ATP mediated receptor interconversion as a model of estrogen action: Isolation of the factor which converts the non-estrogen binding form of the receptor to the lower affinity binding form

Abstract

Two steroid binding states of an estrogen receptor each with different equilibrium constants ( K d values) R x ( K d = 0.06 nM) and R y , ( K d = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third non-estrogen binding form of the receptor, designated R nb , has also been identified. These three forms of the receptor are interconvertible and appear to have a common molecular weight of approx. 66,000 under denaturing conditions. Hydroxytamoxifen binds preferentially and with high affinity to R x ( K d 0.03 nM) and the conversion of R x to R y which is mediated by gamma phosphoryl group of ATP is also inhibitable by hydroxytamoxifen. Thus receptor interconversion, which may have general application to hormone action, potentially explains agonist/antagonist activity. The conversion of the non-estrogen binding form of the receptor ( R nb ) to the lower affinity receptor ( R y ) in chick oviduct cytosol is catalyzed by a reaction requiring the loss of the terminal phosphoryl moiety from ATP. There is a specific requirement for Mg 2+. We now describe that ammonium sulfate fractionation of the cytosol allows the separation of the receptor entities from the “activating factor” ( F y ) that catalyzes the conversion of R nb to R y In the presence of gamma [ 32P]-ATP at 30°C the purified non-steroid binding form of the receptor is phosphorylated. Phosphoamino acid analysis using Partisil-10 SAX anion exchange resin demonstrates that a serine is phosphorylated; and quantitation of the phosphorylation is indicative of one phosphoserine/receptor molecule. Treatment of the receptor with the partially purified activating factor to induce estradiol binding causes a dramatic reduction in phosphorylation.