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We have examined the carbohydrate composition of corticosteroid-binding globulin (CBG) obtained from rat and human serum. Rat CBG contained a carbohydrate composition that was strikingly different from that of human CBG. Like other glycoproteins that circulate in human plasma, human CBG had a carbohydrate composition that was consistent with the presence of biantennary and triantennary oligosaccharide structures. In contrast, the carbohydrate composition of rat CBG indicated the presence of more than one sialic acid residue per antenna. It is not clear whether rat CBG contains a carbohydrate structure with sialic acids attached to both galactose and N-acetylglucosamine on the same antenna, or a terminal disialylated structure (sialic acid linked alpha 2-8 to sialic acid). These structural variations may play a role in the interaction of CBG with its receptor.

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds a number of circulating steroid hormones (testosterone, dihydrotestosterone and estradiol) with high affinity, thus regulating their free concentration in plasma. In addition to binding steroids, SHBG itself binds to receptor sites on plasma membranes with somewhat unusual kinetics. Both the off and on rates are quite slow. The steroid-binding and membrane-binding functions are intertwined in what is clearly an allosteric relationship. Occupation of SHBG's steroid-binding site by a steroid inhibits its ability to bind to its membrane receptor-binding site. This inhibition is not related to a steroid's biological activity. Metabolites of steroids without biological activity, e.g. 2-methoxyestradiol, actively inhibit SHBG's interaction with its membrane receptor. However, if unliganded SHBG is allowed to bind to its receptor on intact cells, and an appropriate steroid hormone then is introduced, adenylate cyclase is activated and intracellular cAMP increases. This function is specific for steroids with biological activity, 2-methoxyestradiol has no activity in this arena. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.

The binding of human sex hormone-binding globulin (SHBG) to a human prostatic cancer cell line (LNCaP) and the results of that binding were examined. Membranes derived from LNCaP cells bound unliganded SHBG on two sets of sites whose affinities were: Ka1 = 3.1 +/- 1.6 x 10(10) M-1 and Ka2 = 8.7 +/- 4.3 x 10(6) M-1. Intact cells also bound SHBG, but even after 6 h, less than 10% of specifically bound SHBG was internalized. This observation speaks against a role for the membrane binding of SHBG in steroid transport across cell membranes. When LNCaP cells were prebound with SHBG, addition of dihydrotestosterone or estradiol resulted in a dose-dependent increase in intracellular cAMP. SHBG in the absence of steroids or dihydrotestosterone in the absence of SHBG was without effect. 2-Methoxyestradiol, a steroid metabolite without biological activity, but which binds to SHBG more tightly than does estradiol, was also without effect. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.

This investigation was undertaken to ascertain whether rat liver cells contained a receptor for corticosteroid-binding globulin (CBG) that could be differentiated clearly from the asialoglycoprotein receptor. To do this, [125I]CBG, [125I] asialo-CBG, and [125I]asialofetuin were used as probes to differentiate the binding activities of the two receptors. On hepatic membranes, CBG bound to a single set of sites with a Kd of 0.74 microM, asialofetuin bound to a single set of sites with a Kd of 0.018 microM, asialo-CBG bound to two sets of sites with Kd values of 0.004 and 1.4 microM and in the presence of 1 microM asialofetium, asialo-CBG bound to a single set of sites with a Kd of 0.53 microM, not different (P greater than 0.2) from the Kd of CBG. Cross-competition studies using the three 125I-labeled ligands and allowing each to compete with the three radioinert ligands indicated the existence of two separate receptors. Desialylation of hepatic membranes differentially affected the binding of CBG and asialofetuin. Finally, whole cells bound CBG specifically, but internalized it to only a minimal extent (less than 10%). This observation does not support a role for the CBG-receptor system in the entry of steroids into cells.

Sex hormone-binding globulin is a plasma glycoprotein that binds certain estrogens and androgens with high affinity. Over the past several years it has been shown that, in addition to functioning as a regulator of the free concentration of a number of steroid hormones, SHBG plays a central role in permitting certain steroid hormones to act without entering the cell. The system is complex. SHBG interacts with a specific, high affinity receptor (R(SHBG)) on cell membranes that appears to transduce its signal via a G protein. The SHBG-R(SHBG) complex causes the activation of adenylyl cyclase and the generation of cAMP within a matter of minutes after exposure to an appropriate steroid. Only steroids that bind to SHBG can activate SHBG-R(SHBG), but not all steroids that bind have this function, e.g. are agonists. All steroids that bind to SHBG but do not activate adenylyl cyclase are antagonists. The signals generated by the steroid-SHBG-R(SHBG) complex generate messages that have effects on the transcriptional activity of classic, intracellular receptors for steroid hormones. These and other downstream effects of this system are reviewed.

