The proximal portion of the COOH terminus of the oxytocin receptor is required for coupling to G(q), but not G(i). Independent mechanisms for elevating intracellular calcium concentrations from intracellular stores

Sarasija Hoare, John A III Copland, Zuzana Strakova, Kirk Ives, Yow Jiun Jeng, Mark R. Hellmich, Melvyn S. Soloff

Research output: Contribution to journalArticle

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Abstract

As the oxytocin receptor plays a key role in parturition and lactation, there is considerable interest in defining its structure/functional relationships. We previously showed that the rat oxytocin receptor transfected into Chinese hamster ovary cells was coupled to both G(q/11) and G(i/o), and that oxytocin stimulated ERK-2 phosphorylation and prostaglandin E2 synthesis via protein kinase C activity. In this study, we show that deletion of 51 amino acid residues from the carboxyl terminus resulted in reduced affinity for oxytocin and a corresponding rightward shift in the dose-response curve for oxytocin-stimulated [Ca2+](i). However, oxytocin- stimulated ERK-2 phosphorylation and prostaglandin E2 synthesis did not occur in cells expressing the truncated receptor. Oxytocin also failed to increase phospholipase A activity or activate protein kinase C, indicating that the mutant receptor is uncoupled from G(q)-mediated pathways. The Δ51 receptor is coupled to G(i), as oxytocin-stimulated Ca2+ transients were inhibited by pertussis toxin, and a Gβγ sequestrant. Preincubation of Δ51 cells with the tyrosine kinase inhibitor, genistein, also blocked the oxytocin effect. A A39 mutant had all the activities of the wild type oxytocin receptor. These results show that the portion between 39 and 51 residues from the COOH terminus of the rat oxytocin receptor is required for interaction with G(q/11), but not G(i/o). Furthermore, an increase in intracellular calcium was generated via a G(i)βγ-tyrosine kinase pathway from intracellular stores that are distinct from G(q)-mediated inositol trisphosphate-regulated stores.

Original languageEnglish (US)
Pages (from-to)28682-28689
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number40
DOIs
StatePublished - Oct 1 1999
Externally publishedYes

Fingerprint

Oxytocin Receptors
Oxytocin
Calcium
Phosphorylation
Dinoprostone
Protein-Tyrosine Kinases
Protein Kinase C
Cells
Genistein
Phospholipases A
Pertussis Toxin
Inositol
Cricetulus
Lactation
Ovary
Parturition
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

The proximal portion of the COOH terminus of the oxytocin receptor is required for coupling to G(q), but not G(i). Independent mechanisms for elevating intracellular calcium concentrations from intracellular stores. / Hoare, Sarasija; Copland, John A III; Strakova, Zuzana; Ives, Kirk; Jeng, Yow Jiun; Hellmich, Mark R.; Soloff, Melvyn S.

In: Journal of Biological Chemistry, Vol. 274, No. 40, 01.10.1999, p. 28682-28689.

Research output: Contribution to journalArticle

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abstract = "As the oxytocin receptor plays a key role in parturition and lactation, there is considerable interest in defining its structure/functional relationships. We previously showed that the rat oxytocin receptor transfected into Chinese hamster ovary cells was coupled to both G(q/11) and G(i/o), and that oxytocin stimulated ERK-2 phosphorylation and prostaglandin E2 synthesis via protein kinase C activity. In this study, we show that deletion of 51 amino acid residues from the carboxyl terminus resulted in reduced affinity for oxytocin and a corresponding rightward shift in the dose-response curve for oxytocin-stimulated [Ca2+](i). However, oxytocin- stimulated ERK-2 phosphorylation and prostaglandin E2 synthesis did not occur in cells expressing the truncated receptor. Oxytocin also failed to increase phospholipase A activity or activate protein kinase C, indicating that the mutant receptor is uncoupled from G(q)-mediated pathways. The Δ51 receptor is coupled to G(i), as oxytocin-stimulated Ca2+ transients were inhibited by pertussis toxin, and a Gβγ sequestrant. Preincubation of Δ51 cells with the tyrosine kinase inhibitor, genistein, also blocked the oxytocin effect. A A39 mutant had all the activities of the wild type oxytocin receptor. These results show that the portion between 39 and 51 residues from the COOH terminus of the rat oxytocin receptor is required for interaction with G(q/11), but not G(i/o). Furthermore, an increase in intracellular calcium was generated via a G(i)βγ-tyrosine kinase pathway from intracellular stores that are distinct from G(q)-mediated inositol trisphosphate-regulated stores.",
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