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GPCRs regulate the assembly of a multienzyme complex for purine biosynthesis

Nature Chemical Biology volume 7, pages 909–915 (2011)Cite this article

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Abstract

G protein–coupled receptors (GPCRs) transmit exogenous signals to the nucleus, promoting a myriad of biological responses via multiple signaling pathways in both healthy and cancerous cells. However, little is known about the response of cytosolic metabolic pathways to GPCR-mediated signaling. Here we applied fluorescent live-cell imaging and label-free dynamic mass redistribution assays to study whether purine metabolism is associated with GPCR signaling. Through a library screen of GPCR ligands in conjunction with live-cell imaging of a metabolic multienzyme complex for de novo purine biosynthesis, the purinosome, we demonstrated that the activation of endogenous Gαi-coupled receptors correlates with purinosome assembly and disassembly in native HeLa cells. Given the implications of GPCRs in mitogenic signaling and of the purinosome in controlling metabolic flux via de novo purine biosynthesis, we hypothesize that regulation of purinosome assembly and disassembly may be one of the downstream events of mitogenic GPCR signaling in human cancer cells.

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Figure 1: Characteristic signatures of purinosome assembly and disassembly observed by fluorescent live-cell imaging and DMR assays.
Figure 2: DMAT and TBB responses after pretreatment of HeLa cells with a GPCR agonist library.
Figure 3: DMR assays characterizing endogenous adrenergic receptors manipulated by corresponding agonists, antagonists and toxins.
Figure 4: OXY, but not SAL, promotes purinosome formation in live HeLa cells.
Figure 5: Activation of α2A-AR, but not β2-AR, influences purinosome assembly.
Figure 6: Gαi signaling for purinosome regulation by α2A-AR activation.

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References

  1. Neves, S.R., Ram, P.T. & Iyengar, R. G protein pathways. Science 296, 1636–1639 (2002).

    Article  CAS  Google Scholar 

  2. Pierce, K.L., Premont, R.T. & Lefkowitz, R.J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002).

    Article  CAS  Google Scholar 

  3. Dorsam, R.T. & Gutkind, J.S. G-protein-coupled receptors and cancer. Nat. Rev. Cancer 7, 79–94 (2007).

    Article  CAS  Google Scholar 

  4. Rozengurt, E. Mitogenic signaling pathways induced by G protein-coupled receptors. J. Cell. Physiol. 213, 589–602 (2007).

    Article  CAS  Google Scholar 

  5. Rajagopal, S., Rajagopal, K. & Lefkowitz, R.J. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat. Rev. Drug Discov. 9, 373–386 (2010).

    Article  CAS  Google Scholar 

  6. Kroemer, G. & Pouyssegur, J. Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell 13, 472–482 (2008).

    Article  CAS  Google Scholar 

  7. Kaelin, W.G. Jr. & Thompson, C.B. Q&A: Cancer: clues from cell metabolism. Nature 465, 562–564 (2010).

    Article  CAS  Google Scholar 

  8. Denkert, C. et al. Metabolite profiling of human colon carcinoma—deregulation of TCA cycle and amino acid turnover. Mol. Cancer 7, 72 (2008).

    Article  Google Scholar 

  9. Christopherson, R.I., Lyons, S.D. & Wilson, P.K. Inhibitors of de novo nucleotide biosynthesis as drugs. Acc. Chem. Res. 35, 961–971 (2002).

    Article  CAS  Google Scholar 

  10. An, S., Kumar, R., Sheets, E.D. & Benkovic, S.J. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science 320, 103–106 (2008).

    Article  CAS  Google Scholar 

  11. An, S., Kyoung, M., Allen, J.J., Shokat, K.M. & Benkovic, S.J. Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2. J. Biol. Chem. 285, 11093–11099 (2010).

    Article  CAS  Google Scholar 

  12. An, S., Deng, Y., Tomsho, J.W., Kyoung, M. & Benkovic, S.J. Microtubule-assisted mechanism for functional metabolic macromolecular complex formation. Proc. Natl. Acad. Sci. USA 107, 12872–12876 (2010).

    Article  CAS  Google Scholar 

  13. Fang, Y., Ferrie, A.M., Fontaine, N.H. & Yuen, P.K. Characteristics of dynamic mass redistribution of epidermal growth factor receptor signaling in living cells measured with label-free optical biosensors. Anal. Chem. 77, 5720–5725 (2005).

    Article  CAS  Google Scholar 

  14. Fang, Y., Li, G. & Ferrie, A.M. Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells. J. Pharmacol. Toxicol. Methods 55, 314–322 (2007).

