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Endocytosis of the seven-transmembrane RGS1 protein activates G-protein-coupled signalling in Arabidopsis

Nature Cell Biology volume 14, pages 1079–1088 (2012)Cite this article

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Abstract

Signal transduction typically begins by ligand-dependent activation of a concomitant partner that is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (regulator of G-protein signalling 1), which is the prototype of a seven-transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase-accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required for both G-protein-mediated sugar signalling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis mechanisms.

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Figure 1: AtRGS1 internalizes in response to sugar.
Figure 2: AGB1 is essential for AtRGS1 internalization.
Figure 3: In vivo and in vitro function of AtWNK8.
Figure 4: Phosphorylation and function of the C terminus of AtRGS1.
Figure 5: AtWNK8 physically interacts with the G-protein βγ subunit.
Figure 6: wnk8 mutant expression and phenotypes.
Figure 7: Model of sustained G-protein activation in Arabidopsis; comparison of activation mechanisms between fast and slow nucleotide exchanging G proteins.

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References

  1. Kohout, T. A. & Lefkowitz, R. J. Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol. Pharmacol. 63, 9–18 (2003).

    Article  CAS  Google Scholar 

  2. Hanyaloglu, A. C. & von Zastrow, M. Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu. Rev. Pharmacol. Toxicol. 48, 537–568 (2008).

    Article  CAS  Google Scholar 

  3. Johnston, C. A. et al. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl Acad. Sci. USA 104, 17317–17322 (2007).

    Article  CAS  Google Scholar 

  4. Urano, D. et al. G protein activation without a GEF in the plant kingdom. PLoS Genet. 8, e1002756 (2012).

    Article  CAS  Google Scholar 

  5. Gookin, T. E., Kim, J. & Assmann, S. M. Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: Computational prediction and in-vivo protein coupling. Genome Biol. 9, R120 (2008).

    Article  Google Scholar 

  6. Moriyama, E. N., Strope, P. K., Opiyo, S. O., Chen, Z. & Jones, A. M. Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors. Genome Biol. 7, R96 (2006).

    Article  Google Scholar 

  7. Jones, J. C. et al. The crystal structure of a self-activating G protein alpha subunit reveals its distinct mechanism of signal initiation. Sci. Signal 4, ra8 (2011).

    Article  Google Scholar 

  8. Chen, J. G. et al. A seven-transmembrane RGS protein that modulates plant cell proliferation. Science 301, 1728–1731 (2003).

    Article  CAS  Google Scholar 

  9. Ullah, H. et al. The β-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. Plant Cell 15, 393–409 (2003).

    Article  CAS  Google Scholar 

  10. Ullah, H. et al. Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science 292, 2066–2069 (2001).

    Article  CAS  Google Scholar 

  11. Trusov, Y. et al. Heterotrimeric G protein gamma subunits provide functional selectivity in Gβγ dimer signaling in Arabidopsis. Plant Cell 19, 1235–1250 (2007).

    Article  CAS  Google Scholar 

  12. Chakravorty, D. et al. An atypical heterotrimeric G protein gamma subunit is involved in guard cell K(+) channel regulation and morphological development in Arabidopsis thaliana. Plant J. 67, 840–851 (2011).

    Article  CAS  Google Scholar 

  13. Chen, J. G., Gao, Y. & Jones, A. M. Differential roles of Arabidopsis heterotrimeric G-protein subunits in modulating cell division in roots. Plant Physiol. 141, 887–897 (2006).

    Article  CAS  Google Scholar 

  14. Chen, J. G. & Jones, A. M. AtRGS1 function in Arabidopsis thaliana. Methods Enzymol. 389, 338–350 (2004).

    Article  CAS  Google Scholar 

  15. Booker, K. S., Schwarz, J., Garrett, M. B. & Jones, A. M. Glucose attenuation of auxin-mediated bimodality in lateral root formation is partly coupled by the heterotrimeric G protein complex. PLoS One 5, e12833 (2010).

    Article  Google Scholar 

  16. Grigston, J. C. et al. d-Glucose sensing by a plasma membrane regulator of G signaling protein, AtRGS1. FEBS Lett. 582, 3577–3584 (2008).

    Article  CAS  Google Scholar 

  17. Pego, J. V. & Smeekens, S. C. Plant fructokinases: A sweet family get-together. Trends Plant Sci. 5, 531–536 (2000).

    Article  CAS  Google Scholar 

  18. Sherson, S. M., Alford, H. L., Forbes, S. M., Wallace, G. & Smith, S. M. Roles of cell-wall invertases and monosaccharide transporters in the growth and development of Arabidopsis. J. Exp. Bot. 54, 525–531 (2003).

    Article  CAS  Google Scholar 

  19. Valpuesta, V. & Botella, M. A. Biosynthesis of l-ascorbic acid in plants: new pathways for an old antioxidant. Trends Plant Sci. 9, 573–577 (2004).

    Article  CAS  Google Scholar 

  20. Chen, Z. et al. Expression analysis of the AtMLO gene family encoding plant-specific seven-transmembrane domain proteins. Plant Mol. Biol. 60, 583–597 (2006).

