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Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast

Nature Biotechnology volume 16, pages 1334–1337 (1998)Cite this article

Abstract

We describe a procedure for isolating agonists for mammalian G protein–coupled receptors of unknown function. Human formyl peptide receptor like-1 (FPRL-1) receptor, originally identified as an orphan G protein–coupled receptor related to the formyl peptide receptor (FPR1), was expressed in Saccharomyces cells designed to couple receptor activation to histidine prototrophy. Selection for histidine prototrophs among transformants obtained with a plasmid-based library encoding random peptides identified six different agonists, each of whose production yielded autocrine stimulation of the receptor expressed in yeast. A synthetic version of each peptide promoted activation of FPRL-1 expressed in human embryonic kidney (HEK293) cells, and five of the peptides exhibited significant selectivity for activation of FPRL-1 relative to FPR1. One selective peptide was tested and found to mobilize calcium in isolated human neutrophils. This demonstrates that stimulation of FPRL-1 results in neutrophil activation and suggests that the receptor functions as a component of the inflammatory response. This autocrine selection protocol may be a generally applicable method for providing pharmacological tools to evaluate the physiological roles of the growing number of mammalian orphan G protein–coupled receptors.

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Figure 1: (A) Strain CY1141 expressing both FPRL-1 and the A5 peptide, the A5 peptide alone or the receptor alone were streaked on LUH-AT plates.
Figure 2: Measurement of changes in intracellular Ca2+ in HEK293/Gα16 cells stably expressing (A) FPRL-1 or (B) FPR1 upon exposure to increasing concentrations of peptides A5 and formylated A5 (f-A5).
Figure 3: Intracellular calcium concentration in human neutrophils exposed to synthetic peptide A5 and formylated A5 (fA5).

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References

  1. Devane, W.A., Hanuss, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G. et al. 1992. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258: 1946–1949.

    Article  Google Scholar 

  2. Meunier, J.-C., Mollereau C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P. et al. 1995. Isolation and structure of the endogenous agonist of opioid receptor-like ORL-1 receptor. Nature 377: 532–535.

    Article  Google Scholar 

  3. Murphy, P., Özçelik T., Kenney, R.T., Tiffany H.L., McDermott D., and Francke U. 1992. A structural homologue of the N-formyl peptide receptor. J. Biol. Chem. 267: 7637–7643.

    Google Scholar 

  4. Ye, R.D., Cavanagh, S.L., Quehenberger, O., Prossnitz, E.R., and Cochrane, C.G. 1992. Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor. Biochem. Biophys. Res. Commun. 184: 582– 589.

    Article  Google Scholar 

  5. Durstin, M., Gao, J.-L., Tiffany, H.L., McDermott, D., and Murphy, P.M. 1994. Differential expression of members of the N-formylpeptide receptor gene cluster in human phagocytes. Biochem. Biophys. Res. Commun. 201: 174–179.

    Article  Google Scholar 

  6. Bao, L., Gerard, N.P., Eddy R. Jr., Shows, T.B., and Gerard, C. 1992. Mapping of genes for the human C5a receptor (C5aR), human fMLP receptor (FPR), and two fMLP receptor homologue orphan receptors (FPRH1, FPRH2) to chromosome 19. Genomics 13: 437–440 .

    Article  Google Scholar 

  7. Showell, H.J., Freer, R.J., Zigmond, S.H., Schiffman, E., Aswanikumar, S., Corcoran, B. et al. 1976. The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils. J. Exp. Med. 143: 1154–1169.

    Article  Google Scholar 

  8. Fiore, S., Maddox, J.F., Perez, H.D., and Serhan, C.N. 1994. Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor. J. Exp. Med. 180: 253–260.

    Article  Google Scholar 

  9. Manfredi, J.P., Klein, C., Herrero, J.J., Byrd, D.R., Trueheart, J., Wiesler, W.T. et al. 1996. Yeast a mating factor structure-activity relationship derived from genetically selected peptide agonists and antagonists of Ste2p. Mol. Cell. Biol. 16: 4700– 4709.