We have studied the concentration and properties of a protein which binds cortisol in human milk in samples obtained from women during the first 100 days after delivery. A filter disk assay was developed both for the measurement of plasma corticosteroid-binding globulin (CBG) and for the cortisol-binding protein in milk. The concentration of CBG in milk, expressed as its capacity to bind cortisol, is highest on the day of delivery, ca, 0.80 mug/dl, falls over the next 10 days to ca. 0.25 mug/dl, and remains at that level thereafter. If the concentration of CBG is expressed relative to the concentration of serum albumin in milk, it increases from day 1 to day 3 and then remains constant. A detailed comparison of CBG derived from milk and plasma showed that the two proteins co-migrated on Sephadex, sucrose gradient ultracentrifugation, and polyacrylamide gel electrophoresis. The two proteins had the same affinity for cotrisol, progesterone, 17-OH-progesterone, and dexamethasone. Furthermore, the binding activity of CBG in milk was neutralized with anti-CBG antibodies raised against CBG isolated from plasma. Unlike CBG, the concentration of cortisol in milk, 0.8-3.5 mug/dl, showed no systematic variation as a function of the postpartum day on which the sample was obtained.

Specific binding sites for corticosteroid-binding globulin (CBG) were detected on membranes prepared from rat spleen. The binding sites are typical of membrane receptors; they are saturable, specific, have high affinity, and require Mg2+ or Ca2+ for binding. There was little specific binding at 4 C, and maximal binding was obtained at 37 C. Scatchard analysis revealed a single set of binding sites with an apparent Kd of 0.84 microM, and a binding capacity of 39 pmol/mg membrane protein. The sites were specific for CBG; binding of [125I]CBG was not inhibited by a 10,000-fold excess of either rat albumin or rat transferrin. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the membrane-bound [125I]CBG revealed the presence of lower mol wt iodinated products which were undetectable in the unbound [125I]CBG fraction. Further, whereas the electrophoretic patterns from uterine, pulmonary, and renal membranes showed that the less mobile band (mol wt, 60K) of the normal CBG doublet appeared to be metabolized to a greater extent than the more mobile band (mol wt, 52K), those from splenic membranes showed equal metabolism of both parts of the doublet.

We isolated testosterone-estradiol-binding globulin TeBG rapidly and in high yield from pooled pregnancy plasma. It showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Both bands stained with three different monoclonal antibodies to TeBG, thus demonstrating their immunological similarity. Freshly drawn, individual sera, from men, women, and pregnant patients were submitted to microaffinity chromatography, a procedure which partially purifies TeBG in approximately 4 hr. The partially purified plasma was submitted to SDS PAGE, followed by immunoblotting. The blotted TeBG exhibited the same two bands seen in the isolated, purified protein. The size heterogeneity observed in TeBG purified to: proteolysis occurring during isolation; a peculiarity of pregnancy plasma; or heterogeneity attendant upon the use of pooled plasma for isolation.

Because it no longer seemed reasonable to us that the sole function of the steroid-binding proteins in plasma was to serve as a buffer reservoir for steroid hormones, we conducted experiments which sought out other possibilities. Both CBG and SHBG bind to cell membranes, and this interaction partakes of the general characteristics of peptide hormone-membrane receptor systems. Additionally, human CBG has the ability to cause an increase in the activity of membrane-bound adenylate cyclase in MCF-7 cells, and this, in turn, results in an increase in cellular cAMP content. Thus, CBG appears to be a protein hormone. As a first consideration, one might presume that because CBG's half-life is measured in days, it would be counted among the hormones which, for the most part, are tonic in their effects, e.g., thyroid hormone. However, two important considerations tend to believe this presumption: (1) CBG which is unoccupied by steroid is not hormonally active (Figure 5): (2) Depending upon the time of day, circulating CBG is approximately 0-60% occupied in normal humans. These observations result in a circumstance in which a substantial portion of circulating CBG is available for activation by bursts of cortisol secretion. It seems prudent to speculate that, because steroids are essential for CBG's activity, the hormonal role of CBG may be entwined with, or complementary to the steroids which it binds. Finally, we should comment on the impact that our model of CBG as a hormone has on the view that only unbound steroid can be hormonally active. First, it should be stated that we have not addressed this question experimentally. Although there is evidence that CBG may be required for cortisol action, we feel that an obligate role for it is not documented adequately. At this time, we believe that CBG's hormonal role is compatible with a hypothesis that encompasses the view that unbound steroid hormones can diffuse into cells in some tissues and that both free and bound steroid can enter cells in others. Obviously, the final word on these important topics, as always, awaits the proper experiments.

