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. 1994 May;14(5):3339–3349. doi: 10.1128/mcb.14.5.3339

The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization.

C J Stefan 1, K J Blumer 1
PMCID: PMC358700  PMID: 8164685

Abstract

To identify functional domains of G-protein-coupled receptors that control pathway activation, ligand discrimination, and receptor regulation, we have used as a model the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. From a collection of random mutations introduced in the region coding for the third cytoplasmic loop of Ste2p, six ste2sst alleles were identified by genetic screening methods that increased alpha-factor sensitivity 2.5- to 15-fold. The phenotypic effects of ste2sst and sst2 mutations were not additive, consistent with models in which the third cytoplasmic loop of the alpha-factor receptor and the regulatory protein Sst2p control related aspects of pheromone response and/or desensitization. Four ste2sst mutations did not dramatically alter cell surface expression or agonist binding affinity of the receptor; however, they did permit detectable responses to an alpha-factor antagonist. One ste2sst allele increased receptor binding affinity for alpha-factor and elicited stronger responses to antagonist. Results of competition binding experiments indicated that wild-type and representative mutant receptors bound antagonist with similar affinities. The antagonist-responsive phenotypes caused by ste2sst alleles were therefore due to defects in the ability of receptors to discriminate between agonist and antagonist peptides. One ste2sst mutation caused rapid, ligand-independent internalization of the receptor. These results demonstrate that the third cytoplasmic loop of the alpha-factor receptor is a multifunctional regulatory domain that controls pathway activation and/or desensitization and influences the processes of receptor activation, ligand discrimination, and internalization.

