Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function.
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 (V(2)R) chimera V(2)ROTR3iC. Substitution of either A6.28K or V6.30K in wild-type V(2)R did not recapitulate the increase in phosphatidylinositide (PI) turnover observed in V(2)ROTR3iC. Hence, the role of K6.30 may be context-specific. Deletion of two NH(2)-terminal OTR3iC segments in the V(2)ROTR3iC 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.
Zhong
Miao
M
Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.
Parish
Bridgette
B
Murtazina
Dilyara A
DA
Ku
Chun-Ying
CY
Sanborn
Barbara M
BM
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D018045:Receptors, Oxytocin / Q000235:genetics* / Q000378:metabolism*; D017483:Receptors, Vasopressin / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014667:Vasopressins / Q000494:pharmacology
D010716:Phosphatidylinositols; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D011993:Recombinant Fusion Proteins; D014667:Vasopressins; D001120:Arginine; D048049:Extracellular Signal-Regulated MAP Kinases; D000409:Alanine