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. Author manuscript; available in PMC: 2011 Feb 2.
Published in final edited form as: Nat Chem Biol. 2009 Aug 23;5(10):734–742. doi: 10.1038/nchembio.206

Figure 4.

Figure 4

PTHR endocytosis and prolonged cAMP responses. (a) HEK293 cells stably expressing GFP-N-PTHR (upper panels) and transfected with Dyn K44A (lower panels) were perfused with s100 nM PTH1–34-TMR for about 20 s and then with buffer alone for the remainder of the experiment. 20 min after ligand exposure and washout, PTH1–34-TMR and GFP-N-PTHR complexes redistributed in endosomes in control cells, but remained exclusively localized at the plasma membrane of Dyn K44A-expressing cells. Scale bars, 5 μm. (b) Averaged cAMP response over a 50-min time course measured by FRET changes from HEK293 cells stably expressing PTHR and transiently expressing Epac-CFP/YFP with or without Dyn K44A. Cells were continuously perfused with control buffer or 100 nM PTH1–34 (arrow). Data represent the mean ± s.e.m. of n = 9. (c) Bars represent the average cAMP response. In these experiments, the ligand was washed out 20 s after application by perfusion to remove the unbound ligand.