    Article  CAS  Google Scholar 

  21. Chen, Z. et al. Two seven-transmembrane domain Mildew Resistance Locus O proteins cofunction in Arabidopsis root thigmomorphogenesis. Plant Cell 21, 1972–1991 (2009).

    Article  CAS  Google Scholar 

  22. Hislop, J. N. & von Zastrow, M. Role of ubiquitination in endocytic trafficking of G-protein-coupled receptors. Traffic 12, 137–148 (2011).

    Article  CAS  Google Scholar 

  23. Hislop, J. N. & Zastrow, M. in Regulated Membrane Trafficking and Proteolysis of GPCRs The G Protein-Coupled Receptors Handbook (ed. Devi, L. A.) 95–105 (Humana Press, 2005).

    Book  Google Scholar 

  24. Marchese, A., Paing, M. M., Temple, B. R. & Trejo, J. G protein-coupled receptor sorting to endosomes and lysosomes. Annu. Rev. Pharmacol. Toxicol. 48, 601–629 (2008).

    Article  CAS  Google Scholar 

  25. Klopffleisch, K. et al. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis. Mol. Syst. Biol. 7, 532 (2011).

    Article  Google Scholar 

  26. Nelson, B. K., Cai, X. & Nebenfuhr, A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136 (2007).

    Article  CAS  Google Scholar 

  27. Moore, M. J., Soltis, P. S., Bell, C. D., Burleigh, J. G. & Soltis, D. E. Phylogenetic analysis of 83 plastid genes further resolves the early diversification of eudicots. Proc. Natl Acad. Sci. USA 107, 4623–4628 (2010).

    Article  CAS  Google Scholar 

  28. Stubbs, M. D. et al. Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1). Biochim. Biophys. Acta 1550, 52–63 (2001).

    Article  CAS  Google Scholar 

  29. Seki, M. et al. Functional annotation of a full-length Arabidopsis cDNA collection. Science 296, 141–145 (2002).

    Article  Google Scholar 

  30. Day, P. W., Wedegaertner, P. B. & Benovic, J. L. Analysis of G-protein-coupled receptor kinase RGS homology domains. Methods Enzymol. 390, 295–310 (2004).

    Article  CAS  Google Scholar 

  31. Lalonde, S. et al. A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2. Front Physiol. 1, 24 (2010).

    Article  CAS  Google Scholar 

  32. Huang, C. L., Cha, S. K., Wang, H. R., Xie, J. & Cobb, M. H. WNKs: protein kinases with a unique kinase domain. Exp. Mol. Med. 39, 565–573 (2007).

    Article  CAS  Google Scholar 

  33. Chen, J. G. et al. RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis. J. Exp. Bot. 57, 2697–2708 (2006).

    Article  CAS  Google Scholar 

  34. Deuschle, K. et al. Rapid metabolism of glucose detected with FRET glucose nanosensors in epidermal cells and intact roots of Arabidopsis RNA-silencing mutants. Plant Cell 18, 2314–2325 (2006).

    Article  CAS  Google Scholar 

  35. Lalonde, S., Wipf, D. & Frommer, W. B. Transport mechanisms for organic forms of carbon and nitrogen between source and sink. Annu. Rev. Plant Biol. 55, 341–372 (2004).

    Article  CAS  Google Scholar 

  36. Lemaire, K., Van de Velde, S., Van Dijck, P. & Thevelein, J. M. Glucose and sucrose act as agonist and mannose as antagonist ligands of the G protein-coupledreceptor Gpr1 in the yeast Saccharomyces cerevisiae. Mol. Cell 16, 293–299 (2004).

    Article  CAS  Google Scholar 

  37. Isshiki, T., Mochizuki, N., Maeda, T. & Yamamoto, M. Characterization of a fission yeast gene, gpa2, that encodes a G alpha subunit involved in the monitoring of nutrition. Genes Dev. 6, 2455–2462 (1992).

    Article  CAS  Google Scholar 

  38. Versele, M., de Winde, J. H. & Thevelein, J. M. A novel regulator of G protein signalling in yeast, Rgs2, downregulates glucose-activation of the cAMP pathway through direct inhibition of Gpa2. Embo J. 18, 5577–5591 (1999).

    Article  CAS  Google Scholar 

  39. Jones, J. C., Temple, B. R., Jones, A. M. & Dohlman, H. G. Functional reconstitution of an atypical G protein heterotrimer and regulator of G protein signaling protein (RGS1) from Arabidopsis thaliana. J. Biol. Chem. 286, 13143–13150 (2011).

    Article  CAS  Google Scholar 

  40. Murakami-Kojima, M., Nakamichi, N., Yamashino, T. & Mizuno, T. The APRR3 component of the clock-associated APRR1/TOC1 quintet is phosphorylated by a novel protein kinase belonging to the WNK family, the gene for which is also transcribed rhythmically in Arabidopsis thaliana. Plant Cell Physiol. 43, 675–683 (2002).

