Pcdna6.0 vector

The VIP1R mutants were cloned into pcDNA6.0 vector (Invitrogen) with a FLAG tag at its N-terminus. The cell seeding and transfection follow the same method as cAMP accumulation assay. After 24 h of transfection, cells were washed once with PBS and digested with 0.2% (w/v) EDTA in PBS. Cells were blocked with PBS containing 5% (w/v) BSA for 15 min at room temperature and then incubated with primary anti-Flag antibody (diluted with PBS containing 5% BSA at a ratio of 1:300, Sigma) for 1 h at room temperature. Thereafter, cells were washed three times with PBS containing 1% (w/v) BSA before incubating with anti-mouse Alexa-488-conjugated secondary antibody (diluted with PBS containing 5% BSA at a ratio of 1:1000, Invitrogen) at 4 °C in the dark for 1 h. After another three times wash, cells were resuspended, and fluorescence intensity was quantified in a BD Accuri C6 flow cytometer system (BD Biosciences) at excitation 488 nm and emission 519 nm. Approximately 10,000 cellular events per sample were collected and data were normalized to WT.

The full-length VIP1R(31–457) and VIP1R mutants was cloned into pcDNA6.0 vector (Invitrogen) with a FLAG tag at its N-terminus (see Supplementary Table 5 for a list of primers used in this study). CHO-K1 cells (ATCC, #CCL-61) were cultured in Ham’s F-12 Nutrient Mix (Gibco) supplemented with 10% (w/v) fetal bovine serum. Cells were maintained at 37 °C in a 5% CO₂ incubator with 100,000 cells per well in a 12-well plate. Cells were grown overnight and then transfected with 1 μg VIP1R constructs by FuGENE^® HD transfection reagent (DNA/FuGENE^® HD ratio of 1:3) in each well. After 24 h, the transfected cells were seeded onto 384-well microtiter plates (3000 cells per well). cAMP accumulation was measured using the LANCE cAMP kit (PerkinElmer) according to the manufacturer’s instructions with different concentrations of peptides. Fluorescence signals were then measured at 620 and 665 nm by an Envision multilabel plate reader (PerkinElmer). Data presented are means ± SEM of at least three independent experiments.

The full-length FSHR and FSHR mutants were cloned into pcDNA6.0 vector (Invitrogen) with a FLAG tag at its N-terminus. CHO-K1 cells (ATCC, #CCL-61) were cultured in Ham’s F-12 Nutrient Mix (Gibco) supplemented with 10% (w/v) fetal bovine serum. Cells were maintained at 37 °C in a 5% CO₂ incubator with 150,000 cells per well in a 12-well plate. Cells were grown overnight and then transfected with 1 μg FSHR constructs by FuGENE® HD transfection reagent in each well for 24 h. cAMP accumulation was measured using the LANCE cAMP kit (PerkinElmer) according to the manufacturer’s instructions. The transfected cells were seeded onto 384-well plates with 2500 cells each well, and then incubated with ligands for 30 min at 37 °C, then Eu and Ulight were added separately before cAMP levels were measured. Fluorescence signals were measured at 615 nm and 665 nm by an Envision multilevel plate reader (PerkinElmer). Data were analyzed using Graphpad Prism8.0, the three-parameter, nonlinear regression equation in Prism suite was used in fitting. The response values were normalized by WT receptors within each individual experiment, with the basal activity for WT as 0, while the fitted E_max of WT as 100. Experiments were performed at least three times, the detail information were attached in the figure legends, each experiment conducted in triplicate. Data were presented as means ± SEM.