Lysine 270 in the third intracellular domain of the oxytocin receptor is an important determinant for Gαq coupling specificity

To identify structural elements important to specific Galpha(q) coupling in the oxytocin receptor (OTR), intracellular domains were exchanged between OTR and Galpha(s)-coupled vasopressin V-2 receptors (V(2)Rs). Substitution of sequence from the second (2i) and third (3i) intracellular domains of V2R into comparable positions in OTR markedly reduced ligand affinity and resulted in a loss of Gaq coupling. Substitution of the 2i domain of OTR into V2R decreased ligand affinity and vasopressin-stimulated adenylyl cyclase activity and only slightly increased phosphatidylinositide turnover. In contrast, substitution of the OTR3i domain into V2R produced a receptor chimera with high ligand affinity, decreased vasopressin-stimulated adenylyl cyclase activity, and markedly enhanced ligand-stimulated phosphatidylinositide turnover. The C-terminal 36 amino acids, but not the N-terminal 13 amino acids, of the OTR3i domain contained the determinants critical for enhanced activation of PLC. Mutation of a single lysine in the C-terminal OTR3i sequence to the corresponding V2R residue (valine) eliminated the enhanced ability of the V2R chimera to stimulate PLC but did not affect maximal adenylyl cyclase stimulation. Furthermore, mutation of this residue (K270) in wild-type OTR completely abolished the ability of the receptor to stimulate phosphatidylinositide turnover, with only a small reduction in ligand affinity. These data demonstrate that OTR K270 is critically important in the stimulation by OTR of phosphatidylinositide turnover and that this determinant can also increase this activity in the V2R chimera. Mutation of K270 also adversely affects the ability of OTR to stimulate ERK1/2 phosphorylation. Therefore, this residue plays an important role in the specificity of OTR/Galpha(q)/PLC coupling.