sign in sign up
<<
Home , Endocrine gland, Endocrine system

1

Introduction to the

OBJECTIVES 1. List the main endocrine of the body. 4. Explain the significance of binding to plasma 2. List the chemical nature of the major . . 3. Describe how the chemical nature influences hormone 5. Describe the major pathways, and synthesis, storage, , transport, clearance, their mechanism for termination, for different classes mechanism of action, and appropriate route of exoge- of hormones and provide a specific example of each. nous hormone administration.

Endocrine glands secrete chemical messengers, called hor- hydroxylations of in hepatocytes and renal mones (Box 1.1), into the extracellular fluid in a highly tubular cells. regulated manner. Secreted hormones gain access to the cir- Numerous extracellular messengers, including prosta- culation, often via fenestrated , and regulate target glandins, growth factors, , and , organs throughout the body. The endocrine system is com- also regulate cellular function. However, these messengers posed of the pituitary , the gland, parathy- act predominantly within the context of a microenviron- roid glands, and adrenal glands (Fig. 1.1). The endocrine ment in an autocrine or paracrine manner, and thus are system also includes the and testis, which carry out a discussed only to a limited extent where needed. gametogenic function that is absolutely dependent on their To function, hormones must bind to specific recep- endogenous endocrine function. In addition to dedicated tors expressed by specifictarget cell types within target endocrine glands, endocrine cells reside as a minor compo- organs. Hormones are also referred to as ligands, in the nent (in terms of mass) in other organs, either as groups of context of binding, and as agonists, in that cells (the islets of Langerhans in the ) or as indi- their binding to the receptor is transduced into a cellular vidual cells spread throughout several glands, including the response. Receptor antagonists typically bind to a receptor gastrointestinal (GI) tract, , , adipose , and lock it in an inactive state, unable to induce a cellular and . In addition, there are several types of hypotha- response. Drugs that bind to and alter the activity of ste- lamic neuroendocrine that produce hormones. roid hormone receptors are referred to as selective receptor The serves as a transitory exchange , but also modulators. For example, Tamoxifen is a mixed functions as an important endocrine structure of pregnancy. receptor agonist/antagonist, and thus is referred to as a The endocrine system also encompasses a range of spe- “selective modulator” or SERM. Loss cific enzymes, either cell-associated or circulating, that per- or inactivation of a receptor leads to hormonal resistance. form the function of peripheral conversion of hormonal Constitutive activation of a receptor leads to unregulated, precursors (see Box 1.1). For example, angiotensinogen hormone-independent activation of cellular processes. from the liver is converted in the circulation to The widespread delivery of hormones in the I by the renal-derived enzyme , followed by conver- makes the endocrine system ideal for the functional coor- sion to the active hormone angiotensin II by the trans- dination of multiple organs and cell types in the following membrane ectoenzyme angiotensin I–converting enzyme contexts: (ACE) that is enriched in the endothelia of the (see 1. Allowing normal development and growth of the Chapter 7). Another example of peripheral conversion of a precursor to an active hormone involves the two sequential 2. Maintaining internal

1 2 CHAPTER 1 Introduction to the Endocrine System

BOX 1.1 A List of Most Hormones and Their Sites of Production Hormones Synthesized and Secreted by Dedicated Endocrine Glands () (GH) Adrenocorticotropic hormone (ACTH) Heart Thyroid-stimulating hormone (TSH) Atrial natriuretic (ANP) Follicle-stimulating hormone (FSH) Kidney (LH) Thyroid Gland Tetraiodothyronine (T ; thyroxine) 4 (T ) 3 Parathyroid Glands (PTH) Islets of Langerhans (Endocrine Pancreas) Intestines Somatostatin Glucagon-like peptide-1 (GLP-1) Epinephrine Glucagon-like peptide-2 (GLP-2) -dependent insulinotropic peptide (GIP; gastrin inhibitory peptide) sulfate (DHEAS) Liver Hormones Synthesized by Insulin-like growth factor-I (IGF-I) Hormones Produced to a Significant Degree by -17 β Peripheral Conversion Lungs Inhibin Angiotensin II Testes Kidney Antimüllerian hormone (AMH) 1α,25-dihydroxyvitamin D Inhibin Adipose, Mammary Glands, Other Organs Hormones Synthesized in Organs with a Primary Estradiol-17β Function Other Than Endocrine Brain () Liver, Other Organs Antidiuretic hormone (ADH; ) Testosterone Corticotropin-releasing hormone (CRH) Genital , , Sebaceous Gland, Other Organs Thyrotropin-releasing hormone 5- (DHT) -releasing hormone (GnRH) Growth hormone–releasing hormone (GHRH) Many Organs Somatostatin T3 CHAPTER 1 Introduction to the Endocrine System 3

Hypothalamus

Pituitary gland

Thyroid gland

Parathyroid glands

Adrenal glands

Pancreas

Ovaries

Testes

Fig. 1.1 Major glands of the endocrine sys- tem. (From Koeppen BM, Stanton BA, ed- itors: Berne and Levy , 6th ed., Philadelphia, 2010, Mosby.)

