Principles and Practice of Endocrinology and Metabolism



General Features of Hormone Systems and Historical Perspective
Target Cell Concept
Hormone Receptors

General Features

Recognition and Coupling Domains of Receptors

Receptor Occupancy and Bioeffect

Agonist-Antagonist Concept

Regulation of Receptors

Structure of Receptors
Classification of Hormones

Mechanism of Action of Group I Hormones

Mechanism of Action of Group II Hormones
Chapter References

Multicellular organisms use intercellular communication mechanisms to ensure their survival by coordinating the responses necessary for adjusting to constantly changing external and internal environments. Two systems comprising several highly differentiated tissues have evolved to serve these functions. One is the nervous system, and the other is the endocrine system, which classically has been viewed as using mobile hormonal messages that are secreted from one gland or tissue to act on a distant tissue. There is an exquisite convergence of these regulatory systems. For example, neural regulation of the endocrine system is important; many neurotransmitters resemble hormones in their synthesis, release, transport, and mechanism of action; and many hormones are synthesized in the nervous system (see Chap. 175). The focus of this chapter is the endocrine system and how hormones work.
The word hormone is derived from a Greek term that means to arouse to activity. Classically defined, a hormone is a substance that is synthesized in one organ and transported by the circulatory system to act on another tissue. However, this original description is too restrictive, because hormones can act on adjacent cells (i.e., paracrine action) and on the cell in which they were synthesized (i.e., autocrine action) without entering the circulation.
Early studies concentrated on defining the endocrine action of hormones by removing or ablating an organ to localize the site of production. An extract of the tissue was then used to restore the function, and this served as a bioassay for subsequent purification of the hormone and the elucidation of physiologic and biochemical actions. This classic era of the study of hormonal action was descriptive. During this period, many hormones were discovered, and their major effects were defined. Because it was assumed that hormones had a unique source and a single or predominant action, they were named for the tissue of origin (e.g., thyroid hormone) or for the action (e.g., growth hormone).
The next era of investigation of hormonal action was characterized by the discovery of many more hormones and by a more detailed analysis of how hormones work. The investigation of their functions was aided by methods and ideas previously exploited by endocrinologists, including the use of radioisotopes, the concept of turnover, improved means of purifying molecules, and the availability of sophisticated analytic machinery. Such studies changed the direction of research in hormonal action from a descriptive (i.e., organ or tissue) to a mechanistic (i.e., molecule or function) approach. Where a molecule worked was no longer as important as how it acted. A single hormone could have hemocrine (i.e., transportation through the blood), paracrine, or autocrine actions but affect the different target cells in a similar way, and some effects could be produced by a variety of hormones. For example, naming a single molecule the “growth hormone” was incorrect, because this hormone is but one of several—including the thyroid hormones, sex hormones, glucocorticoids, insulin, and various growth-promoting polypeptides—that are involved in growth, and growth promotion is only one of the actions of the so-called growth hormone.
The principles of hormone synthesis, storage, secretion, transport, metabolism, and feedback control were established during this period. A major contribution was the elaboration of the concept of hormone receptors, and of the properties of specificity and selectivity of response, how target cells are defined, how responses are modulated, how signals are transduced from the outside of a cell to its interior, and how hormones can be classified according to their mechanism of action.
The techniques of molecular biology and recombinant DNA have been applied to hormonal action with remarkable success. It is now possible to analyze hormonal effects on gene expression and to study which few nucleotides of the 3×109 in each haploid genome confer the response. Another exciting area is the overlapping spectrum of activity of components of hormonal action systems with nonhormonal proteins. Consider the similar features of the guanosine triphosphate (GTP)–binding proteins involved in the hormone-sensitive adenylate cyclase system with the transforming RAS oncogene family of proteins or with transducin, which is the protein that couples photoactivation to the visual response.1,2 The homology of platelet-derived growth factor (PDGF) gene and the v-sis transforming gene is remarkable, as is the similarity between the insulin and epidermal growth factor receptors, both of which have intrinsic tyrosine kinase activity.3,4,5 and 6 Researchers are exploring the molecular bases of endocrine diseases, such as pseudohypoparathyroidism, several types of dwarfism, Graves disease, certain types of extreme insulin resistance, testicular feminization, acromegaly, vitamin D resistance, and hereditary nephrogenic diabetes insipidus, to name a few.7,8,9,10,11,12,13 and 14 This knowledge has challenged many of the earlier concepts of hormonal action and endocrine disease.
There are ~200 types of differentiated cells in humans. Only a few produce hormones, but virtually all of the 75 trillion cells in a human body are targets of one or more of the ~50 known hormones. The concept of target cells is undergoing redefinition. It was thought that hormones affected a single cell type, or only a few kinds of cells, and that a hormone elicited a unique biochemical or physiologic action. For example, it was presumed that thyroid-stimulating hormone (TSH) stimulated thyroid growth and thyroid hormonogenesis; adrenocorticotropic hormone (ACTH, also called corticotropin) enhanced growth and function of the adrenal cortex; glucagon increased hepatic glucose production; and luteinizing hormone (LH) stimulated gonadal steroidogenesis. However, these same hormones also stimulate lipolysis in adipose cells.15 Although the physiologic importance of this effect is unclear, the concept of unique sites of actions of these hormones is untenable. A more relevant example is that of insulin, which effects various responses in different cells and occasionally influences different processes within the same cell. It enhances glucose uptake and oxidation in muscle, lipogenesis in fat, amino acid transport in liver and lymphocytes, and protein synthesis in liver and muscle. These and other examples necessitated a reevaluation of the target cell concept.
With the delineation of specific cell-surface and intracellular hormone receptors, the definition of a target has been expanded to include any cell in which the hormone binds to its receptor, whether or not a biochemical or physiologic response has been determined. This definition also is imperfect, but it has heuristic merit, because it presumes that not all actions of hormones have been elucidated.
The response of a target cell is determined by the differentiated state of the cell, and a cell can have several responses to a single hormone. Cells can respond to a hormone in a hemocrine, paracrine, or autocrine manner. An example is the hormone gastrin-releasing peptide (also called mammalian bombesin). Gastrin-releasing peptide has hemocrine and paracrine actions in the gut but is produced by and stimulates the growth of small cell carcinoma cells of the lung.16
Several factors determine the overall response of a target cell to a hormone. The concentration of a hormone around the target cell depends on the rate of synthesis and secretion of the hormone, the proximity of target and source, the association-dissociation constants of the hormone with specific plasma carrier proteins, the rate of conversion of an inactive or suboptimally active form of the hormone into the active form, and the rate of clearance of the hormone from blood by other tissues or by degradation or excretion. The actual response to the hormone depends on the relative activity and state of occupancy, or both, of the specific hormone receptors on the plasma membrane or within the cytoplasm or nucleus; the metabolism of the hormone within the target cell; the presence of other factors within the target cell that are necessary for the hormone response; and postreceptor desensitization of the cell. Alterations of any of these processes can change the hormonal effect on a given target cell and must be considered in addition to the classic feedback loops.
One of the major challenges in making the hormone-based communication system work is depicted in Figure 4-1. Hormone concentrations are very low in the extracellular fluid, generally in the range of 10-15 to 10-9 M. This is much lower than that of the many structurally similar molecules (e.g., sterols, amino acids, peptides) and other molecules that circulate at concentrations in the 10-5 to 10-3 M range. Target cells must identify the various hormones present in small amounts and differentiate a given hormone from the 106- to 109-fold excess of other, often closely related, molecules. This high degree of discrimination is provided by cell-associated recognition molecules called receptors. Hormones initiate their bioeffects by binding to specific receptors, and because any effective control system must provide a means of stopping a response, hormone-induced actions usually terminate after the effector dissociates from the receptor.