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Selected References

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  1. Blinder D., Jenness D. D. Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae. Mol Cell Biol. 1989 Sep;9(9):3720–3726. doi: 10.1128/mcb.9.9.3720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blumer K. J., Reneke J. E., Thorner J. The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae. J Biol Chem. 1988 Aug 5;263(22):10836–10842. [PubMed] [Google Scholar]
  3. Blumer K. J., Thorner J. Beta and gamma subunits of a yeast guanine nucleotide-binding protein are not essential for membrane association of the alpha subunit but are required for receptor coupling. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4363–4367. doi: 10.1073/pnas.87.11.4363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blumer K. J., Thorner J. Receptor-G protein signaling in yeast. Annu Rev Physiol. 1991;53:37–57. doi: 10.1146/annurev.ph.53.030191.000345. [DOI] [PubMed] [Google Scholar]
  5. Burkholder A. C., Hartwell L. H. The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res. 1985 Dec 9;13(23):8463–8475. doi: 10.1093/nar/13.23.8463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Campbell P. T., Hnatowich M., O'Dowd B. F., Caron M. G., Lefkowitz R. J., Hausdorff W. P. Mutations of the human beta 2-adrenergic receptor that impair coupling to Gs interfere with receptor down-regulation but not sequestration. Mol Pharmacol. 1991 Feb;39(2):192–198. [PubMed] [Google Scholar]
  7. Cartwright C. P., Tipper D. J. In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions. Mol Cell Biol. 1991 May;11(5):2620–2628. doi: 10.1128/mcb.11.5.2620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan R. K., Otte C. A. Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones. Mol Cell Biol. 1982 Jan;2(1):11–20. doi: 10.1128/mcb.2.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chan R. K., Otte C. A. Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones. Mol Cell Biol. 1982 Jan;2(1):21–29. doi: 10.1128/mcb.2.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cheung A. H., Dixon R. A., Hill W. S., Sigal I. S., Strader C. D. Separation of the structural requirements for agonist-promoted activation and sequestration of the beta-adrenergic receptor. Mol Pharmacol. 1990 Jun;37(6):775–779. [PubMed] [Google Scholar]
  11. Cheung A. H., Huang R. R., Strader C. D. Involvement of specific hydrophobic, but not hydrophilic, amino acids in the third intracellular loop of the beta-adrenergic receptor in the activation of Gs. Mol Pharmacol. 1992 Jun;41(6):1061–1065. [PubMed] [Google Scholar]
  12. Cohen G. B., Oprian D. D., Robinson P. R. Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296. Biochemistry. 1992 Dec 22;31(50):12592–12601. doi: 10.1021/bi00165a008. [DOI] [PubMed] [Google Scholar]
  13. Cotecchia S., Exum S., Caron M. G., Lefkowitz R. J. Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2896–2900. doi: 10.1073/pnas.87.8.2896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dietzel C., Kurjan J. Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone. Mol Cell Biol. 1987 Dec;7(12):4169–4177. doi: 10.1128/mcb.7.12.4169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dohlman H. G., Thorner J., Caron M. G., Lefkowitz R. J. Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem. 1991;60:653–688. doi: 10.1146/annurev.bi.60.070191.003253. [DOI] [PubMed] [Google Scholar]
  16. Eriotou-Bargiota E., Xue C. B., Naider F., Becker J. M. Antagonistic and synergistic peptide analogues of the tridecapeptide mating pheromone of Saccharomyces cerevisiae. Biochemistry. 1992 Jan 21;31(2):551–557. doi: 10.1021/bi00117a036. [DOI] [PubMed] [Google Scholar]
  17. Franke R. R., Sakmar T. P., Graham R. M., Khorana H. G. Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin. J Biol Chem. 1992 Jul 25;267(21):14767–14774. [PubMed] [Google Scholar]
  18. Hausdorff W. P., Campbell P. T., Ostrowski J., Yu S. S., Caron M. G., Lefkowitz R. J. A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):2979–2983. doi: 10.1073/pnas.88.8.2979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hausdorff W. P., Hnatowich M., O'Dowd B. F., Caron M. G., Lefkowitz R. J. A mutation of the beta 2-adrenergic receptor impairs agonist activation of adenylyl cyclase without affecting high affinity agonist binding. Distinct molecular determinants of the receptor are involved in physical coupling to and functional activation of Gs. J Biol Chem. 1990 Jan 25;265(3):1388–1393. [PubMed] [Google Scholar]
  20. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jenness D. D., Spatrick P. Down regulation of the alpha-factor pheromone receptor in S. cerevisiae. Cell. 1986 Aug 1;46(3):345–353. doi: 10.1016/0092-8674(86)90655-0. [DOI] [PubMed] [Google Scholar]
  22. Khorana H. G. Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J Biol Chem. 1992 Jan 5;267(1):1–4. [PubMed] [Google Scholar]
  23. Kitamura K., Shimoda C. The Schizosaccharomyces pombe mam2 gene encodes a putative pheromone receptor which has a significant homology with the Saccharomyces cerevisiae Ste2 protein. EMBO J. 1991 Dec;10(12):3743–3751. doi: 10.1002/j.1460-2075.1991.tb04943.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kjelsberg M. A., Cotecchia S., Ostrowski J., Caron M. G., Lefkowitz R. J. Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J Biol Chem. 1992 Jan 25;267(3):1430–1433. [PubMed] [Google Scholar]