• The iodinated antiarrhythmic drug amiodarone frequently causes an elevation of the serum thyroxine (T 4 ) level in patients who remain clinically euthyroid. Less frequently, true iodine-induced hyperthyroidism may occur. The clinical and laboratory distinction between these two conditions is often difficult. Since the serum sex hormone—binding globulin (SHBG) concentration is elevated in hyperthyroidism, this study was carried out to evaluate the serum SHBG concentration as a possible marker of hyperthyroidism in patients receiving amiodarone. Patients treated with amiodarone were divided into three groups: clinically euthyroid with normal serum T 4 and triiodothyronine (T 3 ) concentrations, clinically euthyroid with elevated serum T 4 and normal T 3 concentrations, and clinically hyperthyroid with elevated serum T 4 and T 3 concentrations. The mean serum SHBG concentration was significantly elevated in amiodarone-induced hyperthyroid patients, while it was normal in euthyroid patients treated with amiodarone who had normal or elevated serum T 4 concentrations. The results suggest that the hyperthyroxinemia induced by amiodarone is not associated with excess thyroid hormone action in the liver unless the serum T 3 concentration is also elevated. ( Arch Intern Med 1987;147:1781-1785)

We isolated testosterone-estradiol-binding globulin (TeBG) from human pregnancy plasma and used the isolated protein to obtain a rabbit antiserum to it. Using immunochemical techniques, the antiserum was demonstrated to be both monospecific and to have the ability to remove TeBG-binding activity from human serum. This antiserum was used to develop a RIA for human TeBG. The sensitivity is such that TeBG can be determined in as little as 0.005 microliter serum or plasma, although we routinely use 0.5-1.0 microliter. The correlation between the RIA and an established binding assay, using 63 different specimens, was 0.995.

We used immunological techniques to compare the serum corticosteroid-binding globulins (CBG) and testosterone-estradiol-binding globulins (TeBG) of Old World primates (man, chimpanzee, cynomologus, and rhesus), New World monkeys (squirrel and owl), and prosimians (galago and lemur). Four different antihuman TeBG antisera could not differentiate human and chimpanzee TeBG and recognized the galago and lemur TeBG as similar as well as the rhesus and cynomologus TeBG, as similar. Western blots of serum subjected to sodium dodecyl sulfate gel electrophoresis, with detection by an anti-TeBG antiserum, showed similar patterns of distribution of the two molecular species of TeBG for all of the New World primates and the owl monkey. The abundance of the two TeBG species was reversed in squirrel monkey serum, while lemur and galago displayed only a single band. Four different antihuman CBG antisera grouped together the CBGs of human and chimpanzee, rhesus and cynomologus, and lemur and galago. The squirrel monkey has a CBG with a markedly decreased affinity for cortisol; all four antisera perceived its CBG as much more immunologically distant from the human protein than that of the owl monkey. Indeed, three of the four antisera grouped squirrel monkey CBG with that of the prosimians, while one antiserum saw squirrel monkey CBG as even more distant from the human protein than the CBG of the primitive primates, the prosimians.

We examined the influence of numerous substances on the secretion of corticosteroid-binding globulin (CBG) and testosterone-estradiol binding globulin (TeBG) by a human hepatoma-derived cell line. Thyroxine, at physiologic concentrations, resulted in an increased secretion of TeBG but not CBG. Estrogens, antiestrogens, and androgens were without effect on either of these binding proteins.

Corticosteroid-Binding globulin (CBG) is a plasma protein that binds certain steroid hormones, mainly cortisol and progesterone. It has been demonstrated recently that specific binding sites for this protein exist on cell membranes. In this communication we establish that binding to these sites results in the induction of adenylate cyclase activity and the accumulation of cAMP in MCF-7 cells. These events are critically dependent upon a steroid being bound to CBG. These data are consistent with the hypothesis that CBG is a prohormone which is activated when cortisol is bound to it.