Estrogen receptor in chicken oviduct: Receptor dissociation kinetics and transformation

Abstract

Cytosolic and nuclear estrogen receptor forms of chicken oviduct have been studied by (1) measuring hormone dissociation kinetics and by (2) sucrose density gradient analysis on high salt gradients. Estradiol dissociates from the receptor in chicken oviduct cytosol at 22°C following a two-phase exponential process. The fraction of receptor with a fast dissociation rate ( k = 120 × 10 −3min −1) decreases as a function of the pre-incubation at 22°C: after prolonged pre-incubation only the slowly dissociating ( k = 12.3 × 10 −3min −1) form remains. Dissociation of moxestrol, a synthetic estrogen with a higher affinity, from the cytosol receptor at 30°C is similar, showing a transition of a fast dissociating form ( k = 120 × 10 −3min −1) to a slowly dissociating form ( k = 7.6 × 10 −3min −1) as a result of pre-incubation at 30°C. A concomitant temperature-dependent shift of the estrogen receptor from a 4.8 S to a 6.1 S form was observed with moxestrol but not with estradiol as a ligand. Sodium molybdate (20 mM) and NaSCN (400 mM) inhibit the temperature-dependent increase in sedimentation coefficient, but molybdate allows the formation of a receptor form which shows intermediary dissociation kinetics. Estrogen receptor, precipitated with ammonium sulfate (0–35%) shows monophasic dissociation kinetics of estradiol ( k =39.5 × 10 −3min −1) and for moxestrol ( k = 10.8 × 10 −3min −1), suggesting full receptor activation only with moxestrol as a ligand. Moxestrol-receptor complexes obtained by ammonium sulfate precipitation sediment at 0°C at 4.8 S. Only after subsequent incubation at 30°C a shift from 4.8 S to 5.9 S is observed, suggesting that the formation of the slowly dissociating form of the receptor may precede the formation of a stable transformed receptor complex. The nuclear estrogen receptor with estradiol as a ligand shows biphasic dissociation kinetics at 22°C ( k = 10 × 10 −3min −1; k = 14.0 × 10 −3min −1). The ratio of both components (1:1) does not change after preincubation of the nuclear receptor extract at 22°C. Moxestrol dissociates from the nuclear receptor at 30°C monophasically with a slow rate ( k = 6.1 × 10 −3min −1), suggesting that it is extracted as an activated hormone-receptor complex. Our data show that in vitro the cytosolic estrogen receptor can undergo a temperature-dependent transition from a low to a high affinity form. The in vitro transformation to a form with a higher sedimentation coefficient appears to be dependent on the type of ligand bound to the receptor. The receptor extracted from the nuclei is predominantly present in the higher affinity state.