FIGURE 4-1. Specificity and selectivity of hormone receptors. Many different molecules circulate in the extracellular fluid (ECF), but only a few are recognized by hormone receptors. Receptors must select these molecules from among high concentrations of the other molecules. This simplified drawing shows that a cell may have no hormone receptors, have one receptor, have a receptor but no hormone in the vicinity, or have receptors for several hormones.

A target cell is defined by its ability to bind a given hormone selectively by means of a receptor, an interaction that is often quantitated using radioactive ligands that mimic hormone binding. Several features of this interaction are important. The radioactivity must not alter the bioactivity of the ligand. The binding should be specific, in which case the ligand is displaceable by unlabeled agonist or antagonist. Binding should be saturable. Binding should occur within the concentration range of the expected biologic response.
All receptors, whether for polypeptides or steroids, have at least two functional domains, and most have several more. A recognition domain binds the hormone, and a second region, the coupling domain, generates a signal that links hormone recognition to some intracellular function. The binding of hormone by receptor implies that some region of the hormone molecule has a conformation that is complementary to a region of the receptor molecule. The degree of similarity, or fit, determines the tightness of the association; this is measured as the affinity of binding. If the native hormone has a relative affinity of 1, other natural molecules range between 0 and 1. In absolute terms, this actually spans a binding affinity range of more than a trillion. Ligands with a relative affinity of more than 1 for some receptors have been synthesized and are used to study receptor biology.
Coupling (i.e., signal transduction) occurs in two ways. Polypeptide and protein hormones, and the catecholamines, bind to receptors located in the plasma membrane, and thereby generate signals that regulate various intracellular functions. Steroids, thyroid hormones, retinoids, and other hormones of this class interact with intracellular receptors, and this complex provides the initial signal.
The amino acid sequences of the recognition and coupling domains have been identified in many polypeptide hormone receptors. Hormone analogues with specific amino acid substitutions were used to change binding and alter the bioactivity of the hormone. Steroid hormone receptors also have these two functional domains; one site binds the hormone and the other binds to specific DNA regions. They also have other domains important for their function, which are described later. Several receptors have been characterized by recombinant DNA techniques, and structural analysis shows that these domains are highly homologous. This homology has been used to isolate cDNAs encoding several receptors that had not been obtained through classic protein purification procedures. The investigations have shown that these nuclear receptors are part of a large family of related proteins.17 This family of proteins is thought to regulate gene transcription, often in association with other transcription factors and coregulatory molecules. The ligands for these are called orphan receptors.
The dual functions of binding and coupling ultimately define a receptor, and it is the coupling of hormone binding to signal transduction, called receptor-effector coupling, that provides the first step in the amplification of the hormonal response. This dual purpose also differentiates the target cell receptor from the plasma carrier proteins that bind hormone without generating a signal. It is important to differentiate the binding of hormones to receptors from the association that hormones have with various transport or carrier proteins. Table 4-1 lists several features of these functionally different classes of proteins.

TABLE 4-1. A Comparison of Hormone Receptors with Transport Proteins

The concentrations of hormone required for occupancy of the receptor and for elicitation of a specific biologic response often are similar (Fig. 4-2A). This is especially true for steroid hormones, but some polypeptide hormones also exhibit this characteristic. This tight coupling is remarkable, considering the many steps that must occur between hormone binding and complex responses, such as transport, enzyme induction, cell lysis, or cell replication. When receptor occupancy and bioeffect are tightly coupled, significant changes in the latter occur when receptor occupancy changes. This happens when fewer receptors are available (Fig. 4-3A) or the affinity of the receptor changes but hormone concentration remains constant (see Fig. 4-3B). Otherwise, there is a marked dissociation of binding and effect, and a maximal bioeffect occurs when only a small percentage of the receptors are occupied (see effect 2 in Fig. 4-2B).

FIGURE 4-2. Hormone binding and biologic effect are compared in the absence (A) and presence (B, effect 2) of spare receptors. Some biologic effects in a tissue may be tightly coupled to binding, but others demonstrate the spare receptor phenomenon (e.g., compare effects 1 and 2 in B). (From Granner DK. Characteristics of hormone systems. In: Martin DW Jr, Mayer PA, Rodwell VW, Granner DK, eds. Harper’s review of biochemistry, 20th ed. Los Altos, CA: Lange Medical Publications, 1985:501.)

FIGURE 4-3. Changes of receptor occupancy have large effects on the biologic response when effector and receptor occupancy are tightly coupled. This can occur when the receptor number changes (A) or when the affinity of the receptor for the hormone changes (B). In the hypothetical case shown in (A), a decrease from 20,000 receptors per cell to 10,000 results in a 50% decrease of the maximal response, a Vmax effect. A decrease in affinity (i.e., solid to interrupted line in [B], or rightward shift) means that more hormone is required for a given effect, but the same maximal response can be obtained. This is a Km effect. (From Granner DK. Characteristics of hormone systems. In: Murray RK, Granner DK, Mayer PA, Rodwell VW, eds. Harper’s biochemistry, 21st ed. Norwalk, CT: Appleton & Lange, 1988.)

Receptors not involved in the elicitation of the response are called spare receptors. They are observed in the response of several polypeptide hormones and are thought to provide a means of increasing the sensitivity of a target cell to activation by low concentrations of hormone and to provide a reservoir of receptors. The concept of spare receptors is operational and may depend on which aspect of the response is examined and which tissue is involved. For example, there is excellent agreement between LH binding and cyclic adenosine monophosphate (cAMP) production in rat testis and ovarian granulosa cells (there generally are no spare receptors when any hormone activates adenylate cyclase), but steroidogenesis in these tissues, which is cAMP dependent, occurs when fewer than 1% of the receptors are occupied (see effects 1 and 2 in Fig. 4-2).18 Transcription of the phosphoenolpyruvate carboxykinase gene is repressed when far fewer than 1% of hepatoma cell insulin receptors are occupied, but there is a high correlation between insulin binding and amino acid transport in thymocytes.19 Other examples of the dissociation of receptor binding and biologic effects include the effects of catecholamines on muscle contraction, lipolysis, and ion transport.20 These end-responses presumably reflect a cascade or multiplier effect of the hormone.
Different responses within the same cell can require various degrees of receptor occupancy. For example, successively greater degrees of occupancy of the adipose cell insulin receptor increase, in sequence, lipolysis, glucose oxidation, amino acid transport, and protein synthesis.21
Molecules can be divided into four groups according to their ability to elicit a hormone receptor–mediated response. These classes are agonists, partial agonists, antagonists, and inactive agents (Table 4-2).

