cAMP accumulation assay

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.