ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 325, No. 1, January 1, pp. 47–57, 1996 Article No. 0006 Plasma Membrane Is the Primary Site of Localization of the Nonactivated Estrogen Receptor in the Goat Uterus: Hormone Binding Causes Receptor Internalization Narayanan Karthikeyan and Raghava Varman Thampan 1 Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500 134, India Received June 15, 1995, and in revised form October 2, 1995 the literature (4–9) in that it does not get ‘‘activated’’ Evidence is presented to show that the cellular site during salt-dependent or temperature-dependent trans- of localization of the goat uterine nonactivated estro- formations. As a result, its partner in the heterodimer, gen receptor (naER) is the plasma membrane. Expo- the DNA binding protein, has been named the estrogen sure of purified plasma membrane preparations to es- receptor activation factor (E-RAF) 2 in view of the fact tradiol (20 nM) causes dissociation of the receptor from that it provides the naER with access to the nuclei. the membrane into the medium. This receptor move- The E-RAF has no capacity to bind estradiol. ment takes place in the presence of diethylstilbestrol The naER was identified as possessing a tyrosine while testosterone, progesterone, dexamethasone, and kinase activity capable of affecting both auto- and ca- tamoxifen do not facilitate the dissociation of the sein phosphorylation (3). During the course of the pres- naER from the plasma membrane. Further, tamoxifen ent study an increase in the yield of the purified protein inhibits the estradiol-mediated dissociation of the was noticed when the tissue was homogenized in a me- naER from the plasma membrane. The naER is a glyco- dium containing Triton X-100. This observation, cou- protein and is also a tyrosine kinase. The tyrosine ki- pled with the assumption that, being a tyrosine kinase, nase activity is inhibited in the presence of estrogens the naER could be grouped in a family of proteins that while tamoxifen reverses this estrogen-dependent in- represents growth factors and oncoproteins, prompted hibition.  1996 Academic Press, Inc. us to examine whether naER is a plasma membrane Key Words: nonactivated estrogen receptor; plasma constituent. membrane; glycoprotein; tyrosine kinase. MATERIALS AND METHODS 2,4,6,7- 3 H-estradiol-17b (sp act 101 Ci/mmol) was purchased from Amersham, England. ATP, unlabeled estradiol, diethylstilbestrol, The recent recognition of the involvement of receptor testosterone, progesterone, dexamethasone, tamoxifen, Triton X-100, heterodimers in the action of various hormones which Tyrophostin-25, 4-chloro-1-naphthol, phosphoamino acids, Tween 20, include thyroid hormone, vitamin D 3 , and retinoic acid Nonidet P-40, and methyl-a-D-glucopyranoside were obtained from (1, 2) serve to point out the possibility that alternate Sigma, U.S.A. Phosphocellulose and cellulose cc-31 were purchased from Whatman, England. [ 32 P]Adenosine 5-triphosphate (sp act receptor forms in addition to the regular nuclear recep- 3000 Ci/mol) was purchased from the Board of Radiation and Isotope tor homodimer may be required for the initiation of the Technology, Bhabha Atomic Research Centre, Bombay. Goat anti- gene regulatory events in a given hormone-sensitive rabbit IgG, horseradish peroxidase-coupled goat anti-rabbit IgG, and system. We, in our laboratory, have been focusing our concanavalin A were obtained from Genei, Bangalore. Sepharose 4B attention on an estrogen receptor heterodimer in which and Sephadex G-100 were purchased from Pharmacia, Sweden. All routine chemicals, of reagent grade, were purchased from local com- one monomer binds the hormone while the other binds mercial establishments. to the DNA. This receptor was named the nonactivated estrogen receptor (3) since it did not display a DNA binding property under any circumstance. This protein 2 Abbreviations used: naER, nonactivated estrogen receptor; E- is different from the proteins bearing similar names in RAF, estrogen receptor activation factor; ER, estrogen receptor; hsp90, heat shock protein 90; DES, diethylstilbestrol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HRPO, 1 To whom correspondence should be addressed at Rajiv Gandhi horseradish peroxidase; HAP, hydroxylapatite; PMSF, phenylmeth- ylsulfonyl fluoride; NP-40, Nonidet P-40; SDH, succinic dehydroge- Centre for Biotechnology, Jagathy, Thiruvananthapuram 695 014, Kerala, India. Fax: 0091-471-69472. nase; G-6-Pase, glucose 6-phosphatase; BSA, bovine serum albumin. 47 0003-9861/96 $12.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.