alpha receptor. FEBS Lett. 1999 Jan 29;443(3):357–362. doi: 10.1016/s0014-5793(99)00007-1. [DOI] [PubMed] [Google Scholar]
  22. Satoh S., Chang C. s., Katoh H., Hasegawa H., Nakamura K., Aoki J., Fujita H., Ichikawa A., Negishi M. The key amino acid residue of prostaglandin EP3 receptor for governing G protein association and activation steps. Biochem Biophys Res Commun. 1999 Feb 5;255(1):164–168. doi: 10.1006/bbrc.1998.0161. [DOI] [PubMed] [Google Scholar]
  23. Schuster V. L., Itoh S., Andrews S. W., Burk R. M., Chen J., Kedzie K. M., Gil D. W., Woodward D. F. Synthetic modification of prostaglandin f(2alpha) indicates different structural determinants for binding to the prostaglandin F receptor versus the prostaglandin transporter. Mol Pharmacol. 2000 Dec;58(6):1511–1516. doi: 10.1124/mol.58.6.1511. [DOI] [PubMed] [Google Scholar]
  24. Sugimoto Y., Namba T., Honda A., Hayashi Y., Negishi M., Ichikawa A., Narumiya S. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. J Biol Chem. 1992 Apr 5;267(10):6463–6466. [PubMed] [Google Scholar]
  25. Zhou H., Yan F., Yamamoto S., Tai H. H. Phenylalanine 138 in the second intracellular loop of human thromboxane receptor is critical for receptor-G-protein coupling. Biochem Biophys Res Commun. 1999 Oct 14;264(1):171–175. doi: 10.1006/bbrc.1999.1508. [DOI] [PubMed] [Google Scholar]

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