Sex hormone-binding globulin is synthesized in target cells.

Sex hormone-binding globulin (SHBG) is a multifunctional protein that acts in humans to regulate the response to steroids at several junctures. It was originally described as a hepatically secreted protein that is the major binding protein for sex steroids in plasma, thereby regulating the availability of free steroids to hormone-responsive tissues. SHBG also functions as part of a novel steroid-signaling system that is independent of the classical intracellular steroid receptors. Unlike the intracellular steroid receptors that are ligand-activated transcription factors, SHBG mediates androgen and estrogen signaling at the cell membrane by way of cAMP. We have reviewed the current state of knowledge on the SHBG gene and the role of SHBG in steroid signaling (we shall not address its function as a plasma-binding protein).

Sex hormone-binding globulin: anatomy and physiology of a new regulatory system.

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.

Sex hormone-binding globulin mediates steroid hormone signal transduction at the plasma membrane.

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.

Effects of sex hormone binding globulin (SHBG) on human prostatic carcinoma.

The purpose of this study was to determine what effects sex hormone binding globulin (SHBG) might have on the growth and steroid content of human prostate carcinoma. Two human prostate carcinoma cell lines were used for this study, ALVA-41 and ALVA-101. The first part of the study was to determine the effect of SHBG or albumin on the uptake of [3H]DHT in the cells. In this experiment both SHBG and albumin inhibits the uptake of [3H]DHT into each of the cell lines when studied in vitro. The degree of inhibition was dependent on the binding capacity of the protein. When [3H]thymidine uptake was measured in each of the cell lines following either the addition of SHBG or albumin to the culture media, an increase in uptake and presumably DNA synthesis was noted in the ALVA-41 and ALVA-101 cells for SHBG additions but not for albumin. Further, this stimulation was increased when testosterone was added to the media, however, [3H]thymidine uptake was decreased by high concentrations of dihydrotestosterone (DHT) or if the SHBG was saturated with DHT prior to being added to the media. The cells also demonstrate high affinity cell membrane receptors for SHBG. Finally, using a 3', 550 bp cDNA or SHBG, 1.9 and 2.8 kb mRNAs were detected on Northern analysis of the ALVA-101 and ALVA-41 cells. These data indicate SHBG can inhibit uptake of steroids into the prostate, but also it may act as a stimulus for growth through a SHBG cell surface receptor. In addition, the growth effect may be through an autocrine effect from SHBG or a SHBG-related peptide.

Are corticosteroid-binding globulin and sex hormone-binding globulin hormones?

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.

Sex hormone-binding globulin receptor signal transduction proceeds via a G protein.

The plasma protein, sex hormone-binding globulin (SHBG) binds to a receptor (R(SHBG)) on cell membranes to form an SHBG-R(SHBG) complex. When an appropriate steroid binds to this complex, there is a rapid rise in intracellular cyclic adenosine monophosphate (cAMP). Although the system is moderately well characterized, the molecular cloning of R(SHBG) has not been accomplished and there is a paucity of evidence regarding the mechanism of transmission of the R(SHBG) signal. In this communication, we offer two independent lines of evidence that a G protein is involved in R(SHBG) signal propagation. Exposure of cell membranes containing R(SHBG) to a non-hydrolyzable analog of guanosine triphosphate (guanylyl-5'-imidodiphosphate) caused a substantive decrease in the binding of SHBG to R(SHBG). This behavior is typical of membrane receptors coupled to G proteins and has been used by others as evidence to support that relationship. Another set of experiments involved the assumption that, if R(SHBG)-induced increases in cAMP were diminished when the wild-type alpha subunit of a G protein was replaced with mutants that were inefficient/ineffective in signal transduction, then the idea that G proteins were involved in that signal would be buttressed. Hence, we infected COS-1 cells with a construct containing such mutants, along with a cAMP response element reporter, and demonstrated a marked decrease in R(SHBG)-engendered reporter activity, e.g. cAMP generation.

Androgen and estrogen signaling at the cell membrane via G-proteins and cyclic adenosine monophosphate.