TABLE 4-2. Classification of Steroids According to Their Action as Glucocorticoids

Agonists elicit the maximal response, although different concentrations may be required. In the example of Figure 4-4,1,2 and 3 could be porcine insulin, porcine proinsulin, and guinea pig insulin, respectively. In all systems tested, these insulins have the same rank order of potency, but each elicits a maximal response if present in sufficient concentration. Likewise, 1, 2, and 3 could be dexamethasone, cortisol, and corticosterone (see Table 4-2).Partial agonists evoke an incomplete response even when very large concentrations of the hormone are used, as shown by line B of Figure 4-5. Antagonists generally have no effects themselves, but they competitively inhibit the action of agonists or partial agonists (see lines A through C in Fig. 4-5). Many structurally similar compounds elicit no effect and have no effect on the action of the agonists or antagonists. These are classified as inactive agents and are represented as line D in Figure 4-5.

FIGURE 4-4. Within a class of hormones—glucocorticoids, for example—different molecules may have different potencies. In this case, hormones 1, 2, and 3 are all agonists, but very different concentrations are required to achieve a given biologic response. The binding of steroid to receptor would parallel each of these curves. (From Granner DK. Characteristics of hormone systems. In: Murray RK, Granner DK, Mayer PA, Rodwell VW, eds. Harper’s biochemistry, 21st ed. Norwalk, CT: Appleton & Lange, 1988.)

FIGURE 4-5. Classification of hormones according to their biologic activity. Steroids, for example, can be classified as agonists (line A), partial agonists (line B), antagonists (C in A+C or B+C), or inactive agents (dotted line D). This drawing represents induction of the enzyme tyrosine aminotransferase. (From Granner DK. Characteristics of hormone systems. In: Murray RK, Granner DK, Mayer PA, Rodwell VW, eds. Harper’s biochemistry, 21st ed. Norwalk, CT: Appleton & Lange, 1988.)

Partial agonists also compete with agonists for binding to and activation of the receptor, when they become partial antagonists. The extent of the inhibition of agonist activity caused by partial or complete antagonists depends on the relative concentration of the various steroids. Generally, much higher concentrations of the antagonist are required to inhibit an agonist than are necessary for the latter to exert its maximal effect. Because these concentrations are rarely achieved in vivo, this phenomenon is used for studies of the mechanism of action of hormones in vitro.
The binding of a ligand to the receptor must facilitate a change in this molecule so that it can bind to DNA. This phenomenon was first suggested in studies that used the steroids in Table 4-2.22 The hypothesis assumes that agonists bind to and fully activate the receptor and elicit the maximal biologic response; that partial agonists fully occupy the receptor but afford incomplete activation and therefore a partial response; and that antagonists fully occupy the receptor, but because this complex is unable to bind to DNA, it elicits no intrinsic response but does inhibit the action of agonists.
The number of hormone receptors on or in a cell is in a dynamic state and can be regulated physiologically or be influenced by diseases or therapeutic measures. The receptor concentration and affinity of hormone binding can be regulated.
Some changes can be acute and can significantly affect hormone responsiveness of the cell. For instance, cells exposed to b-adrenergic agonists for minutes to hours no longer activate adenylate cyclase in response to more agonist, and the biologic response is lost. This desensitization occurs by two mechanisms.23 The loss of receptors, called down-regulation, involves the internal sequestration of receptors, segregating them from the other components of the response system, including the regulatory and catalytic subunits of adenylate cyclase. Removal of the agonist results in the return of receptors to the cell surface and restoration of hormonal sensitivity.23 An example of a second form of desensitization of the a-adrenergic system involves the covalent modification of receptor by phosphorylation.24 This cAMP-dependent process entails no change in receptor number and no translocation. Reconstitution experiments show that because the phosphorylated receptor is unable to activate adenylate cyclase, the activation and hormone binding domains are uncoupled.23 Other examples of physiologic adaptation that is accomplished through down-regulation of receptor number by the homologous hormone include insulin, glucagon, thyrotropin-releasing hormone, growth hormone, LH, follicle-stimulating hormone, and catecholamines. A few hormones, such as angiotensin II and prolactin, up-regulate their receptors. The changes in receptor number can occur over a period of minutes to hours and are probably an important means of regulating biologic responses.
How the loss of receptor affects the biologic response elicited at a given hormone concentration depends on whether there are spare receptors (Fig. 4-6). Suppose there is a fivefold reduction in receptor number in a cell. With no spare receptors (see Fig. 4-6A), the maximal response obtained is 20% that of control, hence, the effect is on the Vmax. With spare receptors (see Fig. 4-6B), the maximal response is obtained, but at five times the originally effective hormone concentration, analogous to a Km effect.

FIGURE 4-6. The effect a five-fold loss of receptors has on a biologic system that lacks (A) or has (B) spare receptors. (From Granner DK. Characteristics of hormone systems. In: Martin DW Jr, Mayer PA, Rodwell VW, Granner DK, eds. Harper’s review of biochemistry, 20th ed. Los Altos, CA: Lange Medical Publications, 1985:502.)

The acetylcholine receptor (AChR), which exists in relatively large amounts in the electric organ of Torpedo californica, was the first plasma membrane–associated receptor to be studied in detail. The AChR consists of four subunits: a2, b, d and g.25 The two a subunits bind acetylcholine.26 The technique of site-directed mutagenesis has been used to show which regions of this subunit participate in the formation of the transmembrane ion channel, which is the major function of the AChR.25
Other receptors occur in very small amounts, and recombinant DNA techniques have been used to deduce many of the structures and to find and characterize new receptors. The insulin receptor is a heterotetramer (a2b2) linked by multiple disulfide bonds, in which the extramembrane a subunit binds insulin and the membrane-spanning b subunit transduces the signal through the tyrosine kinase component of the cytoplasmic portion of this polypeptide27 (Fig.4-7). The insulin-like growth factor-I (IGF-I) receptor has a similar structure, and the epidermal growth factor (EGF) and low-density lipoprotein receptors are similar in many respects28,29 and 30 (see Fig. 4-7). Receptors that couple ligand binding to signal transduction through G-protein intermediaries characteristically have seven membrane-spanning domains.31

FIGURE 4-7. Schematic representation of the structures of the low-density lipoprotein (LDL), epidermal growth factor (EGF), and insulin receptors. The amino terminus (NH2) of each is in the extracellular portion of the molecule. The carboxyterminus (COOH) is in the cytoplasm. The open boxes represent cysteine-rich regions that are thought to be involved in ligand binding. Each receptor has a short domain (~25 amino acids) that traverses the plasma membrane (hatched line) and an intracellular domain of variable length. The EGF and insulin receptors have tyrosine kinase activity associated with the cytoplasmic domain (
) and have autophosphorylation sites in this region. The insulin receptor is a heterotetramer connected by disulfide bridges (vertical bars).