3. Regulating the onset of reproductive maturity at BOX 1.2 Characteristics of / and the function of the in Peptide Hormones the adult In the adult, endocrine organs produce and secrete • Synthesized as prehormones or preprohormones their hormones in response to control systems • Stored in membrane-bound secretory vesicles (some­ that are tuned to set-points, or set ranges, of the levels times called secretory granules) of circulating hormones. These set-points are genetically • Regulated at the level of secretion (regulated exocy- determined but may be altered by age, circadian rhythms tosis) and synthesis (24-hour cycles or diurnal rhythms), seasonal cycles, the • Often circulate in blood unbound environment, , inflammation, and other influences. • Usually administered by injection Major forms of are caused by lack of • Hydrophilic and signal through transmembrane receptors hormone (e.g., ), excess of hormone (e.g., ) or dysfunction of receptor (hor- monal resistance). It is important to appreciate that hor- CHEMICAL NATURE OF HORMONES mones often stimulate both the differentiated function and growth of target tissues and organs. This underlies the role Hormones are classified biochemically asproteins/, of hormones in driving neoplastic transformation and can- catecholamines, hormones, and iodothyronines. cer progression (i.e., the existence of hormonally respon- The chemical nature of a hormone determines the following: sive ). The pathogenesis of these and other forms of 1. How it is synthesized, stored, and released in a regulated endocrine disease are discussed in the subsequent chapters. manner The material in this chapter covers generalizations 2. How it is carried in the blood common to all hormones or to specific groups of hor- 3. Its biologic half- (t1/2) and mode of clearance mones. The chemical nature of the hormones and their 4. Its cellular mechanism of action mechanisms of action are discussed. This presentation provides the generalized information necessary to cate- Proteins/Peptides gorize the hormones and to make predictions about the The protein and peptide hormones can be grouped into most likely characteristics of a given hormone. Some structurally related molecules that are encoded by gene of the exceptions to these generalizations are discussed families (Box 1.2). Protein/peptide hormones gain their later. specificity from their primary sequence, which 4 CHAPTER 1 Introduction to the Endocrine System

Pre-growth hormone Signal peptidase

Growth hormone A

Prepro-opiomelanocortin Signal peptidase

Pro-opiomelanocortin Pituitary specific prohormone convertases

&& ACTH Fig. 1.2 Prehormone (A) and preprohormone (B) B processing with specific examples.

confers specific higher-order structures, and from post- hypothalamic neurons produce αMSH, but not ACTH. translational modifications, such as glycosylation. The ability of cells to process the same prohormone into Protein/peptide hormones are synthesized on the polyr- different peptides is due to cell type expression of prohor- ibosome as larger preprohormones. The nascent peptides mone convertases, resulting in cell-specific processing of have at their N terminus a group of 15 to 30 amino acids the prohormone. called the signal peptide, which directs the growing poly- Protein/peptide hormones are stored in the gland as peptide through the endoplasmic reticular membrane membrane-bound secretory vesicles and are released into the cisternae. The signal peptide is enzymatically by exocytosis through the regulated secretory pathway. removed, and the protein is then transported from the cis- This means that hormones are not continually secreted, ternae to the Golgi apparatus, where it is packaged into a but rather that they are secreted in response to a stim- membrane-bound secretory vesicle that buds off into the ulus, through a mechanism of -secretion cou- cytoplasm. Posttranslational modification occurs in the pling. Regulated exocytosis is induced by an elevation of endoplasmic reticulum, Golgi apparatus, and secretory intracellular Ca2+ along with activation of other compo- vesicle. nents (e.g., small G proteins), which interact with vesic- The original gene transcript is called either a prehor- ular and components. This ultimately mone or a preprohormone (Fig. 1.2). Removing the signal leads to the fusion of the secretory vesicular membrane peptide produces either a hormone or a prohormone. A with the cell membrane and exocytosis of the vesicular prohormone is a polypeptide that requires further cleav- contents. age before the mature hormone is produced. Often this Protein/peptide hormones are soluble in aqueous sol- final cleavage occurs while the prohormone is within the vents and, with the notable exceptions of the insulin-like Golgi apparatus or the secretory vesicle. Sometimes pro- growth factors (IGFs) and growth hormone (GH), cir- hormones contain the sequence of multiple hormones. culate in the blood predominantly in an unbound form;