  25. Konopka J. B., Jenness D. D., Hartwell L. H. The C-terminus of the S. cerevisiae alpha-pheromone receptor mediates an adaptive response to pheromone. Cell. 1988 Aug 26;54(5):609–620. doi: 10.1016/s0092-8674(88)80005-9. [DOI] [PubMed] [Google Scholar]
  26. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  27. Kurjan J. Pheromone response in yeast. Annu Rev Biochem. 1992;61:1097–1129. doi: 10.1146/annurev.bi.61.070192.005313. [DOI] [PubMed] [Google Scholar]
  28. Lameh J., Philip M., Sharma Y. K., Moro O., Ramachandran J., Sadée W. Hm1 muscarinic cholinergic receptor internalization requires a domain in the third cytoplasmic loop. J Biol Chem. 1992 Jul 5;267(19):13406–13412. [PubMed] [Google Scholar]
  29. Lechleiter J., Hellmiss R., Duerson K., Ennulat D., David N., Clapham D., Peralta E. Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes. EMBO J. 1990 Dec;9(13):4381–4390. doi: 10.1002/j.1460-2075.1990.tb07888.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Maeda S., Lameh J., Mallet W. G., Philip M., Ramachandran J., Sadée W. Internalization of the Hm1 muscarinic cholinergic receptor involves the third cytoplasmic loop. FEBS Lett. 1990 Sep 3;269(2):386–388. doi: 10.1016/0014-5793(90)81199-x. [DOI] [PubMed] [Google Scholar]
  31. Marsh L., Herskowitz I. STE2 protein of Saccharomyces kluyveri is a member of the rhodopsin/beta-adrenergic receptor family and is responsible for recognition of the peptide ligand alpha factor. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3855–3859. doi: 10.1073/pnas.85.11.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marsh L. Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist. Mol Cell Biol. 1992 Sep;12(9):3959–3966. doi: 10.1128/mcb.12.9.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McCaffrey G., Clay F. J., Kelsay K., Sprague G. F., Jr Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1987 Aug;7(8):2680–2690. doi: 10.1128/mcb.7.8.2680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nakayama N., Miyajima A., Arai K. Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saccharomyces cerevisiae. EMBO J. 1985 Oct;4(10):2643–2648. doi: 10.1002/j.1460-2075.1985.tb03982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Okamoto T., Murayama Y., Hayashi Y., Inagaki M., Ogata E., Nishimoto I. Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell. 1991 Nov 15;67(4):723–730. doi: 10.1016/0092-8674(91)90067-9. [DOI] [PubMed] [Google Scholar]
  36. Ostrowski J., Kjelsberg M. A., Caron M. G., Lefkowitz R. J. Mutagenesis of the beta 2-adrenergic receptor: how structure elucidates function. Annu Rev Pharmacol Toxicol. 1992;32:167–183. doi: 10.1146/annurev.pa.32.040192.001123. [DOI] [PubMed] [Google Scholar]
  37. Raths S. K., Naider F., Becker J. M. Peptide analogues compete with the binding of alpha-factor to its receptor in Saccharomyces cerevisiae. J Biol Chem. 1988 Nov 25;263(33):17333–17341. [PubMed] [Google Scholar]
  38. Reneke J. E., Blumer K. J., Courchesne W. E., Thorner J. The carboxy-terminal segment of the yeast alpha-factor receptor is a regulatory domain. Cell. 1988 Oct 21;55(2):221–234. doi: 10.1016/0092-8674(88)90045-1. [DOI] [PubMed] [Google Scholar]
  39. Robbins L. S., Nadeau J. H., Johnson K. R., Kelly M. A., Roselli-Rehfuss L., Baack E., Mountjoy K. G., Cone R. D. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell. 1993 Mar 26;72(6):827–834. doi: 10.1016/0092-8674(93)90572-8. [DOI] [PubMed] [Google Scholar]
  40. Robinson P. R., Cohen G. B., Zhukovsky E. A., Oprian D. D. Constitutively active mutants of rhodopsin. Neuron. 1992 Oct;9(4):719–725. doi: 10.1016/0896-6273(92)90034-b. [DOI] [PubMed] [Google Scholar]
  41. Rohrer J., Bénédetti H., Zanolari B., Riezman H. Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast. Mol Biol Cell. 1993 May;4(5):511–521. doi: 10.1091/mbc.4.5.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Samama P., Cotecchia S., Costa T., Lefkowitz R. J. A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem. 1993 Mar 5;268(7):4625–4636. [PubMed] [Google Scholar]
  43. Shapiro R. A., Nathanson N. M. Deletion analysis of the mouse m1 muscarinic acetylcholine receptor: effects on phosphoinositide metabolism and down-regulation. Biochemistry. 1989 Oct 31;28(22):8946–8950. doi: 10.1021/bi00448a039. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Sprague G. F., Jr Assay of yeast mating reaction. Methods Enzymol. 1991;194:77–93. doi: 10.1016/0076-6879(91)94008-z. [DOI] [PubMed] [Google Scholar]
  46. Strathern J. N., Higgins D. R. Recovery of plasmids from yeast into Escherichia coli: shuttle vectors. Methods Enzymol. 1991;194:319–329. doi: 10.1016/0076-6879(91)94024-7. [DOI] [PubMed] [Google Scholar]
  47. Valiquette M., Bonin H., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5089–5093. doi: 10.1073/pnas.87.13.5089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weiner J. L., Guttierez-Steil C., Blumer K. J. Disruption of receptor-G protein coupling in yeast promotes the function of an SST2-dependent adaptation pathway. J Biol Chem. 1993 Apr 15;268(11):8070–8077. [PubMed] [Google Scholar]
  49. Zanolari B., Raths S., Singer-Krüger B., Riezman H. Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction. Cell. 1992 Nov 27;71(5):755–763. doi: 10.1016/0092-8674(92)90552-n. [DOI] [PubMed] [Google Scholar]

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