Members of the nuclear receptor superfamily have several functional domains: a ligand-binding domain in the carboxyl-terminal region, an adjacent DNA-binding domain, and one or more trans-activation domains. There may also be dimerization, nuclear translocation, and heat shock protein domains, and regions that allow for interactions with a number of other accessory factor and coregulatory proteins17,32 (Fig. 4-8). The amino acid sequence homology is particularly strong in the various DNA-binding domains, and it was this feature that led to the elucidation of the nuclear receptor superfamily.17

FIGURE 4-8. Nuclear receptor family members have several general domains. The amino-terminal region is most variable and often contains a trans -activating domain (TAD1). The DNA-binding domain (DBD) is most conserved, and this feature led to the discovery that these receptors are part of a large family of DNA-binding proteins. The hormone-or ligand-binding domain (LBD), which affords specificity, is located in the carboxyl-terminal (COOH) region of the molecule and contains a second trans -activating domain (TAD2). Also shown are regions that allow for nuclear translocation, dimerization, and interaction with heat shock protein (Hsp90). Members of this family that have no known ligand are called orphan receptors.

A classification based on the location of receptors and the nature of the signal used to mediate hormonal action within the cell appears in Table 4-3, and general features of each group are listed in Table 4-4.

TABLE 4-3. Hormones and Their Actions: Classification According to Mechanism of Action

TABLE 4-4. General Features of Hormone Groups

The hormones in group I are lipophilic. After secretion, these hormones associate with transport proteins, a process that circumvents the solubility problem while prolonging the plasma half-life by preventing the hormone from being metabolized and excreted. These hormones readily traverse the plasma membrane of all cells and encounter receptors in the cytosol or the nucleus of target cells. The ligand-receptor complex is assumed to be the intracellular messenger in this group.
The second major group consists of water-soluble hormones that bind to the plasma membrane of the target cell. These hormones regulate intracellular metabolic processes through intermediary molecules, called second messengers (the hormone itself is the first messenger), which are generated because of the ligand-receptor interaction. The second-messenger concept arose from the observation of Sutherland33 that epinephrine binds to the plasma membrane of pigeon erythrocytes and increases intracellular cAMP. This was followed by a series of experiments in which cAMP was found to mediate the metabolic effects of many hormones. Hormones that use this mechanism are shown in group IIA. Several hormones, some of which were previously thought to affect cAMP, appear to use cyclic guanosine mono-phosphate (cGMP) (group IIB) or calcium or phosphatidylinositide metabolites (or both) as the intracellular signal (group IIC). The intracellular messenger has been identified as a protein kinase/phosphatase cascade for the hormones listed in group D.
A few hormones fit in more than one category (i.e., some hormones act through cAMP and Ca2+), and assignments change with new information.
A schematic representation of the mechanism of action of group I hormones (see Table 4-3) is shown in Figure 4-9. These lipophilic molecules probably diffuse through the plasma membrane of all cells but encounter their specific, high-affinity receptor only within target cells. The hormone-receptor complex then undergoes an “activation” reaction that causes size, conformation, and surface charge changes that render it able to bind to chromatin. In some cases—with the glucocorticoid receptor, for example—this process involves the disruption of a receptor–heat shock protein complex. Whether the association and activation processes occur in the cytoplasm or nucleus appears to depend on the specific hormone. The hormone-receptor complex binds to specific regions of DNA and activates or inactivates specific genes.34,35 By selectively affecting gene transcription and the production of the respective messenger RNAs (mRNAs), the amounts of specific proteins are changed, and metabolic processes are influenced. The effect of each of these hormones is specific; generally, the hormone affects <1% of the proteins or mRNAs in a target cell.

FIGURE 4-9. A general model of group I hormone action. The hormone binds to intracellular receptors in the cytoplasm or the nucleus and causes a conformational change. The hormone-receptor complex then binds to a specific region on DNA called the hormone response element. This interaction, with the help of various accessory factors and coregulators, results in the activation or repression of a restricted number of genes. The hormone response elements and associated factor elements are called hormone response units. The “bubble” indicates that this region of DNA is open or accessible to the transcription complex. These regions of DNA are often found to be sensitive to digestion by the enzyme DNase I.

The nuclear actions of steroid hormones predominate and are well defined, but direct actions of these hormones in the cytoplasm and on various organelles and membranes also have been described.36 An effect of estrogens, cAMP, and glucocorticoids on mRNA degradation rates has been demonstrated.37,38,39 and 40 Glucocorticoids also affect posttranslational processing of some proteins.41
Although the biochemistry of gene transcription in mammalian cells is not completely understood, a general model of the structural requirements of steroid regulation of gene transcription can be drawn (Fig. 4-10). Steroid-regulated genes must be in regions of “open” or transcriptionally active chromatin (depicted as the bubble in Fig. 4-9), as defined by their susceptibility to digestion by the enzyme DNase I.42 The open or closed conformation of chromatin may be regulated by the extent of acetylation of the histones that combine with DNA to form chromatin (discussed later this section). Genes have at least two separate regulatory regions in the DNA sequence immediately 5′ of the transcription initiation site. The first of these, the basal promoter element (BPE), is generic, because it is present in some form in all genes.43 This is depicted as containing the consensus sequence GTATA (A/T)A(A/T), called the TATA box (see Chap. 3), because this is the structure found most frequently. Another common component of the BPE is the CAAT box; this sequence or some equivalent structure usually is present. The BPE appears to specify the site of RNA polymerase II attachment to DNA and therefore the accuracy of transcript initiation.44

FIGURE 4-10. Structural requirements for hormonal regulation of gene transcription. Transcription starts at the arrow, where 1+ signifies the first nucleotide of the gene that is transcribed or copied. Immediately adjacent, on the 5′ upstream side, is the basal promoter element, which generally consists of a TATA box and other components, such as a CAAT box. Hormone response elements (which bind the liganded receptor) can be anywhere in the 5′ region or in the gene itself. Other DNA elements (accessory factor elements) that cooperate with the hormone response elements to regulate transcription can also be located at various sites. Together these form a hormone response unit.

A second regulatory region is located slightly farther upstream than the BPE, and this may also consist of several discrete elements. This region modulates the frequency of transcript initiation and is less dependent on position and orientation. In these respects, it resembles the transcription enhancer elements found in other genes.45,46 The regulatory region consists of two types of DNA elements in genes that respond to hormones. Hormone response elements (HREs) are short segments of DNA that bind a specific hormone receptor/ligand complex.32,34,35 HREs are often capable of regulating transcription from test promoter/reporter gene constructs, but in most physiologic circumstances, other DNA element/protein complexes are required. The HRE usually is found within 250 nucleotides of the transcription initiation site, but the precise location of the HRE varies from gene to gene.
Identification of an HRE requires that it bind the hormone-receptor complex more avidly than does surrounding DNA or DNA from another source. The HRE also must confer hormone responsiveness. Putative regulatory sequence DNA can be ligated to reporter genes to assess this point. Usually, these fusion genes contain reporter genes not ordinarily influenced by the hormone, and these genes often are not expressed in the tissue being tested. Commonly used reporter genes are firefly luciferase or bacterial chloramphenicol acetyltransferase. The fusion gene is transfected into a target cell, and if the hormone now regulates the transcription of the reporter gene, an HRE is functionally defined. Position, orientation, and base-substitution effects can be precisely described using this technique.
Although HREs do transmit a hormone response in simple promoter test conditions, the situation is much more complex in most naturally occurring genes. The HRE must interact with other elements (and associated binding proteins) to function optimally. Such assemblies of cis-acting DNA elements and trans acting factors are called hormone response units (HRUs).45 An HRU, therefore, consists of one or more HREs and one or more DNA elements with associated accessory factors (Fig. 4-11). In complex promoters—regulated by a variety of hormones—certain accessory factor components of one HRU (glucocorticoid) may be part of that for another (retinoic acid). This arrangement may provide for the hormonal integration of complex metabolic responses.

