Functional characterization of mammalian V2R.

For functional characterization COS-7 cells were transiently transfected with V2R constructs and non-radioactive cAMP assays were performed as described in Materials and Methods. Emax and EC50 values were determined from concentration-response-curves of AVP (1 fM–10 µM) using GraphPad Prism. Data are presented as mean±S.E.M. of independent experiments (number indicated in parentheses), each carried out in duplicate. Cyclic AMP levels of non-stimulated (10.0±2.2 amol/cell) and 10 µM AVP-stimulated (185.9±28.6 amol/cell) human V2R served as reference basal and Emax values and were set 0% and 100%, respectively. Cell surface and total expression levels of V2R orthologs were measured by cell surface and total cellular ELISAs (see Materials and Methods). Specific optical density (OD) readings (OD value of HA-tagged construct minus OD value of GFP-transfected cells) are given as a percentage of human double HA/FLAG-tagged V2R.
#For the cell surface ELISA the non-specific OD value (GFP) was 0.013±0.007 (set 0%) and the OD value of the human V2R was 0.983±0.133 (set 100%).
##For the total cellular ELISA the non-specific OD value (GFP) was 0.061±0.020 (set 0%) and the OD value of the human V2R was 0.814±0.118 (set 100%). The number of independent experiments, each carried out in quadruplicate, is given in parentheses.
*indicates significance differences (p<0.01) to the average basal and AVP-induced activity of all V2R orthologs.