Submitted on November 1, 2005
Accepted on November 19, 2006
Amino Acids in the C-Terminal Region of the Oxytocin Receptor Third Intracellular Domain are Important for Receptor Function
Miao Zhong1, Bridgette Parish2, Dilyara A Murtazina1, Chun-Ying Ku1, and Barbara M. Sanborn1*
1 Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States
2 Biochemistry, University of Texas Medical School, Houston, Houston, Texas, United States
* To whom correspondence should be addressed. E-mail: barbara.sanborn{at}colostate.edu.
Previously, residue K6.30 in the C-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 (PLC) activation in both OTR and the vasopressin V2 receptor 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 N-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 [3H|]-OT. This OTR mutant was unable to activate PI turnover or ERK1/2 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 [3H]-OT and the ability to stimulate PI turnover. Affinity for [3H]-OT and membrane expression were 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 C-terminal region of the third intracellular domain of OTR influence the ability of OTR to activate G protein-mediated actions.