FIGURE 4-11. The hormone response unit (HRU). The HRU is an assembly of DNA elements and bound proteins. An essential component is the hormone response element with ligand-bound receptor (R). Also important are the accessory factor (AF) elements with bound transcription factors. More than two dozen of these accessory factors have been linked to hormone effects on transcription. The AFs can interact with each other or with the nuclear receptors. The components of the HRU communicate with the basal transcription machinery through a coregulator complex. The components of the coregulator complex, some of which are described in the text, provide for the direction and specificity of the hormone response. (From Granner DK. Hormone action. In: Murray RK, Granner DK, Mayes PA, Rodwell VW, eds. Harper’s biochemistry, 25th ed. Nor-walk, CT: Appleton & Lange, 1999.)

The communication between an HRU and the basal transcription apparatus is accomplished by one or more of a class of coregulator molecules (see Fig. 4-11). The first of these described was the cAMP response element binding (CREB) protein, so-called CBP. CBP, through an amino terminal domain, binds to phosphorylated serine 137 of CREB and facilitates transactivation in response to cAMP. It thus is described as a coactivator. CBP and its close relative, p300, interact with a number of signaling molecules, including activator protein-1, signal transducers and activators of transcription, nuclear receptors, and CREB.46
CBP/p300 also binds to the p160 family of coactivators—described in the next paragraph—and to a number of other proteins. It is important to note that CBP/p300 also has intrinsic histone acetyltransferase (HAT) activity. The importance of this is described later in this section. Some of the many actions of CBP/p300 appear to depend on intrinsic enzyme activities and the ability of this protein to serve as a scaffold for the binding of other proteins.
Three other families of coactivator molecules, all of ~160 kDa, have been described. These members of the p160 family of coactivators include (a) SRC-1 and NCoA-1; (b) GRIP 1, TIF2, and NCoA-2; and (c) p/CIP, ACTR, AIB1, RAC3, and TRAM-1.47 The different names for members within a subfamily often represent species variations or minor splice variants. There is ~35% amino acid identity between members of the different subfamilies.
The role of these many coactivators is still evolving. It appears that certain combinations are responsible for specific ligand-induced actions through various receptors. The role of HAT is particularly interesting. Mutations of the HAT domain disable many of these transcription factors. Current thinking holds that these HAT activities acetylate histones and result in the remodeling of chromatin into a transcription-efficient environment.48 In keeping with this hypothesis, histone deacetylation is associated with the inactivation of transcription.
In certain instances, the removal of a corepressor complex through a ligand-receptor interaction results in the activation of transcription. For example, in the absence of hormone, the thyroid or retinoic acid receptors are bound to a corepressor complex containing N-CoR or SMRT and associated proteins, some of which have histone deacetylase activity.47 The target gene is repressed until the binding of hormone to the thyroid receptor results in the dissociation of this complex, and gene activation then ensues.
Most group II hormones are water soluble, have a short plasma half-life and no transport proteins, and initiate a response by binding to a receptor located in the plasma membrane (see Table 4-3 and Table 4-4).
Cyclic AMP (3′,5′ adenylic acid), a ubiquitous nucleotide derived from adenosine triphosphate (ATP) through the action of the enzyme adenylate cyclase (Fig. 4-12), plays a crucial role in the action of several hormones. The intracellular level of cAMP is increased or decreased by various hormones (see Table 4-3). This effect varies from tissue to tissue and sometimes within a given tissue, depending on which specific hormone-receptor interactions occur. For example, epinephrine causes large increases of cAMP in muscle and relatively small changes in liver; the opposite is true of glucagon.
Tissues that respond to several hormones of this group do so through unique receptors converging on a single class of adenylate cyclase molecules. The best example of this is the adipose cell in which epinephrine, ACTH, TSH, glucagon, LH, melanocyte-stimulating hormone, and vasopressin stimulate adenylate cyclase and increase cAMP.15 Combinations of maximally effective concentrations are not additive, and treatments that destroy one receptor response have no effect on the response of other hormones. Some actions of cAMP occur outside the nucleus, presumably by mediating changes in the degree of phosphorylation of critical enzymes involved in metabolic processes, such as glycogenolysis, gluconeogenesis, and lipolysis.49 Effects of cAMP on the transcription of many genes have been described, and this clearly is a major action of this molecule.50

FIGURE 4-12. The formation and metabolism of cyclic adenosine monophosphate (AMP). (ATP, adenosine triphosphate.)

The components of this system in mammalian cells are shown in Figure 4-13. The interaction of the hormone with its receptor causes the activation or inactivation of adenylate cyclase. This process is mediated by two GTP-dependent regulatory protein complexes, designated Gs (stimulatory) and Gi (inhibitory), each of which is composed of three subunits: a, b, and g (see Fig. 4-13). These G proteins are part of a large family with more than 20 members that mediate many biologic processes in addition to hormone responses51 (Table 4-5). Adenylate cyclase, located on the inner surface of the plasma membrane, catalyzes the formation of cAMP from ATP in the presence of magnesium (see Fig. 4-12).

FIGURE 4-13. Components of the hormone receptor–G-protein effector system. Receptors that couple to effectors through G proteins typically have seven membrane-spanning domains. The amino (N) and carboxy (C) termini are shown. In the absence of hormone (left), the heterotrimeric G-protein complex (i.e., a, b, g) is in an inactive, guanosine diphosphate (GDP)–bound form and is probably not associated with the receptor. This complex is anchored to the plasma membrane through prenylated groups on the bg subunits (wavy lines) and perhaps by myristoylated groups on a subunits. On binding of hormone to the receptor, there is a presumed conformational change of the receptor and activation of the G-protein complex. This results from the exchange of GDP and guanosine triphosphate (GTP) on the a subunit, after which a and bg dissociate. The a subunit binds to and activates the effector (E). E can be adenylate cyclase (as), a K+ channel (a1 a0), phospholipase C-b(aq) or other molecule. The bg subunit can also have direct actions on E.

