COUPLING OF OXYTOCIN RECEPTOR TO G-PROTEINS IN RAT MYOMETRIUM DURING LABOR - G(I) RECEPTOR INTERACTION

Occupancy of oxytocin receptor (OTR) binding sites in pregnant rat myo metrial membranes with iodinated oxytocin antagonist (OTA), followed b y detergent solubilization and size selection, showed that radioactivi ty eluted in two distinct peaks: one corresponding in size to the isol ated receptor (similar to 60 kDa) and the other ranging from 240 to 32 0 kDa. The unliganded 240- to 320-kDa fraction contained OTRs coupled to G proteins, as the addition of oxytocin (OT) increased guanosine S- 35-labeled 5'-O-(3-thiotriphosphate) binding up to twofold in a dose-d ependent manner. The effects of OT were blocked by coincubation with O TA. G protein alpha-subunits associated with OTRs in the 240- to 320-k Da peak were identified by immunoadsorption. Significant amounts of bo th G(alpha q/11) and G(alpha i3) were associated with the OTR; a lesse r 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 PLA(2) were also associated with the OTR. The resu lts corroborate the well-established interaction of OTR with G(q) and further show that G(i) coupling might be an important component of OTR signal transduction. To further investigate the interaction of G(i) w ith the OTR, we showed that OT stimulation of guanosine 5'-triphosphat ase activity in intact myometrial membranes was inhibited by pertussis toxin. Pertussis toxin-stimulated ADP ribosylation of G(alpha i) in m yometrial membranes was also decreased by OT treatment. These findings with pertussis toxin strongly indicate that OTR is coupled to G(i) in rat myometrial membranes. The 60-kDa OTR peak (noncoupled receptor) w as demonstrable in the myometrium only before the end of gestation and after parturition and accounted for about one-half the I-129-OTA bind ing 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 th e fraction of receptors coupled to signal transduction components, one of which is G(i).