Previously, residue K6.30 in the COOH-terminal region of the third intracellular domain (3iC) of the oxytocin (OT) receptor (OTR) was identified as important for receptor function leading to phospholipase C activation in both OTR and the vasopressin V 2 receptor (V2R) chimera V2ROTR3iC. Substitution of either A6.28K or V6.30K in wild-type V2R did not recapitulate the increase in phosphatidylinositide (PI) turnover observed in V2ROTR3iC. Hence, the role of K6.30 may be context-specific. Deletion of two NH2-terminal OTR3iC segments in the V2ROTR3iC chimera did not diminish vasopressin-stimulated PI turnover, whereas deletion of RVSSVKL (residues 6.19 -6.25) reduced receptor expression. Deletion of this sequence in wild-type OTR reduced expression by 50% without affecting affinity for [ 3 H]OT. This OTR mutant was unable to activate PI turnover or extracellular signal-regulated kinase 1/2 phosphorylation. The effects of alanine substitution for individual residues in RVSSVKL indicated differential importance for OTR function. The R6.19A substitution lost high-affinity sites for [ 3 H]OT and the ability to stimulate PI turnover. Affinity for [ 3 H]OT and membrane expression was not affected by any other substitutions. OTR-V6.20A and OTR-K6.24A mutants functioned as well as wild-type OTR, whereas OTR S6.21A, S6.22A, and V6.23A mutants exhibited impaired abilities to activate PI turnover (20 -40% of OTR), and the OTR-L6.25A mutant exhibited constitutive activity. In conclusion, specific amino acids in the RVSSVKL segment in the COOH-terminal region of the third intracellular domain of OTR influence the ability of OTR to activate G protein-mediated actions.vasopressin type II receptor; G protein; phospholipase C; extracellular signal-regulated protein kinase-1/2

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