TABLE 4-5. Classes and Functions of G Proteins

What was originally considered a single protein with hormone binding and catalytic domains was found to be a system of extraordinary complexity. Biochemical and genetic studies have established the biochemical uniqueness of the hormone-receptor, GTP-regulatory, and catalytic domains of the adenylate cyclase complex.
Two parallel systems, stimulatory (s) and inhibitory (i), converge on the catalytic molecule (C). Each system consists of a receptor, Rs or Ri, and a regulatory complex, Gs or Gi. Gs and Gi are trimers composed of a, b, and g subunits (see Fig. 4-13) and are so called because they bind and hydrolyze GTP. The as and aiare unique proteins of 39 to 46 kDa; the b subunits are 37-kDa proteins, and the g subunits are 8-kDa proteins.51 The binding of a hormone to Rs or Ri yields a receptor-mediated activation of G, which requires Mg2+-dependent guanosine diphosphate (GDP)/GTP exchange by a and the subsequent dissociation of b and g from a.
The as subunit has GTPase activity; the active form, as × GTP, is inactivated on hydrolysis of the GTP to GDP, and the trimeric Gs complex is reformed.52 Cholera toxin, an irreversible activator of cyclase, causes adenosine diphosphate (ADP) ribo can stimulate adenylate cyclase directly, and in some instances, the bg complex, which is tightly associated and always acts as a unit, augments this action.
Less is known about how hormones inhibit adenylate cyclase activity. Direct inhibitory effects of Gi protein on adenysylation of as on arginine at position 201 in the protein, and in so doing, inactivates the GTPase; as remains in the active form.53,54 The ai also has GTPase activity; however, because GDP does not freely dissociate from ai the latter is reactivated by an exchange of GTP for GDP. Pertussis toxin irreversibly activates adenylate cyclase by promoting the ADP ribosylation ofai on a cysteine residue, which prevents this subunit from being activated by ligand-bound receptor.55
The exact mechanism of activation and inactivation of the adenylate cyclase moiety has not been established.55a The as form can stimulate adenylate cyclase directly, and in some instances, the bg complex, is tightly associated and always acts as a unit, augments this action.
Less is known about how hormones inhibit adenylate cyclase activity. Direct inhibitory effects of Gi protien on adenylate cyclase have been difficult to detect. One hypothesis is that the bg complex that is liberated from Gi, which exists in most cells in great abundance over Gs, could bind to and inactivate as. This would effectively shut off the stimulatory signals. Alternatively,bg may directly inhibit some forms of adenylate cyclase. The a subunits and the bg complex have actions independent of those on adenylate cyclase. Some forms of as stimulate Ca2+ channels and inhibit K+ channels. Similarly, some forms of ai stimulate K+ channels and inhibit Ca2+ channels. Other subunits, particularly those of the Gq family (see Table 4-5), are involved in the activation of members of the phospholipase C group of enzymes. This is important in the generation of the intracellular signals of inositol triphosphate and diacylglycerol. The bg complex also can stimulate K+ channels and activate certain isoforms of phospholipase C.51 Members of the family of G proteins mediate a variety of important processes (see Table 4-5). Transducin, the protein that is important in coupling light to photoactivation in the retina, is a member of the G-protein family, as are the proteins involved in smell and taste.56 The products of the RAS oncogenes, which are involved in regulating cell growth, are members of the larger superfamily.57 The G proteins are themselves a family within the large superfamily of GTPases and can be classified according to sequence homology into at least four subfamilies, as illustrated in Table 4-5. There are more than 20 a subunits, and there are at least four b subunits and six g subunits. There are at least five adenylate cyclase molecules.58 There may well be more members of each group.
Pseudohypoparathyroidism, an “experiment of nature,” is a syndrome characterized by hypocalcemia and hyperphos-phatemia, the biochemical hallmarks of hypoparathyroidism, and by several congenital defects (see Chap. 60). Individuals with pseudohypoparathyroidism do not have defective parathyroid function; they secrete large amounts of bioactive parathyroid hormone. Some have target organ resistance on the basis of a postreceptor defect. They are partially deficient in G protein (probably only the as subunit) and fail to couple binding to adenylate cyclase stimulation.58,59 The observation that patients with pseudohypoparathyroidism often have defective responses to other hormones, including TSH, glucagon, and b-adrenergic agents, is not surprising.60 Many G-protein–linked endocrinopa-thies have now been described (see Chap. 60). (See Chap ref 60a.)
Approximately 40% of persons with acromegaly have a G-protein–linked disease. These individuals have one of two mutations in the as subunit that affects the intrinsic GTPase activity of this protein. One mutation, at arginine position 201, affects the same site that is ADP ribosylated by cholera toxin.12 The constitutive overproduction of cAMP results in excessive release of growth hormone and somatotropic cell adenomas. In this way, as-subunit functions as an oncogene. Other a-subunit mutations that affect the GTPase domain result in tumors of the adrenal cortex and ovary.61
In prokaryotic cells, cAMP binds to a specific protein, catabolite regulatory protein, which binds directly to DNA and influences gene expression.62 The analogy of this to steroid hormone action is apparent. In eukaryotic cells, cAMP binds to protein kinase A (PKA), which is a heterotetrameric molecule consisting of two regulatory subunits (R) and two catalytic subunits (C).63 Cyclic AMP binds to the regulatory subunits and yields the following reaction:

