G Protein-Coupled Receptor Assay Protocols

Arginine vasopressin (AVP) was purchased from Sigma-Aldrich. The OPC analogues (OPC-51803 (OPC5), OPC16b, OPC16g, OPC16j, OPC19a, OPC19b, OPC23b, OPC23d, OPC23h and OPC23i) and OPC4 (OPC41061) were kindly provided by Otsuka Pharmaceutical Company. Plasmids for the NanoBiT-β-arrestin-recruitment assay and GloSensor-based cAMP assay were previously described [19 (link), 20 (link)]. For HiBiT-based luciferase-fragment complementation assay, human full-length V2R were N-terminally fused to a HiBiT cassette (HiBiT-V2R), which contained an interleukin 6 (IL6)-derived signal sequence followed by a HiBiT sequence and a linker at the N terminus (MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVSGWRLFKKISGGSGGGGSG; HiBiT tag underlined; gene synthesized with codon optimization). Unless otherwise indicated, all the constructs were inserted into the pCAGGS expression plasmid vector. The V2R mutant constructs (V88^2.53M, Y128^3.41S, L161^4.47P, T273^6.37M, S329^8.47R and S333^8.51del) were generated by an in-house-modified QuikChange Site-Directed Mutagenesis Kit.