Extended Data Fig. 3: Actions of Pitt8 on PTH signaling.
(a, b) Binding isotherms showing competitive inhibition of radio-labeled peptide ligand binding to PTHR by PTH or Pitt8, using plasma membrane extracts from HEK293 cells expressing recombinant PTHR. Inhibition of [125I]-LAPTH binding to G protein-independent conformational R0 state of PTHR (that is, in the absence of G proteins) by PTH1-34 (black) or Pitt8 (pink) (a). Inhibition of [125I]-M-PTH1-15 binding to G protein-dependent RG conformational state of PTHR (that is, in the presence of G proteins) by M-PTH1-15 (black) or Pitt8 (pink). Mean ± s.d. of N = 6 experiments. (c) Concentration-response curves for cAMP production by PTH alone or together with Pitt8. Data are mean ± s.e.m. of N = 3 independent experiments. (d, e) Averaged cAMP time-courses following brief stimulation with 1 nM PTH without (Ctrl, black) or with 10 µM Pitt12 measured by FRET changes from HEK293 cells stably expressing PTHR and a FRET-based cAMP sensor EpacCFP/YFP in the absence (d) or presence of the dominant-negative dynamin mutant (DynK44A) tagged with RFP (e). (f) Time course of β-arrestin 2 interaction with PTHR measured by FRET in HEK293 cells transiently expressing PTHRCFP and βarr-2YFP following brief stimulation with 10 nM PTH without (Ctrl) or with 10 µM Pitt8. Cells were continuously perfused with control buffer or PTH alone or together with Pitt8 (horizontal bar). Data are the mean ± s.e.m. of N = 3 experiments with n = 52 (DMSO) and 47 (Pitt8) cells examined. (g) Time courses of Ca2+ release in response to PTH (100 nM) with or without Pitt8 (10 µM) in live HEK-293 cells expressing recombinant PTHR. Data are the mean ± s.e.m. of N = 3 experiments with n = 32 (DMSO) and 32 (Pitt8) cells examined.
