Proc. Nail. Acad. Sci. USA Vol. 83, pp. 3253-3256, May 1986 Biochemistry Specific binding of human corticosteroid-binding globulin to cell membranes (plasma steroid hormone-binding proteins/membrane receptors/steroid hormone action/prostate) DANIEL J. HRYB*, M. SAEED KHANt, NICHOLAS A. ROMAS*, AND WILLIAM ROSNERtt Departments of *Urology and tMedicine, St. Luke's-Roosevelt Hospital Center, and Columbia University, College of Physicians and Surgeons, 428 West 59th Street, New York, NY 10019 Communicated by Seymour Lieberman, January 21, 1986 ABSTRACT Specific binding sites for corticosteroid-bind- All of the foregoing observations have appeared in a ing globulin were detected on membranes prepared from fragmented way, and no cohesive focus has forced their human prostates. The binding sites are typical of membrane assembly into a meaningful physiological model. We felt that receptors: they are saturable and specific and have high ifthey all were both correct and ofphysiological importance, affinity. There was little specific binding at 40C and 230C. then a membrane receptor for CBG should exist. The Maximal specific binding was obtained at 370C. Scatchard existence of such a receptor would provide both a specific analysis revealed the presence of a single set of binding sites mechanism whereby CBG could be internalized into cells via with an apparent dissociation constant of 8.7 x 10-7 M and a receptor-mediated endocytosis, and a center around which to binding capacity of 22 pmol/mg of membrane protein. The bring together the aforementioned disparate observations. sites were specific for corticosteroid-binding globulin; binding We therefore set out to determine whether there are specific was not inhibited by human testosterone/estradiol-binding cell membrane binding sites for CBG and found that CBG globulin, by albumin, or by transferrin. The density of specific binds saturably and specifically to human prostatic mem- binding sites in membranes obtained from several organs from branes, as well as to membranes from other tissues. A the rhesus monkey is consistent with the hypothesis that preliminary report of some aspects of this study has been corticosteroid-binding globulin is involved in the transport of presented.§ steroid hormones into target tissues. MATERIALS AND METHODS The steroid hormones cortisol and progesterone are secreted Prostatic tissue was obtained from men undergoing transure- into the circulation by the adrenal cortex and ovary, respec- thral prostatectomy (prostate < 65 g) or open prostatectomy tively. They are not in simple solution in the plasma but rather (prostate > 65 g) for benign prostatic hyperplasia and, on two are largely bound to the same trace protein, corticosteroid- occasions, from open prostatectomy on men undergoing binding globulin (CBG) (1-3). The current model of steroid surgery for bladder cancer. Plasma membranes (100,000 x g hormone action envisions that the free (i.e., non-protein- pellet) were prepared by differential centrifugation (21). The bound) steroids diffuse passively across the capillary and cell final 100,000 x g pellet (prostatic membranes) was resus- membranes to the cell's interior where they initiate hormone pended in 1 volume of 25 mM Tris Cl, pH 7.4/10 mM action (4). CaCl2/0.2% bovine serum albumin (buffer A) and either used Over the years, evidence has appeared that indicates it for binding experiments immediately or stored at -700C. might be necessary to modify this model. None of it, Results were the same whether membranes were prepared however, has been either sufficiently convincing or error-free from fresh tissue or from tissue frozen for 2-4 weeks. to upset the free-hormone model. For instance, Rosner and Membranes (100,000 x g fraction) were also prepared from Hochberg (5) provided biochemical evidence years ago that the kidneys, liver, spleen, and uterus of a single adult female CBG-bound cortisol was as active, or possibly more active, rhesus monkey (Macaca mulatta) which, as part of a differ- than free cortisol in the induction of tyrosine aminotrans- ent protocol, had been treated with estradiol valerate (2 ferase activity in rat liver. Others provided biochemical mg/day for 10 days). The rhesus monkey was anesthetized evidence for the presence of CBG or a CBG-like binder in with sodium barbital, after which the desired organs were various glucocorticoid and progesterone target organs: pitu- removed. Membranes were prepared as above. Membrane itary (6, 7), kidney (8), uterus (9-11), muscle (12), lung (13), protein was determined by washing an aliquot of the mem- breast cancer (14), and lymphocytes (15). A CBG-like mol- branes with 0.3 M sucrose and then treating it with 0.1 M ecule has also been identified in plasma-membrane fractions NaOH at 70'C for 30 min. The membrane solution was then from liver (16) and pituitary cells (17). Some of the biochem- centrifuged at 16,000 x g and the protein content