For example, the protein, (POMC), therefore they tend to have short biologic half- (t1/2). contains the amino acid sequences of adrenocortico- Protein hormones are removed from the circulation by (ACTH) and α-melanocyte-stimulating receptor-mediated endocytosis and lysosomal turnover hormone (αMSH). However, the pituitary corticotrope of hormone receptor complexes (see later). Many protein produces ACTH only, whereas keratinocytes and specific hormones are small enough to appear in the urine in a CHAPTER 1 Introduction to the Endocrine System 5

CH2CHCOOH BOX 1.3 Characteristics of HO Catecholamines NH2 • Derived from enzymatic modification of tyrosine Tyrosine • Stored in membrane-bound secretory vesicles • Regulated at the level of secretion (regulated exo- cytosis) and through the regulation of the enzymatic

HO CHCH2NH2 pathway required for their synthesis HO • Transported in blood free or only loosely associated OH with proteins • Often administered as an aerosol puff for opening Norepinephrine bronchioles, and several specific analogs (agonists and antagonists) can be taken orally • Hydrophilic and signal through transmembrane G- HO CHCH2NHCH3 ­protein-coupled receptors called adrenergic receptors HO OH

Epinephrine BOX 1.4 Characteristics of Steroid Fig. 1.3 Structure of the catecholamines, norepinephrine and Hormones epinephrine, and their precursor, tyrosine. • Derived from enzymatic modification of cholesterol • Cannot be stored in secretory vesicles because of physiologically active form. For example, follicle-stimu- lipophilic nature lating hormone (FSH) and luteinizing hormone (LH) are • Regulated at the level of the enzymatic pathway present in urine. Pregnancy tests using urine are required for their synthesis based on the presence of the placental LH-like hormone, • Transported in the blood bound to transport proteins human chorionic gonadotropin (hCG). (binding globulins) Proteins/peptides are readily digested if administered • Signal through intracellular receptors (nuclear hor- orally. Hence, they must be administered by injection or, mone receptor family) in the case of small peptides, through a mucous membrane • Can be administered orally (sublingually or intranasally). Because proteins/peptides do not cross cell membranes readily, they signal through transmembrane receptors. Steroid Hormones Steroid hormones are made by the , ova- Catecholamines ries, testes, and placenta (Box 1.4). Steroid hormones from Catecholamines are synthesized by the these glands fall into five categories:progestins, mineralo- and neurons and include norepinephrine, epinephrine, corticoids, , , and and dopamine (Fig. 1.3; Box 1.3). The primary hor- (Table 1.1). Progestins and the corticoids are 21-carbon monal product of the adrenal medulla is epinephrine, , whereas androgens are 19-carbon steroids and and to a lesser extent, norepinephrine. Epinephrine is estrogens are 18-carbon steroids. Steroid hormones also produced by enzymatic modifications of the amino acid include the active metabolite of vitamin D, which is a sec- tyrosine. Epinephrine and other catecholamines are osteroid (see Chapter 4). ultimately stored in secretory vesicles that are part of Steroid hormones are synthesized by a series of enzy- the regulated secretory pathway. Epinephrine is hydro- matic modifications ofcholesterol (Fig. 1.4). The enzy- philic and circulates either unbound or loosely bound matic modifications of cholesterol are of three general to albumin. Epinephrine and norepinephrine are sim- types: hydroxylations, dehydrogenations/hydrogenations, ilar to protein/peptide hormones in that they signal and breakage of carbon-carbon bonds. The purpose of through membrane receptors, called adrenergic recep- these modifications is to produce a cholesterol derivative tors. Catecholamines have short biologic half-lives that is sufficiently unique to be recognized by a specific (a few minutes) and are inactivated by . Thus progestins bind to the progesterone recep- enzymes. Inactivated forms diffuse out of cells and are tor (PR), bind to the excreted in the urine. receptor (MR), glucocorticoids bind to the 6 CHAPTER 1 Introduction to the Endocrine System