    Article  CAS  Google Scholar 

  41. Park, H. Y. et al. EMF1 interacts with EIP1, EIP6 or EIP9 involved in the regulation of flowering time in Arabidopsis. Plant Cell Physiol. 52, 1376–1388 (2011).

    Article  CAS  Google Scholar 

  42. Wang, Y. et al. The plant WNK gene family and regulation of flowering time in Arabidopsis. Plant Biol (Stuttg) 10, 548–562 (2008).

    Article  CAS  Google Scholar 

  43. Wang, Y., Suo, H., Zhuang, C., Ma, H. & Yan, X. Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis. J. Plant Physiol. (2011).

  44. Kumar, K., Rao, K. P., Biswas, D. K. & Sinha, A. K. Rice WNK1 is regulated by abiotic stress and involved in internal circadian rhythm. Plant Signal Behav. 6, 316–320 (2011).

    Article  CAS  Google Scholar 

  45. An, S. W. et al. WNK1 Promotes PIP2 synthesis to coordinate growth factor and GPCR-G(q) signaling. Curr. Biol. 21, 1979–1987 (2011).

    Article  CAS  Google Scholar 

  46. Huang, C. L., Yang, S. S. & Lin, S. H. Mechanism of regulation of renal ion transport by WNK kinases. Curr. Opin. Nephrol. Hypertens. 17, 519–525 (2008).

    Article  Google Scholar 

  47. Reiter, E., Ahn, S., Shukla, A. K. & Lefkowitz, R. J. Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors. Annu. Rev. Pharmacol. Toxicol. 52, 179–197 (2011).

    Article  Google Scholar 

  48. Tesmer, J. J., Berman, D. M., Gilman, A. G. & Sprang, S. R. Structure of RGS4 bound to AlF4–activated G(i α1): stabilization of the transition state for GTP hydrolysis. Cell 89, 251–261 (1997).

    Article  CAS  Google Scholar 

  49. Lambright, D. G. et al. The 2.0 A crystal structure of a heterotrimeric G protein. Nature 379, 311–319 (1996).

    Article  CAS  Google Scholar 

  50. Sparkes, I. A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1, 2019–2025 (2006).

    Article  CAS  Google Scholar 

  51. Grefen, C. et al. A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J. 64, 355–365 (2010).

    Article  CAS  Google Scholar 

  52. Friedman, E. J. et al. Acireductone dioxygenase 1 (ARD1) is an effector of the heterotrimeric G Protein {β} subunit in Arabidopsis. J. Biol. Chem. 286, 30107–30118 (2011).

    Article  CAS  Google Scholar 

  53. Abramoff, M. D., Magalhaes, P. J. & Ram, S. J. Image processing with ImageJ. Biophoton. Internat. 11, 36–42 (2004).

    Google Scholar 

  54. Anderson, C. W., Baum, P. R. & Gesteland, R. F. Processing of adenovirus 2-induced proteins. J. Virol. 12, 241–252 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Hanna, S. L., Sherman, N. E., Kinter, M. T. & Goldberg, J. B. Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry. Microbiology 146 (Pt 10), 2495–2508 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Smalley for determining the phosphorylation sites of AtRGS1, N. Nakamichi, T. Eulgem and H. Ma (Pennsylvania State University, USA) for supplying experimental materials, K. Williamson and M. Mathew for assistance in protein purification and gene cloning, A. Urano for assisting in experiments and H. Dohlman for helpful discussions. This work was supported by grants from the NIGMS (R01GM065989) and NSF (MCB-0723515 and MCB-0718202) to A.M.J. The Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy through grant DE-FG02-05er15671 to A.M.J. financially supported the genotyping of the materials in this study.

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  1. Daisuke Urano and Nguyen Phan: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

    Daisuke Urano, Nguyen Phan, Jing Yang, Jirong Huang, Jeffrey Grigston, J. Philip Taylor & Alan M. Jones

  2. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, North Carolina 27599, Chapel Hill, USA

    Janice C. Jones

  3. Department of Pharmacology, University of North Carolina at Chapel Hill, North Carolina 27599, Chapel Hill, USA

    Alan M. Jones

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Contributions

A.M.J. and J.H. made the initial observation of glucose-dependent AtRGS1 internalization. J.H., J.G. and J.P.T. characterized the sugar dependence of endocytosis of wild-type and mutant AtRGS1 (Fig. 1). N.P. quantified endocytosis of AtRGS1 in mutant genotypes and performed FRET analyses and co-localization of AtRGS1 with compartment markers. J.C.J. assisted in the initial intrinsic tryptophan fluorescence measurement. J.Y. assisted in the real-time PCR experiments. D.U. designed most of the experiments, performed all of the biochemical and physiological analyses, performed some of the BiFC experiments and analyses, performed all the gene expression profiling and constructed all the expression vectors. A.M.J., D.U. and N.P. wrote the manuscript. All authors edited the manuscript.

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Correspondence to Alan M. Jones.

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Urano, D., Phan, N., Jones, J. et al. Endocytosis of the seven-transmembrane RGS1 protein activates G-protein-coupled signalling in Arabidopsis. Nat Cell Biol 14, 1079–1088 (2012). https://doi.org/10.1038/ncb2568

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