The R2C2 complex has no enzymatic activity, but the binding of cAMP by R dissociates R from C, thereby activating the latter. The active C subunit catalyzes the transfer of the g-phosphate of ATP (Mg2+) to a serine or threonine residue in various proteins. The consensus phosphorylation sites of PKA are -Arg-Arg-X-Ser- and -Lys-Arg-X-X-Ser-, in which X can be any amino acid.64
Protein kinase activities originally were described as cAMP dependent or cAMP independent. This area has also become considerably more complex, because protein phosphorylation is recognized as a general regulatory mechanism. Dozens of protein kinases have been described. All are unique molecules and show considerable variability with respect to subunit composition, molecular weight, autophosphorylation, Km for ATP, and substrate specificity.65
The effects of cAMP in eukaryotic cells are thought to be mediated by protein phosphorylation and dephosphorylation.66 The effect of cAMP, including such diverse processes as steroido-genesis, secretion, ion transport, carbohydrate and fat metabolism, enzyme induction, gene regulation, and cell growth and replication, could be conferred by a specific protein kinase, a specific phosphatase, or by specific substrates for phosphorylation. Many substrates have been identified. For example, the transcription factor, CREB, mediates many of the effects of cAMP on gene transcription.67,68 CREB binds to the cAMP response element in the unphosphorylated state but is much more active in stimulating transcription after it has been phosphorylated by PKA.69 Phosphorylation on serine 133 allows CREB to bind to the coactivator CBP.46 This complex is associated with enhanced rates of transcription of target genes.
Reactions caused by hormones in class IIA can be terminated in several ways, including the hydrolysis of cAMP by phosphodesterases.70 The presence of these hydrolytic enzymes ensures a rapid turnover of the signal (i.e., cAMP), and there is a rapid termination of the biologic process after the hormonal stimulus is removed. The cAMP phosphodiesterases exist in low- and high-Km forms and are themselves subject to regulation by hormones and by intracellular messengers such as calcium, probably acting through calmodulin.71,72 and 73 Inhibitors of phosphodiesterase, most notably xanthine derivatives, increase intracellular cAMP and mimic or prolong the actions of hormones.
Another means of controlling hormonal action is the regulation of the protein dephosphorylation reaction. There are two classes of phosphoprotein phosphatases. One group attacks phosphate on threonine or serine residues, another attacks phosphotyrosine. The threonine or serine protein phosphatases have been divided into two groups. Type 1 enzymes dephosphorylate the b subunit of phosphorylase kinase and are inhibited by small heat- and acid-stable proteins (i.e., inhibitors 1 and 2). The type 2 enzymes dephosphorylate the a subunit of phosphorylase kinase preferentially and are insensitive to the inhibitor proteins.74 There are a few members of each group, but there are many more protein kinases than there are phosphatases.
The regulatory role of phosphatases has been best worked out in the case of glycogen metabolism.75 Certain phosphatases may attack specific residues; for example, some phosphatases remove phosphate from tyrosine residues.76
Some cAMP leaves cells and can be readily detected in extracellular fluids. The actions of glucagon on liver and of vasopressin or parathyroid hormone on the kidney are reflected in elevated levels of cAMP in plasma and urine, respectively.77 This led to diagnostic tests of target organ responsiveness. Extracellular cAMP has little or no bioactivity in mammals, but it is an extremely important intercellular messenger in lower eukaryotes and prokaryotes.
Cyclic GMP is made from GTP by the enzyme guanylate cyclase, which exists in soluble and membrane-bound forms.78,79 and 80 Each of these isozymes has unique kinetic, physiochemical, and antigenic properties. For some time, cGMP was thought to be the functional counterpart of cAMP, but it became apparent that cGMP had a unique place in hormonal action. The atriopeptins, a family of peptides produced in cardiac atrial tissues, cause natriuresis, diuresis, vasodilation, and inhibition of aldosterone secretion (see Chap. 178). These peptides, such as atrial natriuretic factor, bind to and activate the membrane-bound form of guanylate cyclase. This causes an increase of cGMP, as much as 50-fold in some cases, which is thought to mediate these effects. Other evidence links cGMP to vasodilation. A series of compounds, including nitric oxide, nitroprusside, nitroglycerin, sodium nitrite, and sodium azide, cause smooth muscle relaxation and are potent vasodilators. These agents increase cGMP by activating nitric oxide synthase, which in turn produces nitric oxide. Nitric oxide activates the soluble form of guanylate cyclase. Inhibitors of cGMP phosphodiesterase enhance and prolong these responses. The increased cGMP activates cGMP-dependent protein kinase, which phosphorylates several smooth muscle proteins, including the myosin light chain. Presumably, this is involved in relaxation of smooth muscle and vasodilation. A cGMP phosphodiesterase attenuates these responses. Sildenafil (Viagra) is a potent phosphodiesterase inhibitor. The use of this compound in erectile dysfunction is based on the fact that it prolongs the accumulation and action of cGMP on penile smooth musculature.
Ionized calcium is an important regulator of various cellular processes, including muscle contraction, stimulus-secretion coupling, the blood clotting cascade, enzyme activity, and membrane excitability. It also is an intracellular messenger of hormonal action.81,82
Calcium Metabolism. The extracellular calcium concentration is ~1.2 mM and is rigidly controlled. The intracellular free concentration of this ion (Ca2+) is much lower, ~100 to 200 nM, and the concentration associated with intracellular organelles is in the range of 1 to 20 µM. Despite this 5000- to 10,000-fold concentration gradient and a favorable transmembrane electrical gradient, Ca2+ is restrained from entering the cell. Resting (basal) Ca2+ concentrations are determined by the activities of several Ca2+ exchangers and/or Ca2+ pumps located in the plasma membrane and endoplasmic reticulum membrane. Hormones can raise cytosolic Ca2+ via three mechanisms: (a) activation of ligand-gated Ca2+ channels in plasma membrane; (b) depolarization of the plasma membrane that activates inward-directed voltage-gated Ca2+ channels in the plasma membrane; and (c) activation of phospholipase c (PLC) through a Gq (G protein) mechanism, or by tyrosine phosphorylation, to generate inositol trisphosphate (IP3), which stimulates release from intracellular Ca2+ reservoirs. Ca2+ concentrations are rapidly returned to the basal concentrations by a reversal of one or more of these mechanisms, as a sustained elevation of Ca2+ is toxic to cells.
Two observations led to the current understanding of how Ca2+ serves as an intracellular messenger of hormonal action. The first was the ability to quantitate the rapid changes of intracellular Ca2+ concentration that are implicit in a role for Ca2+ as an intracellular messenger. Such evidence was provided by various techniques, including the use of quin-2 or fura-2, fluorescent Ca2+ chelators.83 Rapid changes of Ca2+ in the submicroolar range can be quantitated using these compounds. The second important observation linking Ca2+ to hormonal action involved the definition of the intracellular targets of Ca2+ action. The discovery of a Ca2+-dependent regulator of phosphodiesterase activity provided the basis for understanding how Ca2+and cAMP interact within cells.73
Calmodulin. The major calcium-dependent regulatory protein is calmodulin, a 17-kDa protein that is homologous to the muscle protein troponin C in structure and function.84,85 Calmodulin has four Ca2+-binding sites, and full occupancy of these leads to a marked conformational change of the protein. This conformational change is linked to the ability of calmodulin to activate enzymes. Calmodulin can be a constituent subunit of complex proteins. For example, it is the d subunit of phosphorylase b kinase.86 The interaction of Ca2+ with calmodulin and the resultant change of activity of the latter are similar conceptually to the binding of cAMP to protein kinase and the subsequent activation of this molecule.
Calmodulin participates in regulating various protein kinases and enzymes of cyclic nucleotide generation and degradation.86a As shown in Figure 4-14, the Ca2+-calmodulin complex activates specific calmodulin-dependent protein kinases and a multifunctional calmodulin-dependent protein kinase. Other enzymes are regulated directly (adenylate cyclase, cyclic nucleotide phosphodiesterase, phosphorylase kinase) or indirectly (Ca2+/Mg2+-ATPase, glycerol-3-phosphate dehydrogenase, glycogen synthase, guanylate cyclase, myosin kinase, nicotinamide-adenine dinucleotide (NAD) kinase, phospholipase A2, pyruvate carboxylase, pyruvate dehydrogenase, and pyruvate kinase) by Ca2+, probably through calmodulin.

FIGURE 4-14. Many hormone effects are mediated by phosphatidy-linositide metabolites and by ionic calcium (Ca2+). Certain hormone-receptor interactions are coupled through a G-protein complex to a membrane-associated enzyme, phospholipase C. This enzyme catalyzes the hydrolysis of phosphatidylinositol bisphosphate (PIP2)into diacylglyceride (DAG) and inositol triphosphate (IP3).These intracellular messengers activate specific enzymes. DAG directly activates protein kinase C. IP33 combines with a specific receptor on Ca2+ cellular organelles (e.g., the endoplasmic reticulum [ER]), releasing Ca2+into the cytoplasm. Ca2+, in combination with calmodulin (Cam), activates enzymes such as the specific Cam-kinase and a multifunctional Cam-kinase. Ca2+-Cam also binds to and changes the activity of a number of other proteins. (Prot, protein.) (Courtesy of John Exton.)