TABLE 1.1 Steroid Hormones No. of Family Carbons Specific Hormone Primary Site of Synthesis Primary Receptor Progestin 21 Progesterone Ovary placenta Progesterone receptor (PR) Glucocorticoid 21 Cortisol, Corticosterone Adrenal cortex Glucocorticoid receptor (GR) Mineralocorticoid 21 Aldosterone, 11- Adrenal cortex Mineralocorticoid receptor (MR) Deoxycorticosterone 19 Testosterone, Testis Androgen receptor (AR) Dihydrotestosterone Estrogen 18 Estradiol-17β, Estriol Ovary placenta Estrogen receptor (ER)

receptor (GR), androgens bind to the androgen receptor are easily released into the blood when a downstream ste- (AR), estrogens bind to the estrogen receptor (ER), and roidogenic enzyme within a given pathway is inactive or the active vitamin D metabolite binds to the vitamin D absent (Fig. 1.5). In comparing the ultrastructure of a pro- receptor (VDR). tein hormone–producing cell to that of a steroidogenic The complexity of action is increased cell, protein hormone–producing cells store the product in by the expression of multiple forms of each receptor. secretory granules and have extensive rough endoplasmic Additionally, there is some degree of nonspecificity reticula. In contrast, steroidogenic cells store the precur- between steroid hormones and the receptors they bind to. sor (cholesterol esters) in the form of lipid droplets, but do For example, glucocorticoids bind to the MR with high not store the product. Steroidogenic enzymes are localized affinity, and progestins, glucocorticoids, and androgens to smooth endoplasmic reticulum membrane and within can all interact with the PR, GR, and AR to some degree. An mitochondria, and these two organelles are numerous in appreciation of this “cross-talk” is important to the phy- steroidogenic cells. sician who is prescribing synthetic steroids. For example, An important feature of steroidogenesis is that steroid medroxyprogesterone acetate (a synthetic progesterone hormones often undergo further modifications (apart from given for hormone replacement therapy in postmenopausal those involved in deactivation and ) after their women) binds well to the AR as well as the PR. As discussed release from the original steroidogenic cell. This is referred subsequently, steroid hormones are lipophilic and pass to as peripheral conversion. For example, estrogen syn- through cell membranes easily. Accordingly, classic steroid thesis by the ovary and placenta requires at least two cell hormone receptors are localized intracellularly and act by types to complete the pathway of cholesterol to estrogen regulating . More recently, membrane and (see Chapters 10 and 11). This means that one cell secretes juxtamembrane receptors have been discovered that medi- a precursor, and a second cell converts the precursor to ate rapid, nongenomic actions of steroid hormones. estrogen. There is also considerable peripheral conversion Steroidogenic cell types are defined as cells that can con- of active steroid hormones. For example, the testis secretes vert cholesterol to pregnenolone, which is the first reac- sparingly little estrogen. However, adipose, muscle, and tion common to all steroidogenic pathways. Steroidogenic other tissues express the enzyme for converting testos- cells have some capacity for cholesterol synthesis but often terone (a potent androgen) to estradiol-17β. Peripheral obtain cholesterol from circulating cholesterol-rich lipo- conversion of steroids plays an important role in several proteins (low-density lipoproteins and high-density lipo- endocrine disorders (e.g., see Fig. 1.5). proteins; see Chapter 3). Pregnenolone is then further Steroid hormones are hydrophobic, and a significant modified by six or fewer enzymatic reactions. Because of fraction circulates in the blood bound to transport pro- their hydrophobic nature, steroid hormones and precur- teins (see later). These include albumin, but also the spe- sors can leave the steroidogenic cell easily and so are not cific transport proteins,–binding globulin stored. Thus steroidogenesis is regulated at the level of (SHBG) and -binding globulin (CBG) (see uptake, storage, and mobilization of cholesterol and at the later). Excretion of hormones typically involves inacti- level of steroidogenic enzyme gene expression and activity. vating modifications followed by glucuronide or sulfate Steroids are not regulated at the level of secretion of the conjugation in the liver. These modifications increase preformed hormone. A clinical implication of this mode of the water solubility of the steroid and decrease its affin- secretion is that high levels of steroid hormone precursors ity for transport proteins, allowing the inactivated steroid CHAPTER 1 Introduction to the Endocrine System 7

