Journal: The Journal of Biological Chemistry

Article Title: Rewiring PTH receptor signaling: Hormone dimerization restores endosomal signaling lost in hypocalcemia-linked PTH mutant

doi: 10.1016/j.jbc.2025.110913

Figure Lengend Snippet: Progression of the active ligand–bound PTH 1 R conformations toward their inactive states by weighted ensemble (WE) simulations and FRET recordings in live cells. A and B , the structural features of the TM3–TM6 distance ( A ) and TM6 bending angle ( B ) adopted for visualizing the trajectories of receptor conformations from active toward inactive states. C – E , WE simulations show the progression of the active to inactive receptor conformations when bound by PTH WT ( C ), PTH R25C ( D ), and PTH dimer ( E ). The PTH R25C –PTH 1 R gradually progresses toward its inactive state. In contrast, the PTH dimer –PTH 1 R conformations undergo more constrained changes, indicating the active state’s preferential sampling (or retention). -ln(P) represents the free energy. F – H , the inset shows a schematic of the FRET-based PTH 1 R activation sensor (PTH 1 R CFP/YFP ) with YFP ( yellow ) fused to ICL3 and CFP ( blue ) attached to the receptor C-terminal tail. The graph shows averaged time courses of PTH 1 R activation by recording changes in the FRET ratio in HEK293 cells expressing PTH 1 R CFP/YFP . Cells were continuously perfused with control buffer or 1 μM agonist ( horizontal bar ). Means ± SD from N = 10 (PTH), N = 6 (PTH R25C ), and N = 5 (PTH dimer ).

Article Snippet: HEK293 (American Type Culture Collection) cells were grown in Dulbecco's modified Eagle's medium (DMEM) (low glucose; Life Technologies, 10567022) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Life Technologies, 10438-034), 100 units/ml penicillin, 100 μg/ml streptomycin (Life Technologies, 15140122) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Sampling, Activation Assay, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Rewiring PTH receptor signaling: Hormone dimerization restores endosomal signaling lost in hypocalcemia-linked PTH mutant

doi: 10.1016/j.jbc.2025.110913

Figure Lengend Snippet: PTH 1 R signaling. A , time courses and integrated responses (area under the curve) of ligand (100 nM) induced intracellular Ca 2+ elevation in HEK293 cells expressing recombinant PTH 1 R. Bars represent the mean values ± SD with n = 200 cells from three independent experiments. B and C , time courses ( B ) and integrated ( C ) responses of cAMP production after brief stimulation with 1 nM PTH, PTH R25C , or PTH dimer in HEK293 cells stably expressing recombinant PTH 1 R without (control) or with dynamin K44A expression (DynK 44A ). The percentage of cAMP responses is relative to the response in the presence of forskolin (FSK). Bars represent the mean values ± SD from n = 24 (PTH), n = 36 (PTH R25C ), n = 69 (PTH dimer ) cells in controls and n = 40 (PTH), n = 18 (PTH R25C ), n = 43 (PTH dimer ) cells in Dyn K44A from three independent experiments. D and E , schematic representing the experiment to measure internalization of PTH 1 R SEP ( E ). Time courses showing the internalization profiles for cells stimulated with PTH, PTH R25C , and PTH dimer . Mean values ± SD with n = 15 cells from three independent experiments ( F ). F and G , time courses in cAMP production ( F ) and corresponding integrated values ( G ) in MC3T3 cells expressing the native PTH 1 R. Means ± SD from n = 24 (PTH), n = 24 (PTH R25C ), n = 27 (PTH dimer ) cells from four independent experiments. Data are the mean ± SD with ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, and no significant (ns) by one-way ANOVA with Dunnett’s ( A ) and Tukey’s multiple comparison test ( G ).

Article Snippet: HEK293 (American Type Culture Collection) cells were grown in Dulbecco's modified Eagle's medium (DMEM) (low glucose; Life Technologies, 10567022) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Life Technologies, 10438-034), 100 units/ml penicillin, 100 μg/ml streptomycin (Life Technologies, 15140122) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Recombinant, Stable Transfection, Control, Comparison