    Article  CAS  Google Scholar 

  15. Schröder, R. et al. Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat. Biotechnol. 28, 943–949 (2010).

    Article  Google Scholar 

  16. Fang, Y. Label-free receptor assays. Drug Discov. Today Technol. 7, e5–e11 (2011).

    Article  Google Scholar 

  17. Duncan, J.S. et al. An unbiased evaluation of CK2 inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets. Mol. Cell. Proteomics 7, 1077–1088 (2008).

    Article  CAS  Google Scholar 

  18. Pagano, M.A. et al. The selectivity of inhibitors of protein kinase CK2: an update. Biochem. J. 415, 353–365 (2008).

    Article  CAS  Google Scholar 

  19. Bylund, D.B. et al. International Union of Pharmacology nomenclature of adrenoceptors. Pharmacol. Rev. 46, 121–136 (1994).

    CAS  PubMed  Google Scholar 

  20. Morris, A.J. & Malbon, C.C. Physiological regulation of G protein-linked signaling. Physiol. Rev. 79, 1373–1430 (1999).

    Article  CAS  Google Scholar 

  21. Baker, J.G. The selectivity of beta-adrenoceptor antagonists at the human β1, β2 and β3 adrenoceptors. Br. J. Pharmacol. 144, 317–322 (2005).

    Article  CAS  Google Scholar 

  22. Barbieri, J.T. & Cortina, G. ADP-ribosyltransferase mutations in the catalytic S-1 subunit of pertussis toxin. Infect. Immun. 56, 1934–1941 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Gill, D.M. & Meren, R. ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. Proc. Natl. Acad. Sci. USA 75, 3050–3054 (1978).

    Article  CAS  Google Scholar 

  24. Hanyaloglu, A.C. et al. Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to beta-arrestin-dependent internalization. J. Biol. Chem. 276, 18066–18074 (2001).

    Article  CAS  Google Scholar 

  25. Musnier, A., Blanchot, B., Reiter, E. & Crepieux, P. GPCR signalling to the translation machinery. Cell. Signal. 22, 707–716 (2010).

    Article  CAS  Google Scholar 

  26. Rebholz, H. et al. CK2 negatively regulates Gαs signaling. Proc. Natl. Acad. Sci. USA 106, 14096–14101 (2009).

    Article  CAS  Google Scholar 

  27. Torrecilla, I. et al. Phosphorylation and regulation of a G protein-coupled receptor by protein kinase CK2. J. Cell Biol. 177, 127–137 (2007).

    Article  CAS  Google Scholar 

  28. Gao, Y. & Wang, H.Y. Casein kinase 2 is activated and essential for Wnt/β-catenin signaling. J. Biol. Chem. 281, 18394–18400 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was funded by US National Institutes of Health grant GM24129 (S.J.B.).

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Author notes

  1. Songon An

    Present address: Current address: Department of Chemistry and Biochemistry, University of Maryland, Baltimore, Maryland, USA.,

  2. Florence Verrier and Songon An: These authors contributed equally to the study.

Authors and Affiliations

  1. Science and Technology Division, Biochemical Technologies, Corning Inc., Corning, New York, USA

    Florence Verrier, Ann M Ferrie, Haiyan Sun, Huayun Deng & Ye Fang

  2. Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA

    Songon An & Stephen J Benkovic

  3. Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA

    Minjoung Kyoung

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  1. Florence Verrier
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Contributions

F.V. and S.A. contributed equally to the study. F.V. performed DMR assays and analyzed DMR data. S.A. performed fluorescent live-cell imaging and initial DMR assays, and analyzed data. A.M.F. prepared the compound library and performed initial DMR assays. H.S. performed quantitative real-time PCR. M.K. designed and modified a fluorescent microscope for image collection. H.D. carried out DMR assays related to part of Figure 3b, part of supplementary DMR assays. Y.F. and S.J.B. conceived and designed the study and analyzed data. S.A., Y.F. and S.J.B. wrote the manuscript.

Corresponding authors

Correspondence to Ye Fang or Stephen J Benkovic.

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Competing interests

F. Verrier, A.M. Ferrie, H. Sun, H. Deng and Y. Fang are employees and shareholders of Corning, Inc.

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Verrier, F., An, S., Ferrie, A. et al. GPCRs regulate the assembly of a multienzyme complex for purine biosynthesis. Nat Chem Biol 7, 909–915 (2011). https://doi.org/10.1038/nchembio.690

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