Estradiol receptor has proteolytic activity that is responsible for its own transformation.

Abstract

We have investigated the effect of various protease inhibitors and substrates on the hormone-and temperature-dependent binding of partially purified estradiol-receptor complex to isolated nuclei. Only serine protease substrates and inhibitors significantly depressed estradiol receptor transformation. At 20°C, we observed 50% inhibition with about 3 IAM aprotinin or with 1.4 mM dilsopropyl fluorophosphate. Aprotinin also blocked those size and charge modifications of receptor that are characteristic of the transformation process. The estradiol receptor was able to bind to aprotininagarose only under transforming conditions; i.e., the interaction was hormone-and temperature-dependent and inhibited by molybdate. Diisopropyl fluorophosphate, a covalent reagent for serine esterases, competitively inhibited the binding and specifically eluted the estradiol-receptor complex that had been bound to aprotinin-agarose. These results indicate that estradiol receptor transformation is due to the effect of a serine protease and that the receptor itself is endowed with this catalytic activity, which is triggered by the steroid.Transformation of steroid receptors has been widely studied in cell-free systems and has been defined as that process which confers affinity to the steroid-receptor complex for nuclei and DNA. In all systems so far investigated, transformation is accompanied by a decrease in the molecular size and the net electric charge of the receptor. Receptor transformation is an irreversible first-order reaction, inhibited by millimolar concentration of molybdate, and is triggered by the specific steroid (for review, see refs. 1-3). Evidence for the physiological role of this reaction (4-7) supports the theory that transformation is the critical step in steroid hormone action. Numerous mechanisms have been proposed to explain the molecular basis ofthis event, but none has been definitively accepted.We report here that the receptor-transforming activity of exogenous alkaline phosphatase is due to a contaminating proteolytic activity of the enzyme preparation, that serine protease inhibitors and substrates impair transformation of the estradiol receptor, and that estradiol receptor acquires upon transformation a serine binding site for aprotinin. Therefore, we suggest that estradiol receptor transformation is due to the action of a serine protease activated by the hormone and that the catalytic protein is the hormone receptor itself. MATERIALS AND METHODS[2,4,6,7-3H]Estradiol-17P Ci/mmol; 1 Ci = 37 GBq) was from New England Nuclear. Nonradioactive estradiol-17,B was from Calbiochem. Antipain dihydrochloride, soybean trypsin inhibitor (SBTI), pepstatin A, trypsin inhibitor type 11-0 (ovomucoid) from chicken egg white, leupeptin hemisulfate, a1-antitrypsin, tryptophan methyl ester (TrpOMe), phosphoramidone, phenylmethylsulfonyl fluoride, aprotinin, calf intestinal alkaline phosphatase types I-S and VII-S, and p-nitrophenyl phosphate were from Sigma. Nbenzoyl-L-arginine methyl ester (Bz-Arg-OMe), diisopropyl flu...

Coordinated action of IgE and a B-cell-stimulatory factor on the CD23 receptor molecule up-regulates B-lymphocyte growth.

“…From the amino acid sequence deduced from the cDNA clone, it has been proposed that the B-cell IgE receptor contains a potential arginine cleavage site (14,15), and it may be here that the actions of the BCGF and the functional antibodies are being focused. One possible mechanism for the two agonists is that their binding generates an autoproteolytic activity in the receptor molecule, as was recently described for the estradiol receptor following binding to its ligand (34). While we have no information on the fragment of CD23 that presumably remains bound to the cell after cleavage, the released extracellular fragment has been associated with a number of biological activities.…”

Evaluation of estradiol binding in human benign prostatic hyperplasia

“…Earlier studies had indicated that the binding of steroid hormones (48) and nerve growth factor (49) to their respective receptors were inhibited by PMSF. Another serine protease inhibitor, diisopropylfluoro phosphate (DIFP), has been shown to bind to partially purified estrogen receptor from calf uterus (50).…”

-Fetoprotein as a Biologic Response Modifier: Relevance to Domain and Subdomain Structure

“…It is of special interest to the present review that the human estrogen receptor (ER) also has a similar site, which regulates estrogen binding and which structurally resembles the substratebinding site of chymotrypsin (104). More recently, these studies were extended showing that estradiol receptor (ER) transformation was due to an effect of serine protease intrinsic activity of the ER (105) and that aprotinin (a protease inhibitor) can inhibit estrogen binding to that nuclear receptor (106).…”