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Sustained cyclic AMP production by parathyroid hormone receptor endocytosis

Abstract

Cell signaling mediated by the G protein–coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems—PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine—can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP1−36 action was restricted to the cell surface, whereas PTH1−34 had moved to internalized compartments where it remained associated with the PTHR and Gαs, potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised.

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Figure 1: Time courses of early reactions in the signaling cascade of PTHR.
Figure 2: Coupling, activation and trafficking of Gs proteins.
Figure 3: PTHR endocytosis and cAMP responses mediated by PTH1−34 and PTHrP1−36.
Figure 4: PTHR endocytosis and prolonged cAMP responses.
Figure 5: Mode of activation of the PTHR by long-acting and short-acting signaling ligands.

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References

  1. Lamb, T.D. Gain and kinetics of activation in the G-protein cascade of phototransduction. Proc. Natl. Acad. Sci. USA 93, 566–570 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lohse, M.J. et al. Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors. Trends Pharmacol. Sci. 29, 159–165 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Pugh, E.N. Jr. & Lamb, T.D. Amplification and kinetics of the activation steps in phototransduction. Biochim. Biophys. Acta 1141, 111–149 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Drake, M.T., Shenoy, S.K. & Lefkowitz, R.J. Trafficking of G protein-coupled receptors. Circ. Res. 99, 570–582 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Dean, T., Vilardaga, J.P., Potts, J.T. Jr. & Gardella, T.J. Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor. Mol. Endocrinol. 22, 156–166 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Okazaki, M. et al. Prolonged signaling at the parathyroid hormone receptor by peptide ligands targeted to a specific receptor conformation. Proc. Natl. Acad. Sci. USA 105, 16525–16530 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Horwitz, M.J. et al. Direct comparison of sustained infusion of human parathyroid hormone-related protein-(1–36) [hPTHrP-(1–36)] versus hPTH-(1–34) on serum calcium, plasma 1,25-dihydroxyvitamin D concentrations, and fractional calcium excretion in healthy human volunteers. J. Clin. Endocrinol. Metab. 88, 1603–1609 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Horwitz, M.J. et al. Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)2 vitamin D. J. Bone Miner. Res. 20, 1792–1803 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Castro, M., Nikolaev, V.O., Palm, D., Lohse, M.J. & Vilardaga, J.P. Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism. Proc. Natl. Acad. Sci. USA 102, 16084–16089 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dean, T. et al. Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein receptor: selectivity of a modified PTH(1–15) radioligand for GalphaS-coupled receptor conformations. Mol. Endocrinol. 20, 931–943 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Berlot, C.H. A highly effective dominant negative alpha s construct containing mutations that affect distinct functions inhibits multiple Gs-coupled receptor signaling pathways. J. Biol. Chem. 277, 21080–21085 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Vilardaga, J.P., Bunemann, M., Krasel, C., Castro, M. & Lohse, M.J. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat. Biotechnol. 21, 807–812 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Hein, P., Frank, M., Hoffmann, C., Lohse, M.J. & Bunemann, M. Dynamics of receptor/G protein coupling in living cells. EMBO J. 24, 4106–4114 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hein, P. et al. Gs activation is time-limiting in initiating receptor-mediated signaling. J. Biol. Chem. 281, 33345–33351 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Nikolaev, V.O., Bunemann, M., Hein, L., Hannawacker, A. & Lohse, M.J. Novel single chain cAMP sensors for receptor-induced signal propagation. J. Biol. Chem. 279, 37215–37218 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Syme, C.A., Friedman, P.A. & Bisello, A. Parathyroid hormone receptor trafficking contributes to the activation of extracellular signal-regulated kinases but is not required for regulation of cAMP signaling. J. Biol. Chem. 280, 11281–11288 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Sneddon, W.B. et al. Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50). J. Biol. Chem. 278, 43787–43796 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Shenoy, S.K., McDonald, P.H., Kohout, T.A. & Lefkowitz, R.J. Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science 294, 1307–1313 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Chauvin, S., Bencsik, M., Bambino, T. & Nissenson, R.A. Parathyroid hormone receptor recycling: role of receptor dephosphorylation and beta-arrestin. Mol. Endocrinol. 16, 2720–2732 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Ferrari, S.L., Behar, V., Chorev, M., Rosenblatt, M. & Bisello, A. Endocytosis of ligand-human parathyroid hormone receptor 1 complexes is protein kinase C-dependent and involves β-arrestin2. Real-time monitoring by fluorescence microscopy. J. Biol. Chem. 274, 29968–29975 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Xia, Z. & Liu, Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81, 2395–2402 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

J.-P.V. thanks M.J. Lohse (University of Wuerzburg) and N.O. Nikolaev (University of Wuerzburg) for generously sharing their plasmid encoding Epac-CFP/YFP, C. Berlot (Weis Center for Research) for the plasmid encoding Gαs-CFP, M. Bünemann (University of Wuerzburg) for the Gγ2-CFP construct and A. Bisello (University of Pittsburgh) for Dyn K44A. The authors thank D. Altschuler for the use of his FRET imaging microscope. This work was supported by start-up funds from the Department of Medicine, Massachusetts General Hospital, and the Department of Pharmacology and Chemical Biology, University of Pittsburgh (to J.-P.V.). M.C. is at the Department of Pharmacology, School of Pharmacy, University of Santiago de Compostella (Spain) and received a fellowship from the Xunta de Galicia (Spain) for a research sabbatical at the J.-P.V. laboratory at the Endocrine Unit of the Massachusetts General Hospital. R.B. was supported by US National Institutes of Health grant DK38451 and received an investigator award from the National Kidney Foundation. J.-P.V. thanks P. Friedman and G. Romero for critically commenting on the manuscript.

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Authors

Contributions

S.F. and T.N.F. performed most of the experiments with the support of M.C., B.W. and R.B.; J.-P.V. designed and supervised the experiments and wrote the manuscript with the support of T.J.G., J.T.P., S.F. and T.N.F.

Corresponding author

Correspondence to Jean-Pierre Vilardaga.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Methods (PDF 16769 kb)

Supplementary Video 1

A 3D view of TMR-labeled ligands and GFPN-PTHR in live HEK-293 cells by spinning disc confocal microscopy 20 min after ligand wash out. PTH(1?34)TMR (red ) and GFPN-PTHR (green) co-localized within endocytic compartments. (MOV 330 kb)

Supplementary Video 2

A 3D view of TMR-labeled ligands and GFPN-PTHR in live HEK-293 cells by spinning disc confocal microscopy 20 min after ligand wash out. In contrast, PTHrP(1–36)TMR alone is detected as small puntae at internalized sites. (MOV 284 kb)

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Ferrandon, S., Feinstein, T., Castro, M. et al. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat Chem Biol 5, 734–742 (2009). https://doi.org/10.1038/nchembio.206

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