21 the predominant iodothyronine released by the thyroid is 22 23 18 20 26 24 T4 (3,5,3′,5′-tetraiodothyronine, also called thyroxine), 25 which acts as a circulating precursor of the active form, 12 17 27 19 11 13 16 CD14 15 T3 (3,5,3′-triiodothyronine). Thus peripheral conversion 8 1 9 through specific 5 -deiodination plays an important role in 2 10 ′ 35A B 7 thyroid function (see Chapter 6). cross 4 6 cell membranes by both diffusion and transport systems. A They are stored extracellularly in the thyroid as an integral part of the glycoprotein molecule thyroglobulin (see Chapter 6). Thyroid hormones are sparingly soluble in blood and are Cholesterol transported in blood bound to thyroid hormone–binding

globulin (TBG). T4 and T3 have long half-lives of 7 days and 24 hours, respectively. Thyroid hormones are similar to ste- roid hormones in that the thyroid hormone receptor (TR) HO is intracellular and acts as a . In fact, the TR belongs to the same gene family that includes ste- CH3 H roid hormone receptors and VDRs. Thyroid hormones can O Progesterone C O Estradiol be administered orally and sufficient hormone is absorbed intact to make this an effective mode of therapy. TRANSPORT OF HORMONES IN THE HO O CIRCULATION A significant amount of steroid and thyroid hormones is H transported in the blood bound to plasma proteins that CH2OH O Cortisol Testosterone are produced in a regulated manner by the liver. Protein C O and polypeptide hormones are generally transported free HO OH in the blood. There exists an equilibrium among the con- centrations of bound hormone, free hormone, and plasma O transport protein. O The free hormone is the biologically active form for tar- CH2OH Aldosterone get organ action, feedback control, and clearance by uptake H C O and . Consequently, in evaluating hormonal O C HO status, one must sometimes determine free hormone levels rather than total hormone levels alone. This is particularly important because hormone transport proteins themselves B O are regulated by altered endocrine and disease states. Protein binding serves several purposes. It prolongs the Fig. 1.4 Cholesterol (A) and steroid hormone derivatives (B). circulating t of the hormone. The bound hormone rep- (From Koeppen BM, Stanton BA, editors: Berne and Levy Phys- 1/2 resents a “reservoir” of hormone and as such can serve to iology, 6th ed., Philadelphia, 2010, Mosby.) buffer acute changes in hormone secretion. In addition, ste- roid and thyroid hormones are lipophilic and hydrophobic. hormone to be excreted by the kidney. Steroid compounds Binding to transport proteins prevents these hormones from are absorbed fairly readily in the GI tract and therefore simply partitioning into the cells near their site of secretion often may be administered orally. and allows them to be transported throughout the circulation. Thyroid Hormones CELLULAR RESPONSES TO HORMONES Thyroid hormones are classified as iodothyronines (Fig. 1.6) that are made by the coupling of iodinated tyrosine residues Hormones regulate essentially every major aspect of cel- through an ether linkage (Box 1.5; also see Chapter 6). Their lular function in every . Hormones control specificity is determined by the thyronine structure, but the growth and proliferation of cells. Hormones regulate also by exactly where the thyronine is iodinated. Normally, the differentiation of cells through genetic and epigenetic 8 CHAPTER 1 Introduction to the Endocrine System

Normal testis Null mutation of 17-HSD type 3 (testis-specific enzyme)

Cholesterol Cholesterol

Androstenedione Predominant secreted product of testis

17-HSD type 3 17-HSD type 3 Peripheral conversion to androgens & Testosterone estrogens

Predominant secreted Disorder of sexual development product of testis (XY, sterile, female phenotype, hyperplastic testes) Fig. 1.5 Example of the effect of an enzyme defect on steroid hormone precursors in blood.

I I BOX 1.5 Characteristics of Thyroid 3 ′ 3 NH2 Hormones

HO O CH2CHCOOH • Derived from the iodination of tyrosines, which are 5′ 5 coupled to form iodothyronines • Lipophilic, but stored in thyroid follicle cells by cova- I I lent attachment to thyroglobulin Thyroxine (T4) 3,5,3′,5′-Tetraiodothyronine • Regulated at the level of synthesis, iodination, and secretion II • Transported in blood tightly bound to proteins • Signal through intracellular receptors (nuclear hor- NH2 mone receptor family) HO O CH2CHCOOH • Can be administered orally

