Z. Strakova et Ms. Soloff, COUPLING OF OXYTOCIN RECEPTOR TO G-PROTEINS IN RAT MYOMETRIUM DURING LABOR - G(I) RECEPTOR INTERACTION, American journal of physiology: endocrinology and metabolism, 35(5), 1997, pp. 870-876
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).