Engineering a V2 Vasopressin Receptor Agonist- and Regulator of G-Protein-Signaling-Sensitive G Protein

It is extremely difficult to detect guanine nucleotide exchange or hydrolysis stimulated by receptors which couple to Gsα. Furthermore, Gsα is largely resistant to the GTPase-activating properties of RGS proteins. Coexpression of the vasopressin V2 receptor with a series of chimeric G protein α subunits in which the C-terminal 6–12 amino acids of Gi1α were replaced with the equivalent sequence of Gsα allowed robust vasopressin-stimulated [35S]GTPγS binding. Vasopressin did not stimulate the GTPase activity of fusion proteins between the V2 receptor and either Gsα or Gi1α. However, it produced a concentration-dependent stimulation of Vmax for a V2 receptor-Gi1α/Gs6α fusion protein. This construct bound [3H]vasopressin with high affinity and this was competed by other ligands with rank order anticipated for the V2 receptor. RGS1 enhanced vasopressin stimulation of V2 receptor-Gi1α/Gs6α in a concentration-dependent manner. RGS-GAIP was substantially less potent. Enzyme kinetic analysis demonstrated that RGS1 increased both Vmax of the GTPase activity and the observed Km for GTP, consistent with RGS1 accelerating the rate of GTP hydrolysis of the chimeric G protein, whereas the agonist vasopressin accelerates guanine nucleotide exchange. This approach provides a sensitive assay for V2 receptor agonist ligands and may be amenable to many other Gsα-coupled receptors.