I 3,5,3′-Triiodothyronine (T3) Hormones regulate the expression and function of cytoso- Fig. 1.6 Structure of thyroid hormones, which are iodinated lic, membrane, and secreted proteins, and a specific hor- thyronines. mone may determine the level of its own receptor, or the receptors for other hormones. Although hormones can exert coordinated, pleiotro- changes and their ability to survive or undergo programmed pic control on multiple aspects of cell function, any given cell death. Hormones influence cellular metabolism, ionic hormone does not regulate every function in every cell composition, and transmembrane potential. Hormones type. Rather, a single hormone controls a subset of cellular orchestrate several complex cytoskeletal-associated events, functions in only the cell types that express receptors for including cell shape, migration, division, exocytosis, recy- that hormone (i.e., the target cells). Thus selective recep- cling/endocytosis, and cell-cell and cell-matrix adhesion. tor expression determines which cells will respond to a CHAPTER 1 Introduction to the Endocrine System 9 given hormone. Moreover, the differentiated epigenetic whereas constitutive activation or overexpression of state of a specific cell will determine how it will respond components can provoke a cellular response in a hor- to a hormone. Thus the specificity of hormonal responses mone-independent, unregulated manner. resides in the structure of the hormone itself, the receptor C. Amplification of the initial hormone receptor bind- for the hormone, and the cell type in which the receptor is ing–induced signal, usually by inclusion of an enzy- expressed. hormone concentrations are extremely matic step within a signaling pathway. Amplification low (picomolar to nanomolar range). Therefore a recep- can be so great that maximal response to a hormone is tor must have a high affinity, as well as specificity, for its achieved upon hormone binding to a fraction of avail- cognate hormone. able receptors. Hormone receptors fall into two general classes: trans- D. Activation of multiple divergent or convergent path- membrane receptors and intracellular receptors that ways from one hormone receptor–binding event. For belong to the nuclear hormone receptor family. example, binding of insulin to its receptor activates three separate signaling pathways. Transmembrane Receptors E. Antagonism by constitutive and regulated negative Most hormones are proteins, peptides, or catecholamines feedback reactions. This means that a signal is dampened that cannot pass through the cell membrane. Thus these hor- or terminated by opposing pathways. Gain of function of mones must interact with transmembrane protein receptors. opposing pathways can result in hormonal resistance. Transmembrane receptors are proteins that contain three Signaling pathways use several common modes of domains (proceeding from outside to inside the cell): (1) an informational transfer (i.e., intracellular messengers and extracellular domain that harbors a high-affinity binding site signaling events). These include the following: for a specific hormone; (2) one or more hydrophobic, trans- 1. Conformational shifts. Many signaling components membrane domains that span the cell membrane; and (3) a are proteins and have the ability to toggle between two cytosolic domain that is linked to signaling proteins. (or more) conformational states that alter their activity, Hormone binding to a transmembrane receptor induces a stability, or intracellular location. As discussed previ- conformational shift in all three domains of the receptor pro- ously, signaling begins with hormone receptor binding tein. This hormone receptor binding–induced conformational that induces a conformational change in the receptor change is referred to as a signal. The signal is transduced into (Fig. 1.7). The other modes of informational transfer the activation of one or more intracellular signaling mol- discussed later either regulate or are regulated by con- ecules. Signaling molecules then act on effector proteins, formational shifts in transmembrane receptors and in which, in turn, modify specific cellular functions. The combi- downstream signaling proteins. nation of hormone receptor binding (signal), activation of sig- 2. Covalent phosphorylation of proteins and lipids naling molecules (transduction), and the regulation of one or (Fig. 1.8). Enzymes that phosphorylate proteins or lipids more effector proteins is referred to as a signal transduction are called kinases, whereas those that catalyze dephos- pathway (also called simply a signaling pathway), and the phorylation are called phosphatases. Protein kinases and final integrated outcome is referred to as thecellular response. phosphatases can be classified as either tyrosine-specific Signaling pathways linked to transmembrane receptors kinases and phosphatases or serine/threonine-specific are usually characterized by the following: kinases and phosphatases. There are also mixed function A. Receptor binding followed by a conformational shift kinases and phosphatases that recognize all three resi- that extends to the cytosolic domain. The conforma- dues. An important lipid kinase is phosphatidylinositol- tional shift may result in one or more of the following: 3-kinase (PI3K; see later). The phosphorylated state of a 1. Activation of a guanine exchange function of a signaling component can alter the following: receptor. a. Activity. Phosphorylation can activate or deactivate 2. Homodimerization and/or heterodimerization of a substrate, and proteins often have multiple sites receptors to other receptors or coreceptors within of phosphorylation that induce quantitative and/or the membrane. qualitative changes in the protein’s activity. 3. Recruitment and activation of signaling proteins by b. Stability. For example, phosphorylation of proteins the cytosolic domain. can induce their subsequent ubiquitination and pro- B. Multiple, hierarchal steps in which downstream effector teasomal degradation. proteins are dependent on and driven by upstream recep- c. Subcellular location. For example, the phos- tors and signaling molecules and effector proteins. This phorylation of some nuclear transcription factors means that loss or inactivation of one or more compo- induces their translocation to and retention in the nents within the pathway leads to hormonal resistance, cytoplasm. 10 CHAPTER 1 Introduction to the Endocrine System

