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. 1999 May 17;18(10):2756–2763. doi: 10.1093/emboj/18.10.2756

A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus.

N S Olesnicky 1, A J Brown 1, S J Dowell 1, L A Casselton 1
PMCID: PMC1171357  PMID: 10329622

Abstract

In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones. We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype. Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating. A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor. Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function.

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Selected References

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  1. Bourne H. R. How receptors talk to trimeric G proteins. Curr Opin Cell Biol. 1997 Apr;9(2):134–142. doi: 10.1016/s0955-0674(97)80054-3. [DOI] [PubMed] [Google Scholar]
  2. Bölker M., Kahmann R. Sexual pheromones and mating responses in fungi. Plant Cell. 1993 Oct;5(10):1461–1469. doi: 10.1105/tpc.5.10.1461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bölker M., Urban M., Kahmann R. The a mating type locus of U. maydis specifies cell signaling components. Cell. 1992 Feb 7;68(3):441–450. doi: 10.1016/0092-8674(92)90182-c. [DOI] [PubMed] [Google Scholar]
  4. Caldwell G. A., Naider F., Becker J. M. Fungal lipopeptide mating pheromones: a model system for the study of protein prenylation. Microbiol Rev. 1995 Sep;59(3):406–422. doi: 10.1128/mr.59.3.406-422.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Caldwell G. A., Wang S. H., Xue C. B., Jiang Y., Lu H. F., Naider F., Becker J. M. Molecular determinants of bioactivity of the Saccharomyces cerevisiae lipopeptide mating pheromone. J Biol Chem. 1994 Aug 5;269(31):19817–19825. [PubMed] [Google Scholar]
  6. Casselton L. A., Olesnicky N. S. Molecular genetics of mating recognition in basidiomycete fungi. Microbiol Mol Biol Rev. 1998 Mar;62(1):55–70. doi: 10.1128/mmbr.62.1.55-70.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clare J. J., Romanos M. A., Rayment F. B., Rowedder J. E., Smith M. A., Payne M. M., Sreekrishna K., Henwood C. A. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene. 1991 Sep 15;105(2):205–212. doi: 10.1016/0378-1119(91)90152-2. [DOI] [PubMed] [Google Scholar]
  8. Conklin B. R., Bourne H. R. Structural elements of G alpha subunits that interact with G beta gamma, receptors, and effectors. Cell. 1993 May 21;73(4):631–641. doi: 10.1016/0092-8674(93)90245-l. [DOI] [PubMed] [Google Scholar]
  9. Dowell S. J., Bishop A. L., Dyos S. L., Brown A. J., Whiteway M. S. Mapping of a yeast G protein betagamma signaling interaction. Genetics. 1998 Dec;150(4):1407–1417. doi: 10.1093/genetics/150.4.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dube P., Konopka J. B. Identification of a polar region in transmembrane domain 6 that regulates the function of the G protein-coupled alpha-factor receptor. Mol Cell Biol. 1998 Dec;18(12):7205–7215. doi: 10.1128/mcb.18.12.7205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hagen D. C., McCaffrey G., Sprague G. F., Jr Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1418–1422. doi: 10.1073/pnas.83.5.1418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herskowitz I. MAP kinase pathways in yeast: for mating and more. Cell. 1995 Jan 27;80(2):187–197. doi: 10.1016/0092-8674(95)90402-6. [DOI] [PubMed] [Google Scholar]
  13. Jensen R., Sprague G. F., Jr, Herskowitz I. Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus. Proc Natl Acad Sci U S A. 1983 May;80(10):3035–3039. doi: 10.1073/pnas.80.10.3035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kang Y. S., Kane J., Kurjan J., Stadel J. M., Tipper D. J. Effects of expression of mammalian G alpha and hybrid mammalian-yeast G alpha proteins on the yeast pheromone response signal transduction pathway. Mol Cell Biol. 1990 Jun;10(6):2582–2590. doi: 10.1128/mcb.10.6.2582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. King K., Dohlman H. G., Thorner J., Caron M. G., Lefkowitz R. J. Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit. Science. 1990 Oct 5;250(4977):121–123. doi: 10.1126/science.2171146. [DOI] [PubMed] [Google Scholar]
  16. Konopka J. B., Margarit S. M., Dube P. Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6764–6769. doi: 10.1073/pnas.93.13.6764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Krüger J., Loubradou G., Regenfelder E., Hartmann A., Kahmann R. Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis. Mol Gen Genet. 1998 Nov;260(2-3):193–198. doi: 10.1007/s004380050885. [DOI] [PubMed] [Google Scholar]
  18. Kurjan J. The pheromone response pathway in Saccharomyces cerevisiae. Annu Rev Genet. 1993;27:147–179. doi: 10.1146/annurev.ge.27.120193.001051. [DOI] [PubMed] [Google Scholar]
  19. Leberer E., Thomas D. Y., Whiteway M. Pheromone signalling and polarized morphogenesis in yeast. Curr Opin Genet Dev. 1997 Feb;7(1):59–66. doi: 10.1016/s0959-437x(97)80110-4. [DOI] [PubMed] [Google Scholar]
