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Lysine 270 in the Third Intracellular Domain of the Oxytocin Receptor is an Important Determinant for G_q Coupling Specificity

Department of Biochemistry and Molecular Biology (M.Y., W.W., M.Z., B.M.S.), University of Texas Medical School Houston, Houston, Texas 77030; and Department of Human Genetics (A.P., O.L.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Barbara Sanborn, Department of Biochemistry and Molecular Biology, University of Texas Medical School, P.O. Box 20708, Houston, Texas 77225. E-mail: .

To identify structural elements important to specific G_q coupling in the oxytocin receptor (OTR), intracellular domains were exchanged between OTR and G_s-coupled vasopressin V₂ receptors (V₂Rs). 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 G_q 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/G_q/PLC coupling.

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