Hormone

Extracellular domain

Transmembrane domain

cytosolic domain

Fig. 1.7 Example of hormone-induced conformational change in transmembrane receptor. This often pro- motes dimerization of receptors as well as conformational changes in the cytosolic domain that unmasks a specific activity (e.g., guanine nucleotide exchange factor activity, tyrosine kinase activity).

ATP Tyrosine kinase ADP

N N O C C OH C COP OϪ Pi CO CO O

Tyrosine phosphatase or Activity or Stability Fig. 1.8 Phosphorylation/dephosphorylation Alter subcellular location in signal transduction pathways. In this case Recruitment of proteins phosphotyrosine is shown.

d. Recruitment and clustering of other signaling pro- degree of acetylation. Acetyl transferases drive acetyla- teins. For example, phosphorylation of the cytosolic tion, whereas deacetylases drive deacetylation. A major domain of a transmembrane receptor often induces deacetylase family is comprised of the seven sirtuins the recruitment of signaling proteins to the receptor (SIRTs). where they are phosphorylated. Recruitment hap- 4. Noncovalent guanosine nucleotide triphosphate pens because the recruited protein harbors a domain (GTP) binding to GTP-binding proteins (G pro- that specifically recognizes and binds to the phos- teins). G proteins represent a large family of molec- phorylated residue. Another important example of ular switches, which are latent and inactive when recruitment by phosphorylation is the recruitment bound to GDP, and active when bound to GTP (Fig. of the protein kinase Akt/PKB to the cell membrane, 1.9). G proteins are activated by guanine nucleotide where it is phosphorylated and activated by the pro- exchange factors (GEFs), which promote the dissoci- tein kinase, PDK1. In this case Akt/PKB and PDK1 ation of GDP and binding of GTP. G proteins have are recruited to the cell membrane by the phos- intrinsic GTPase activity. GTP is normally hydrolyzed phorylated membrane lipid, phosphatidylinositol to GDP within seconds by the , thereby ter-

3,4,5-triphosphate (PIP3). minating the transducing activity of the G protein. 3. Covalent acetylation/deacetylation of proteins. Another G-protein termination mechanism (which Acetylation (as well as phosphorylation) of histones represents a target for drug development to treat cer- and other chromatin proteins imparts epigenetic reg- tain endocrine diseases) is the family of proteins called ulation by altering chromatin structure and accessibil- regulators of G-protein signaling (RGS proteins), ity in a regulated and, in some cases, heritable manner. which bind to active G proteins and increase their Many extranuclear proteins are also regulated by their intrinsic GTPase activity. CHAPTER 1 Introduction to the Endocrine System 11

GEF

Effector G protein GDP G protein GTP protein (inactive) (active)

Fig. 1.9 G proteins in signal transduction path- Intrinsic GTPase ways. GEF, Guanine nucleotide exchange factor; RGS protein RGS, regulator of G-protein signaling.

CNG AC HCN

ATP cAMP Ionic current (e.g., Kϩ) RR PDE PKA CC E cAMP AMP R RAP GDP RAP GTP CC

Activation of Protein phosphorylation effector proteins Fig. 1.10 Cyclic AMP/PKA in signal transduction path- (membrane, cytosolic, ways. AC, Adenylyl cyclase; PDE, ; & nuclear proteins) R & C, regulatory and catalytic subunits, respectively, of protein kinase A (PKA); E, Epac (exchange protein acti- vated by cAMP); CNG, cyclic nucleotide–gated channel; Cellular response HCN, hyperpolarization-induced cyclic nucleotide–mod- ulated channel.