of the ical demonstrations have been supported by the immuno- supernatant was ascertained (22). cytochemical visualization of intracellular CBG in liver (18), CBG, containing 1 mol of cortisol per mol of CBG, was uterus (2), kidney (2), and lymphocytes (15); but some of purified from pregnancy plasma by affinity and hydroxylap- these reports have been flawed by the omission of critical atite chromatography (23, 24). Its purity was verified by controls and/or a lack ofevidence that the observations were polyacrylamide gel electrophoresis, both under nondenatur- not due to contamination with plasma (19). More recently, ing conditions and in the presence of NaDodSO4, and by however, in a well-controlled study, guinea pig CBG has been immunoelectrophoresis against antiserum to whole human specifically localized immunohistochemically inside those pituitary cells (corticotrophs) which secrete corticotropin (20). Abbreviations: CBG, corticosteroid-binding globulin; 1251I-CBG, 125I_ labeled CBG; TEBG, testosterone/estradiol-binding globulin. tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge §Hryb, D. J., Khan, M. S., Romas, N. & Rosner, W., the 67th payment. This article must therefore be hereby marked "advertisement" Annual Meeting of. the Endocrine Society, June 19-21, 1985, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Baltimore, MD, abstr. 244. 3253 Downloaded by guest on September 30, 2021 3254 Biochemistry: Hryb et al. Proc. Natl. Acad. Sci. USA 83 (1986) serum. It was iodinated, using chloramine-T, to a specific indicates that there was minimal binding at 4°C or at 23°C. activity of0.1-0.3 mol of'251 per mol of CBG (24). To be sure Binding increased with time at 37°C and began to plateau at6 hr, that the iodinated CBG was native, over 90% was shown to which was chosen as the incubation time for subsequent be specifically bound by a monospecific anti-CBG antiserum experiments. These data are shown again in the main figure, before using it in receptor binding experiments (data not where total and nonspecific binding are also indicated. shown). Human testosterone/estradiol-binding globulin Fig. 2 is a representative Scatchard analysis (28) of the (TEBG) and albumin were isolated as described (25-27). specific binding of CBG to prostatic membranes at 37°C. The Human transferrin was purchased from Sigma. data are fit best by a straight line and indicate that CBG is Binding took place in 1.5-ml Eppendorf polypropylene bound to a single set of sites. The inset is a saturation plot centrifuge tubes that were pretreated with 1.0 ml of buffer A using the same data as was used for the Scatchard analysis. for 1 hr at 370C to decrease test tube blanks. 125I-labeled CBG Using the computer program LIGAND (29), we analyzed (125I-CBG, 250,000-300,000 cpm per tube, about 2 nM), plus four experiments simultaneously. The analysis yielded an or minus 14 ,M radioinert CBG, was incubated with 100-200 association constant (Ka) of 1.16 ± 0.26 (SEM) x 106 M-1 and ,ug of prostatic membrane protein in 25 mM Tris Cl, pH a binding capacity of 22 pmol/mg of membrane protein. 7.4/25 mM CaCl2/0.2% bovine serum albumin, in a final To determine the ligand specificity of the receptor, the volume of 0.25 ml. All assays were done in triplicate, and ability ofradioinert CBG to compete with the specific binding membrane-bound 125I-CBG was separated from free by of '25I-CBG to prostatic membranes was compared with that centrifugation for 10 min at 40C. After the supernatants were of TEBG and two other human plasma proteins, transferrin aspirated, the tip ofeach tube was cut 0.5 cm from the bottom and albumin (Fig. 3). None of these proteins significantly and the 1251I therein was measured in a gamma counter at an inhibited specific CBG binding to the membranes, attesting to efficiency of 72%. Test tubes without membranes served as the specificity of the CBG-membrane interaction. blanks. Specific binding was calculated as the difference Human and rhesus monkey (Macaca mulatta) CBGs are between total binding and nonspecific binding, which was immunologically very similar (30). Therefore, in order to that binding seen in the presence of 14 AM radioinert CBG. study the tissue distribution of specific CBG binding, and in Generally, about 2% of the added 125I-CBG was specifically the absence of the ready availability of fresh human tissues, bound. There were no statistically significant differences in we examined CBG binding in membranes freshly prepared binding between membranes prepared from prostates ob- from the kidney, spleen, liver, and uterus ofa rhesus monkey tained by either transurethral or open prostatectomy. (Fig. 4). All four tissues examined bound substantially more CBG than did human prostate. RESULTS DISCUSSION The dependence of the binding of 125I-CBG to human prostatic membranes as a function oftime and temperature is The studies reported here demonstrate that human prostatic shown in Fig.