cAMP accumulation assay

AD293 cells were plated at a density of 5 × 10⁴/well in 24-well plates 1 day before transfection. Cells were then transiently transfected using Lipofectamine 2000 reagent (Life Technologies) with 50 ng of cDNA encoding GPCR, 200 ng of CRE-driven fly luciferase reporter and 10 ng of thymidine kinase promoter-driven Renilla luciferase, which was used as an internal transfection control at a ratio of 2:1 (Lipofectamine 2000 reagent:DNA). The CRE-luciferase we used here is the pGL4 CRE reporter, originally obtained from Promega as a component of a cAMP assay kit. Detection of cAMP was performed using the Dual-Luciferase reporter assay system from Promega according to the manufacturer's instructions with an EnVision plate reader (PerkinElmer Life Sciences). Renilla luciferase was used for normalization. To test the response of the GCGR to exogenous GCG and of VIP1R to exogenous VIP(1–28), we co-expressed 50 ng of membrane-tethered GCG(1–29) plasmid with 50 ng of GCGR and 50 ng of membrane-tethered VIP(1–28) plasmid with 50 ng of VIP1R according to the literature (16, 24). Membrane-tethered GCG(1–29) and VIP(1–28) plasmids refer to the constructs that are the fusions of glucagon(1–29) and VIP(1–28) to a long flexible linker (a 5×NG linker followed by a MYC tag (EQKLISEEDL), then a 9×NG linker), and the single transmembrane domain of CD8 with sequence ALCWVGIGIGVLAAGVLVVTAIVYVV (25). All experiments were performed in triplicates, each transfected independently.