    Article  Google Scholar 

  10. Stevenson, B.J., Rhodes, N., Errede, B., and Sprague, G.F. Jr. 1992. Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev. 6:1293–1304 .

    Article  CAS  Google Scholar 

  11. Konteatis, Z.D., Siciliano, S.J., Van Riper, G., Molineaux, C.J., Pandya, S., Fischer, P. et al. 1994. Development of C5a receptor antagonists: differential loss of functional responses. J. Immunol. 153: 4200–4205.

    Google Scholar 

  12. Chen, J., Bernstein, H.S., Chen, M., Wang, L., Ishii, M., Turck, C.W. et al. 1995. Tethered ligand library for discovery of peptide agonists. J. Biol. Chem. 270: 23398–23401.

    Article  Google Scholar 

  13. Romano, M., Maddox, J.F., and Serhan, C.N. 1996. Activation of human monocytes and the acute monocytic leukemia cell line (THP-1) by lipoxins involves unique signaling pathways for lipoxin A4 versus lipoxin B4. Evidence for differential Ca2+ mobilization. J. Immunol. 157: 2149–2154.

    Google Scholar 

  14. Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Maenhaut, C. et al. 1989. Selective amplification and cloning of four new members of the G protein-coupled receptor family. Science 244: 569–572.

    Article  Google Scholar 

  15. Feng, Y., Broder, C.C., Kennedy, P.E., and Berger, E.A. 1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272: 872–877.

    Article  Google Scholar 

  16. Crass, T., Raffetseder, U., Martin, U., Grove, M., Klos, A., Köhl, J. et al. 1996. Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells. Eur. J. Immunol. 26: 1944–1950.

    Article  Google Scholar 

  17. Rose, A.B. and Broach, J.R. 1990. Propagation and expression of cloned genes in yeast: 2-micron circle-based vectors. Methods Enzymol. 185: 234–279.

    Article  Google Scholar 

  18. Offermanns, S. and Simon, M.I. 1995 . G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. 270: 15175 –15180.

    Article  Google Scholar 

  19. Kaiser, C., Michaelis, S., and Mitchell, A. 1994. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  20. Kloptowski, T. and Wiater, A. 1965. Synergism of aminotriazole and phosphate on the inhibition of yeast imidazoleglycerolphosphate dehydratase. Arch. Biochem. Biophys. 112: 562–566.

    Article  Google Scholar 

  21. Struhl, K. and Davis, R.W. 1977. Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazoleglycerolphosphate dehydratase (his3). Proc. Natl. Acad. Sci. USA 74: 5255 –5259.

    Article  Google Scholar 

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Acknowledgements

We thank Philip Murphy for providing plasmids encoding FPR and FPRL-1; William T. Wiesler for Cp1584 and β-galactosidase assays; Anna Farescal and Dwight Morrow for conducting peptide searches; Martin McKinney and Lorna Nuñez for technical assistance; and Nan-Xin Qian for providing the HEK293 cell line expressing Gα16. We also thank David R. Webb for a critical reading of the manuscript.

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Authors and Affiliations

  1. Cadus Pharmaceutical Corporation, Tarrytown , 10591-6705, NY

    Christine Klein, Jeremy I. Paul, Karen Sauvé, Mary M. Schmidt, Loretta Arcangeli, John Ransom, Joshua Trueheart, John P. Manfredi & Andrew J. Murphy

  2. Department of Molecular Biology, Princeton University, Princeton, 08544, NJ

    James R. Broach

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  1. Christine Klein
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Correspondence to Christine Klein.

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Klein, C., Paul, J., Sauvé, K. et al. Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast. Nat Biotechnol 16, 1334–1337 (1998). https://doi.org/10.1038/4310

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