Androgens and estrogens are well-known to initiate their actions by binding to specific intracellular receptors. The steroid-receptor interaction, the receptors, and the details of transcriptional activation consequent to the binding of these steroids with their respective receptors have been, and continue to be, intensively studied. More recently, it has become increasingly apparent that steroids may interact with cells by other than this classic pathway. This communication will deal with activation by sex hormones of a signal transduction pathway that originates at the cell membrane and utilizes cyclic adenosine monophosphate (cAMP) as a second messenger. The system consists of three components, an agonist steroid, sex hormone-binding globulin (SHBG), and a membrane receptor (R(SHBG)) for SHBG. SHBG is a well-characterized plasma protein that has two binding sites, one binds certain estrogens and androgens, and the other binds to R(SHBG). The characteristics of this novel signal transduction system, from the interaction of SHBG with R(SHBG), to the intermediacy of G-proteins, to cAMP generation, to downstream effects of the second messenger will be reviewed.

Histidase mRNA. Nature of translational products, tissue specificity, and differential development in male and female rat liver.

Histidase is expressed in only two tissues of the rat, liver and epidermis. Hepatic histidase synthesis and catalytic activity undergo complex sex-specific developmental courses. To determine whether changes in functional histidase mRNA levels underlie this developmental pattern, total cellular RNA was translated in a rabbit reticulocyte cell-free lysate system. Adult liver total cellular RNA directed the synthesis of three translational products immunoreactive with anti-native histidase: a polypeptide of Mr = 75,000 (75K), which corresponds to the in vivo synthesized histidase subunit, and two higher molecular weight proteins, a major and a minor peptide of Mr = 150,000 (150K) and 140,000 (140K), respectively. These latter peptides do not seem to be aggregates or dimers of the 75K polypeptide or precursors which are post-translationally hydrolyzed to Mr = 75,000; their origin and function remain to be clarified. In contrast to in vitro translation of hepatic total cellular RNA, Western blot analysis of liver cytosol confirms the presence of only the 75K histidase subunit, with no evidence of anti-histidase immunoreactive peptides of Mr = 140,000-150,000 synthesized in vivo. Quantitation of the radioactivity in the immunocomplexed 75K histidase subunit, translated using total RNA from livers of fetuses, 19-day-old males, 35-day-old males, adult males and females, and adult kidney and brain (0, 0.007, 0.010, 0.016, 0.031, 0, and 0%, respectively, of total released proteins) indicates that, in general, levels of functional histidase subunit mRNA reflected histidase catalytic activity (0, 0.20, 0.50, 1.01, 3.00, 0 and 0 units/g of tissue) during tissue differentiation and sex specific development. The above data indicate that initial expression and subsequent increases in synthesis and activity of histidase during hepatic differentiation, postnatal development, and sex hormone regulation are due to pretranslationally controlled augmentation in the levels of functional mRNA which specifies the histidase subunit. In tissues which do not express histidase no functional histidase mRNA is evident. The levels of the RNA which translate the combined 140-150K histidase-like polypeptides (0, 0.007, 0.014, 0.035, 0.034, 0, and 0% of released proteins) also paralleled the increase in enzymatic activity during tissue differentiation and development; however, no difference between males and females was evident. The significance of these observations awaits elucidation of the nature of these RNA(s).

Testosterone-estradiol-binding globulin binds to human prostatic cell membranes.

Specific binding sites for human testosterone-estradiol-binding globulin have been found on human prostatic cell membranes. Scatchard analysis reveals both a high and a low affinity binding site for [125I]testosterone-estradiol-binding globulin. The high affinity site is specific for testosterone-estradiol-binding globulin, whereas the low affinity site also binds human corticosteroid-binding globulin and human transferrin.

The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones.

