Coupling of oxytocin receptor to G proteins in rat myometrium during labor: Gi receptor interaction

May 1, 1997The American journal of physiology

Oxytocin receptor interaction with inhibitory proteins in rat uterine muscle during labor

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Abstract

At term, there is about a fivefold increase in oxytocin receptor (OTR) binding in pregnant rat myometrial membranes.

Two distinct peaks of radioactivity were observed: one at approximately 60 kDa corresponding to unliganded OTRs, and another ranging from 240 to 320 kDa associated with OTRs coupled to G proteins.
The addition of oxytocin increased binding of a specific labeled compound up to twofold in a dose-dependent manner, indicating active signaling through the coupled receptors.
Significant amounts of G proteins, specifically G alpha q/11 and G alpha i3, were found associated with the OTR, suggesting their roles in receptor signaling.
Oxytocin stimulation of a specific enzyme activity in myometrial membranes was inhibited by pertussis toxin, indicating a coupling of OTR to Gi proteins.
The presence of the 60-kDa OTR was noted before the end of gestation and after parturition, while the majority of receptors shifted to the larger complex at term.

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Occupancy of oxytocin receptor (OTR) binding sites in pregnant rat myometrial membranes with iodinated oxytocin antagonist (OTA), followed by detergent solubilization and size selection, showed that radioactivity eluted in two distinct peaks: one corresponding in size to the isolated receptor (approximately 60 kDa) and the other ranging from 240 to 320 kDa. The unliganded 240- to 320-kDa fraction contained OTRs coupled to G proteins, as the addition of oxytocin (OT) increased guanosine 35S-labeled 5'-O-(3-thiotriphosphate) binding up to twofold in a dose-dependent manner. The effects of OT were blocked by coincubation with OTA. G protein alpha-subunits associated with OTRs in the 240- to 320-kDa peak were identified by immunoadsorption. Significant amounts of both G alpha q/11 and G alpha i3 were associated with the OTR; a lesser amount of G alpha s was complexed. Using the same approach but with antibodies to effector enzymes, we observed that phospholipase C beta 1 (PLC beta 1) and PLA2 were also associated with the OTR. The results corroborate the well-established interaction of OTR with Gq and further show that Gi coupling might be an important component of OTR signal transduction. To further investigate the interaction of Gi with the OTR, we showed that OT stimulation of guanosine 5'-triphosphatase activity in intact myometrial membranes was inhibited by pertussis toxin. Pertussis toxin-stimulated ADP ribosylation of G alpha i in myometrial membranes was also decreased by OT treatment. These findings with pertussis toxin strongly indicate that OTR is coupled to Gi in rat myometrial membranes. The 60-kDa OTR peak (noncoupled receptor) was demonstrable in the myometrium only before the end of gestation and after parturition and accounted for about one-half the 125I-OTA binding activity. At term, there was about a fivefold increase in binding and almost a complete shift to the 240- to 320-kDa-size complex. Thus the established increased sensitivity of the myometrium to OT at term could be the result of both upregulation of OTRs and an increase in the fraction of receptors coupled to signal transduction components, one of which is Gi.

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Coupling of oxytocin receptor to G proteins in rat myometrium during labor: Gi receptor interaction

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