Along with its effects on enzymes and ion transport, Ca2+-calmodulin regulates the activity of many structural elements in cells. These include the actin-myosin complex of smooth muscle, which is under b-adrenergic control, and various microfila-ment-mediated processes in noncontractile cells, including cell motility, conformation changes, the mitotic apparatus, granule release, and endocytosis. Calcium regulates the transcription of the FOS gene by phosphorylating CREB, probably through the mechanism described in Figure 4-14. This is an interesting example of the convergence of two different signal transduction pathways: the cAMP system and the Ca2+ system.87
Calcium as a Mediator of Hormonal Action. A role for ionized calcium in hormonal action is suggested by observations that the effect of many hormones is blunted in Ca2+-free media or when intracellular calcium is depleted; can be mimicked by agents that increase cytosolic Ca2+, such as the Ca2+ ionophore A23187; and involves changes of cellular calcium flux. These processes have been studied in some detail in the pituitary, smooth muscle, platelets, and salivary gland, but most is known about how vasopressin and a-adrenergic catecholamines regulate glycogen metabolism in the liver.
Phosphorylase activation results from the conversion of phosphorylase b to phosphorylase a through the action of the enzyme phosphorylase b kinase. This enzyme contains calmodulin as its d subunit, and its activity is increased through Ca2+ concentration ranges of 0.1 to 1.0 µM. Addition of a1 agonists or vasopressin to isolated hepatocytes causes a threefold increase of cytosolic Ca2+ (from 0.2 to 0.6 µM) within a few seconds. This change precedes and equals the increase in phosphorylase a activity, and the hormone concentrations required for both processes are comparable.88,89 This effect on Ca2+ is inhibited by a1-antagonists, and removal of the hormone causes a prompt decline of cytosolic Ca2+ and phosphorylase a. The initial source of the Ca2+ appears to be intracellular organelle reservoirs, which seem to be sufficient for the early effects of the hormones. More prolonged action appears to require enhanced influx, inhibition of Ca2+ efflux, or both through the Ca2+ pump. The latter may depend on concomitant increases of cAMP.
Role of Phosphatidylinositide Metabolism in Ca2+-Dependent Hormonal Action. Some signal must provide communication between the hormone receptor on the plasma membrane and the intracellular Ca2+ reservoirs. This is accomplished by the products of phosphatidylinositide metabolism.82 Phosphatidylinositol 4,5-bisphosphate is hydrolyzed to 1,4,5-triphosphate and diacylglycerol through the action of (IP3) phospholipase C (see Fig. 4-14). These two signals activate different pathways. IP3 binds to a receptor on the surface of intracellular organelles that serve as Ca2+ repositories. The binding of IP3 to this receptor (which is similar to the ryanodine receptor) opens these Ca2+ channels, and cytosolic free Ca2+increases.82 Ca2+ enters into cells from the extracellular fluid. Diacylglycerol activates protein kinase C, and this enzyme alters metabolic processes by phosphorylating various substrate proteins.
Steroidogenic agents, including ACTH and cAMP in the adrenal cortex; angiotensin II, K+, serotonin, ACTH, and dibutyryl cAMP in the zona glomerulosa of the adrenal; LH in the ovary; and LH and cAMP in the Leydig cells of the testes have been associated with increased amounts of phosphatidic acid, phosphatidylinositol, and polyphosphatidylinositides in the respective target tissues.90
Other examples include the addition of thyrotropin-releasing hormone to pituitary cells, which is followed within 15 seconds by a marked increase of inositol degradation by phospholipase C. The intracellular levels of inositol diphosphate and triphosphate increase markedly, mobilizing intracellular calcium. This activates calcium-dependent protein kinase, which phosphorylates several proteins, one of which appears to be involved in TSH release.91 Calcium also appears to be the intracellular mediator of gonadotropin-releasing hormone action on LH release, which probably involves calmodulin.92 The roles that Ca2+ and phosphoinositide breakdown products may play in hormonal action are shown in Figure 4-14. In this scheme, the phosphoinositide products are the second messengers, and Ca2+ is a tertiary messenger. Hormones that couple through G proteins generate signals by this mechanism, as do some hormones that initiate signal transduction by the activation of an intrinsic tyrosine kinase activity in the receptor. It is likely that several examples of the complex networking of intracellular messengers will be discovered.
This important group of hormones had been listed under the category of “intracellular mediator unknown.” A major breakthrough came with the discovery that the EGF receptor contained an intrinsic tyrosine kinase activity that was activated on the binding of the ligand, EGF.93 Shortly thereafter, the insulin and IGF-I receptors were also found to contain intrinsic, ligand-activated tyrosine kinase activity.27,94 Several receptors, generally those involved in binding ligands involved in growth control, have intrinsic tyrosine kinase activity or associate with proteins that are tyrosine kinases.95 Another distinguishing feature of this class of hormone action is that these kinases preferentially phosphorylate tyrosine residues, and tyrosine phosphorylation is infrequent (<0.03% of total amino acid phosphorylation) in mammalian cells.4 Some of the hormones, such as EGF, that activate tyrosine kinases activate phospholipase C and exert effects through Ca2+ and IP3 or diacylglycerol. Investigators have considered that tyrosine kinase activation could also initiate a phosphorylation and dephosphorylation cascade that involved the action of one or several other protein kinases and the counterbalancing actions of phosphatases. Considerable evidence supports this hypothesis. Two mechanisms are used to initiate this cascade. Some hormones—such as growth hormone, prolactin, erythropoietin, and the cytokines—initiate their action by activating tyrosine kinase, but this activity is not an integral part of the hormone receptor. The hormone-receptor interaction somehow activates cytoplasmic protein tyrosine kinases (PTKs), such as Tyk-2, Jak-1, or Jak-2.96 These PTKs phosphorylate one or more cytoplasmic proteins, which then associate with other docking proteins through binding to Src homology 2 domains, ~100 amino acid–long segments that are referred to as SH2 domains. Further steps must be elucidated, but it is presumed that hormones of this class mediate their effects, at least in part, through a kinase cascade that is initially activated by PTK.
Activation of the intrinsic tyrosine kinase activity of the insulin receptor results in the phosphorylation of a substrate, called the insulin receptor substrate (IRS), on tyrosine residues. At least four IRS proteins have been identified. Phosphorylated IRS proteins bind to the SH2-domains of a variety of proteins that presumably are directly or indirectly involved in mediating different effects of insulin. For example, the binding of IRS-2 to PI-3 kinase results in the activation of the latter, and this links insulin action to phosphoinositide metabolism and thereby to many physiologic processes, including translocation of the glucose transporter and the regulation of genes involved in metabolism. The growth-promoting effects of insulin, and of IGF-I, appear to result from the phosphorylation of IRS-1 by the insulin receptor. Phosphorylated IRS-1 binds to another SH2 domain–containing protein, GRB-2, which, in turn, activates the MAP kinase pathway. The result of this interaction is the activation of a cascade of threonine or serine kinases.97 The exact role of the many docking proteins, kinases, and phosphatases in insulin action remains to be established. It is particularly important to link these various pathways to the well-established physiologic and biochemical actions of this hormone, and to decipher how the specificity of hormone action is achieved by the many hormones that use one or more of the components of this complex array of signaling proteins.

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