  20. Michaelis S., Herskowitz I. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol Cell Biol. 1988 Mar;8(3):1309–1318. doi: 10.1128/mcb.8.3.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Milano C. A., Allen L. F., Rockman H. A., Dolber P. C., McMinn T. R., Chien K. R., Johnson T. D., Bond R. A., Lefkowitz R. J. Enhanced myocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor. Science. 1994 Apr 22;264(5158):582–586. doi: 10.1126/science.8160017. [DOI] [PubMed] [Google Scholar]
  22. Milligan G., Marshall F., Rees S. G16 as a universal G protein adapter: implications for agonist screening strategies. Trends Pharmacol Sci. 1996 Jul;17(7):235–237. doi: 10.1016/0165-6147(96)10026-2. [DOI] [PubMed] [Google Scholar]
  23. Moore T. D., Edman J. C. The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol. 1993 Mar;13(3):1962–1970. doi: 10.1128/mcb.13.3.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mumberg D., Müller R., Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995 Apr 14;156(1):119–122. doi: 10.1016/0378-1119(95)00037-7. [DOI] [PubMed] [Google Scholar]
  25. Nomoto S., Nakayama N., Arai K., Matsumoto K. Regulation of the yeast pheromone response pathway by G protein subunits. EMBO J. 1990 Mar;9(3):691–696. doi: 10.1002/j.1460-2075.1990.tb08161.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. O'Shea S. F., Chaure P. T., Halsall J. R., Olesnicky N. S., Leibbrandt A., Connerton I. F., Casselton L. A. A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus. Genetics. 1998 Mar;148(3):1081–1090. doi: 10.1093/genetics/148.3.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Offermanns S., Simon M. I. G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J Biol Chem. 1995 Jun 23;270(25):15175–15180. doi: 10.1074/jbc.270.25.15175. [DOI] [PubMed] [Google Scholar]
  28. Price L. A., Kajkowski E. M., Hadcock J. R., Ozenberger B. A., Pausch M. H. Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway. Mol Cell Biol. 1995 Nov;15(11):6188–6195. doi: 10.1128/mcb.15.11.6188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Regenfelder E., Spellig T., Hartmann A., Lauenstein S., Bölker M., Kahmann R. G proteins in Ustilago maydis: transmission of multiple signals? EMBO J. 1997 Apr 15;16(8):1934–1942. doi: 10.1093/emboj/16.8.1934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  31. Scheer A., Cotecchia S. Constitutively active G protein-coupled receptors: potential mechanisms of receptor activation. J Recept Signal Transduct Res. 1997 Jan-May;17(1-3):57–73. doi: 10.3109/10799899709036594. [DOI] [PubMed] [Google Scholar]
  32. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spalding T. A., Burstein E. S., Brauner-Osborne H., Hill-Eubanks D., Brann M. R. Pharmacology of a constitutively active muscarinic receptor generated by random mutagenesis. J Pharmacol Exp Ther. 1995 Dec;275(3):1274–1279. [PubMed] [Google Scholar]
  34. Spalding T. A., Burstein E. S., Wells J. W., Brann M. R. Constitutive activation of the m5 muscarinic receptor by a series of mutations at the extracellular end of transmembrane 6. Biochemistry. 1997 Aug 19;36(33):10109–10116. doi: 10.1021/bi970565g. [DOI] [PubMed] [Google Scholar]
  35. Spellig T., Bölker M., Lottspeich F., Frank R. W., Kahmann R. Pheromones trigger filamentous growth in Ustilago maydis. EMBO J. 1994 Apr 1;13(7):1620–1627. doi: 10.1002/j.1460-2075.1994.tb06425.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tam A., Nouvet F. J., Fujimura-Kamada K., Slunt H., Sisodia S. S., Michaelis S. Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing. J Cell Biol. 1998 Aug 10;142(3):635–649. doi: 10.1083/jcb.142.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tseng C. C., Lin L. A point mutation in the glucose-dependent insulinotropic peptide receptor confers constitutive activity. Biochem Biophys Res Commun. 1997 Mar 6;232(1):96–100. doi: 10.1006/bbrc.1997.6231. [DOI] [PubMed] [Google Scholar]
  38. Vaillancourt L. J., Raper C. A. Pheromones and pheromone receptors as mating-type determinants in basidiomycetes. Genet Eng (N Y) 1996;18:219–247. doi: 10.1007/978-1-4899-1766-9_13. [DOI] [PubMed] [Google Scholar]
  39. Vaillancourt L. J., Raudaskoski M., Specht C. A., Raper C. A. Multiple genes encoding pheromones and a pheromone receptor define the B beta 1 mating-type specificity in Schizophyllum commune. Genetics. 1997 Jun;146(2):541–551. doi: 10.1093/genetics/146.2.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wach A. PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast. 1996 Mar 15;12(3):259–265. doi: 10.1002/(SICI)1097-0061(19960315)12:3%3C259::AID-YEA901%3E3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  41. Wendland J., Vaillancourt L. J., Hegner J., Lengeler K. B., Laddison K. J., Specht C. A., Raper C. A., Kothe E. The mating-type locus B alpha 1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes. EMBO J. 1995 Nov 1;14(21):5271–5278. doi: 10.1002/j.1460-2075.1995.tb00211.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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