Sex hormone-binding globulins (SHBG) is a plasma glycoprotein that binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to investigate the role of steroids in the interaction of SHBG with its receptor. Because the probe for the interaction of SHBG with its receptor is 125I-SHBG, we first showed that 125I-SHBG binds [3H]dihydrotestosterone (DHT) at 4 degrees C and 37 degrees C with KD values similar to those published previously for pure radioinert SHBG. 125I-SHBG could be prevented from binding to its receptor by a variety of steroids whose relative inhibitory activity (dihydrotestosterone much greater than 2-methoxyestradiol greater than testosterone greater than estradiol much greater than methyltrienolone greater than cortisol) was almost identical to their relative ability to bind to SHBG. Because significant binding of [3H]DHT to the SHBG receptor could not be demonstrated, steroid inhibition of SHBG binding must be noncompetitive. If steroids bound to SHBG prevent binding to the SHBG receptor, then liganded SHBG should have a higher apparent KD for its receptor than unliganded SHBG. This is the case. The KD was 0.86 +/- 0.25 nM for the high affinity receptor site using liganded SHBG and 0.19 +/- 0.024 nM for unliganded SHBG. Thus, only liganded SHBG assumes a conformation that prohibits interaction with the SHBG receptor. However, when unliganded SHBG was prebound to its receptor, it retained its ability to bind [3H] DHT. The model that emerges from these observations is as follows. Unliganded SHBG can bind either steroids or receptor in a reversible reaction; SHBG bound to a steroid cannot bind to the receptor, but unliganded SHBG that first binds to the receptor can subsequently bind steroids.

Specific binding of human corticosteroid-binding globulin to cell membranes.

Specific binding sites for corticosteroid-binding globulin were detected on membranes prepared from human prostates. The binding sites are typical of membrane receptors: they are saturable and specific and have high affinity. There was little specific binding at 4 degrees C and 23 degrees C. Maximal specific binding was obtained at 37 degrees C. Scatchard analysis revealed the presence of a single set of binding sites with an apparent dissociation constant of 8.7 X 10(-7) M and a binding capacity of 22 pmol/mg of membrane protein. The sites were specific for corticosteroid-binding globulin; binding was not inhibited by human testosterone/estradiol-binding globulin, by albumin, or by transferrin. The density of specific binding sites in membranes obtained from several organs from the rhesus monkey is consistent with the hypothesis that corticosteroid-binding globulin is involved in the transport of steroid hormones into target tissues.

Delineation and synthesis of the membrane receptor-binding domain of sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein which binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to identify, isolate, sequence, and synthesize the region of SHBG that interacts with its membrane receptor. To accomplish this, highly purified human SHBG was digested with trypsin. The SHBG-derived tryptic peptides were separated by high performance liquid chromatography. They were evaluated for their ability to compete with 125I-SHBG for binding to the SHBG receptor solubilized from human prostatic membranes. Only a single peptide, corresponding to residues 48-57 of the known sequence of human SHBG, inhibited receptor binding. A synthetic decapeptide with this amino acid sequence also competitively inhibited SHBG binding.

Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate.

The sex hormone-binding globulin (SHBG) receptor was solubilized from the membranes of human prostate glands with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid). The binding activity of the soluble receptor was measured by allowing it to bind to 125I-SHBG and precipitating the complex with polyethylene glycol-8000. The binding activity was stable for at least 4 months at -20 degrees C and had a half-life of 23 days at 4 degrees C. Like the membrane-bound receptor, Scatchard analysis revealed two sets of binding sites for the soluble one. At equilibrium (24 h), the high affinity site had an association constant (KA) of 6.8 x 10(8) M-1 and a binding capacity of 1.4 pmol/mg protein, whereas the low affinity site had a KA of 4.7 x 10(6) M-1 and a binding capacity of 43 pmol/mg protein. At 37 degrees C, the association rate constant (k1) was 8.37 x 10(5) M-1 min-1 and the dissociation rate constant (k2) was 3.43 x 10(-4) min-1. The soluble receptor was retarded on Sepharose CL-6B and had an apparent Mr = 167,000.

The effect of extracts of the roots of the stinging nettle (Urtica dioica) on the interaction of SHBG with its receptor on human prostatic membranes.

Extracts from the roots of the stinging nettle (Urtica dioica) are used in the treatment of benign prostatic hyperplasia. The mechanisms underlying this treatment have not been elucidated. We set out to determine whether specific extracts from U. dioica had the ability to modulate the binding of sex hormone-binding globulin to its receptor on human prostatic membranes. Four substances contained in U. dioica were examined: an aqueous extract; an alcoholic extract; U. dioica agglutinin, and stigmasta-4-en-3-one. Of these, only the aqueous extract was active. It inhibited the binding of 125I-SHBG to its receptor. The inhibition was dose related, starting at about 0.6 mg/ml and completely inhibited binding at 10 mg/ml.