Journal: bioRxiv

Article Title: LHR and Gαs trafficking drive sustained cAMP signalling from endosomes to control steroidogenesis

doi: 10.1101/2025.04.30.651471

Figure Lengend Snippet: (A) HEK293A cells transiently expressing LHR and treated with 100 nM hCG-mNG (cyan) during 60 seconds before ligand washout. At times 5 min and 15 min, arrows show colocalization between hCG-mNG positive endosomes and APPL1-mCherry (magenta). (B) Bystander BRET assay measuring hCG-induced LHR-RLuc8 endocytosis in HEK293A cells. Area under the curve (AUC) of LHR endocytosis between 0 and 30 minutes of continuous 30 nM hCG stimulation, with or without 30µM Dyngo4a. N=3 experiments performed in triplicates, means ± SEM. (C, D) BRET measurements of time courses of cAMP using the biosensor NLuc-Epac-VV. LHR was stimulated either by 3 nM hCG (C) or 3 nM LH (D) for 60 seconds, with or without 30µM Dyngo4a. N=3 (n=9) , means ± SEM. (E, F) Dose-responses curves of the AUC between 0 and 60 minutes of cAMP production stimulated by hCG (E) or LH (F), with or without 30µM Dyngo4a. N=3 (n=9) , means ± SEM. (G) EC50 of cAMP response induced by 60-second stimulation with hCG or LH, calculated based on the 0-60 minutes AUC dose-responses. EC50 were calculated on data from three independent experiments performed in triplicates, and are indicated in molar. (H) Luminescence complementation assay monitoring the activation of Gs and the formation of a complex with LHR. Increased luminescence represents the recruitment of Nb37-SmBiT to the LHR-LgBiT by activated endogenous Gαs. N=3 (n=9) , means ± SEM. (I) CRE-driven luciferase gene expression after 6 hours continuous stimulation of HEK293A cells expressing LHR with different hCG concentrations, in presence of DMSO (control), 30µM Dyngo4a or 30µM PitStop2. Data were expressed in percentage of the control’s maximum. N=3 (n=9) , means ± SEM. Comparisons to DMSO for a given condition are indicated with corresponding colour stars and hashes. # p-value = 0.068.

Article Snippet: Dyngo4a was purchased at MedChemExpress (#HY-13863) and used at a final concentration of 30µM.

Techniques: Expressing, Bioluminescence Resonance Energy Transfer, Activation Assay, Luciferase, Gene Expression, Control

Journal: bioRxiv

Article Title: LHR and Gαs trafficking drive sustained cAMP signalling from endosomes to control steroidogenesis

doi: 10.1101/2025.04.30.651471

Figure Lengend Snippet: (A) Gαs-YFP cellular localization over time after 30 nM hCG stimulation of HEK293A cells transiently expressing LHR. Representative pictures of Gαs-YFP cellular localization without ligand stimulation (left) and after 5 minutes (middle) or 15 minutes (right) hCG stimulation. Scale bar = 10 µm. Fluorescence intensity across cell width was quantified before (0min, in black) and after 5 minutes (5min, in red) hCG stimulation. (B) BRET assay measuring the dissociation of YFP-Gαs from the plasma membrane, upon 30 nM hCG stimulation with or without of 30µM Dyngo4a. N=3 (n=9) , means ± SEM. (C, D) Representative images from live cell imaging showing colocalization of YFP-Gαs (cyan) with either APPL1-mCherry (C) or Rab5-mScarlet-I (D) (magenta), before (top) or after 15 minutes (bottom) 30 nM hCG stimulation. Yellow arrows in insets (bottom right) show endosomes positive for both markers. Scale bar = 10 µm. (E) BRET assay comparing the endocytosis of the WT Gαs-YFP and the Myr-Gαs-YFP variant in HEK293ΔGs cells, following 30 nM hCG stimulation. N=3 (n=9) , means ± SEM. (F) BRET assay comparing the cAMP kinetics profiles of HEK293ΔGs cells transiently expressing LHR and co-expressing either WT-Gαs or Myr-Gαs, stimulated with 0.3nM hCG during 60 seconds. N=1 (n=3) representative from N=3 independent experiments , means ± SEM. (G) Quantification of the area under the curve from 0-30 minutes cAMP response. N=3 , means ± SEM.

Article Snippet: Dyngo4a was purchased at MedChemExpress (#HY-13863) and used at a final concentration of 30µM.

Techniques: Expressing, Fluorescence, Bioluminescence Resonance Energy Transfer, Clinical Proteomics, Membrane, Live Cell Imaging, Variant Assay

Journal: bioRxiv

Article Title: LHR and Gαs trafficking drive sustained cAMP signalling from endosomes to control steroidogenesis

doi: 10.1101/2025.04.30.651471

Figure Lengend Snippet: (A) Stimulation of mLTC-1 cells with 100 nM hCG-mNeonGreen for 120 seconds, before washout. Cellular localization of hCG-mNeonGreen before stimulation (no hCG-mNG), and 1, 5 or 15 minutes after stimulation. Scale bar = 10 µm. (B, C) Cytosolic kinetics of cAMP signalling in mLTC-1 cells, induced by 60-second 10nM hCG stimulation with or without 30µM Dyngo4a (B) or 30µM PitStop2 (C). N=3 (n=9) , means ± SEM. (D, E) Measurement of cAMP (D) and progesterone (E) concentrations in extracellular medium of mLTC-1 cells stimulated for 1 minute with 1nM hCG, in presence of DMSO (control), 30µM Dyngo4a or 30µM PitStop2. Supernatants were collected 30, 60, 120, 180 or 360 minutes after hCG stimulation. The 0 minutes condition corresponds to unstimulated cells for which supernatant was collected at 360 minutes. N=3 (n=6) , means ± SEM. (F) Dose-response curves of cAMP concentration in extracellular medium of mLTC-1 cells continuously stimulated for 3 hours with different hCG doses, in presence of DMSO, 30µM Dyngo4A or 30µM PitStop2. N=3 (n=6) , means ± SEM. Comparisons to DMSO for a given condition are indicated in corresponding colours. (G) Progesterone concentration in extracellular medium of mLTC-1 cells stimulated for 3 hours with 0.1nM hCG, in presence of DMSO, 30µM Dyngo4a or 30µM PitStop2. Progesterone dosages were performed on the same samples as cAMP dosages represented in (F). N=3 (n=6) , means ± SEM. # p-value = 0.066. (H) Progesterone concentration in extracellular medium of mLTC-1 cells transiently overexpressing mCherry (control) or Nb37-CAAX-mScarlet (80ng/well of 48-well plate, roughly equivalent to 30ng/well of 96-well plate). N=3 (n=6) , means ± SEM. (I) Measurement of testosterone concentration in extracellular medium of mouse primary Leydig cells stimulated for 3 hours with 0.1nM hCG, in presence of DMSO, 30µM Dyngo4a or 30µM PitStop2. Data were expressed as the percentage of control condition’s maximal response. N=4 (n=12) , means ± SEM.

Article Snippet: Dyngo4a was purchased at MedChemExpress (#HY-13863) and used at a final concentration of 30µM.

Techniques: Control, Concentration Assay