Endocrinology, Vol 123, 2540-2548, Copyright © 1988 by Endocrine Society

ARTICLES

Estradiol-stimulated proteolytic cleavage of the estrogen receptor in mouse uterus

T Horigome, F Ogata, TS Golding and KS Korach
Receptor Biology Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.

Increased proteolytic degradation of the estrogen receptor (ER) was detected in uterine cytosol of estradiol-treated ovariectomized mice compared to saline controls. Estradiol had no direct effect on the proteinase activity or susceptibility of the ER to the enzyme. The proteolytic activity gradually increased after a single injection of estradiol with early increases at 2 and 8 h followed by a progressive increase which reached a maximum at 36 h. The proteinase(s) activity resulted in cleavage of the native ER form of 65,000 (65 K ER) to a product of limited proteolysis having an apparent molecular weight of 54,000 (54 K ER). The pH optimum for this proteinase activity was 6.0. The proteinase was inhibited by 2.5 mM p-chloromercuribenzoic acid and 2.5 mM p-chloromercuriphenylsulfonate and partially inhibited by 2.5 mM iodoacetamide but not by 1 mg/ml leupeptin, 0.1 mg/ml antipain, 0.1 mg/ml chymostatin, 0.1 mg/ml pepstatin, 0.1 mg/ml E-64, 2.5 mg/ml soybean trypsin inhibitor, 2.5 mM phenylmethylsulfonylfluoride, 2.5 mM diisopropylfluorophosphate, and 10 mM EGTA. The results suggested that the proteinase(s) had a thiol group essential for its activity. Estrogen receptor in the mouse uterine cytosol fraction appears to be degraded sequentially in two steps in which 65 K ER is cleaved to a 54 K ER which upon longer incubation is further degraded to a 37 K form. The second step was inhibited by leupeptin, antipain, chymostatin, E- 64, and p-chloromercuribenzoic acid. A possible function of the 54 K ER under physiological conditions is discussed since the 54 K ER was also found in nuclear samples. This form of the ER still retains the ability to bind estradiol and DNA.

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