5. Noncovalent binding of cyclic nucleotide monophos- a. cAMP binds to the regulatory subunit of protein phates to their specific effector proteins (Fig. 1.10). kinase A (PKA; also called cAMP-dependent pro- Cyclic adenosine monophosphate (cAMP) is gener- tein kinase). Inactive PKA is a heterotetramer ated from adenosine triphosphate (ATP) by adeny- composed of two catalytic subunits and two regula- lyl cyclase, which is primarily a membrane protein. tory subunits. cAMP binding causes the regulatory Adenylyl cyclase is activated and inhibited by the G subunits to dissociate from the catalytic subunits, proteins, Gs-α and Gi-α, respectively (see later). There thereby generating two molecules of active catalytic are three general intracellular effectors of cyclic AMP PKA subunits (PKAc). PKAc phosphorylates numer- (cAMP): ous proteins on serine and threonine residues. 12 CHAPTER 1 Introduction to the Endocrine System

Natriuretic peptide

Arginine O2 eNOS

NO Citrulline GTP cGMP

R PDE PKG GMP sGC C cGMP R GTP cGMP cGMP C R PDE R GMP C C

Protein Protein phosphorylation phosphorylation

Cellular response Cellular response ( smooth muscle tone)

Fig. 1.11 Membrane-bound and soluble guanylyl cyclases. R and C, Regulatory and catalytic subunits, re- spectively, of protein kinase G (PKG). eNOS, endothelial nitric oxide synthase; NO, nitric oxide; sGC, soluble guanylyl cyclase.

Substrates of PKAc include numerous cytosolic pro- cGMP is produced from GTP by guanylyl cyclase, teins as well as transcription factors, most notably which exists in both transmembrane and soluble forms cAMP-responsive element–binding protein (CREB (Fig. 1.11). The transmembrane form of guanylyl cyclase is protein). a hormone receptor, receptor (NPR-A b. A second effector of cAMP is Epac (exchange pro- and NPR-B), for the natriuretic peptides (atrial = ANP; tein activated by cAMP), which has two isoforms. brain = BNP; C-type = CNP). The soluble form of guany- Epac proteins act as GEFs (see earlier) for small lyl cyclase is activated by another messenger, nitric oxide G proteins (called Raps). Raps in turn control a (NO). Nitric oxide is produced from molecular oxygen wide array of cell functions, including formation and arginine by the enzyme nitric oxide synthase (NOS). of cell-cell junctional complexes and cell-matrix In vascular endothelial cells, endothelial NOS (eNOS) adhesion, Ca2+ release from intracellular stores activity is the target of vasodilatory neuronal signals (e.g., (especially in cardiac muscle), and in the augmen- acetylcholine) and certain hormones (estrogen). NO then tation of glucose-dependent insulin secretion by diffuses into vascular smooth muscle and activates soluble glucagon-like peptide-1 in pancreatic islet β cells guanylyl cyclase to produce cGMP. cGMP activates pro- (see Chapter 3). tein kinase G (PKG), which phosphorylates and regulates c. cAMP (and cyclic guanosine monophosphate numerous proteins. In vascular smooth muscle, this leads [cGMP], discussed later) also binds directly to and to relaxation and vasodilation. As discussed earlier, cGMP regulates ion channels. These are of two types: also regulates ion channels. cAMP and cGMP are degraded cyclic nucleotide gated (CNG) channels and to AMP and GMP, respectively, by ­hyperpolarization-activated cyclic nucleotide (see Figs. 1.10 and 1.11), thereby terminating their signal- modulated (HCN) channels. For example, norepi- ing function. Phosphodiesterases represent a large fam- nephrine, which acts through a Gs-coupled recep- ily of proteins and display cell-specific expression. cAMP tor, increases heart rate in part through increasing a phosphodiesterases are inhibited by caffeine and other depolarizing inward K+ and Na+ current via an HCN methylxanthines. cGMP is degraded by cGMP phospho- at the sinoatrial node. diesterases, of which one isoform is inhibited by sildenafil CHAPTER 1 Introduction to the Endocrine System 13

OH C CC DAG PLC I CC PKC Protein P1P2 phosphorylation

Ca2ϩ Ca2ϩи CaM Effector IP3 Ca2ϩи CBP proteins

IP3иR Cellular response

Ca2ϩ

SER

Fig. 1.12 IP3 (inositol 1,4,5-triphosphate) and DAG (diacylglycerol) in signaling pathways. CaM, Calmodulin;

CBP, -binding proteins; IP3R, IP3 receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospho- C; SER, smooth endoplasmic reticulum.

(Viagra). In some contexts, cAMP and cGMP can modu- This leads to an increase in Ca2+ binding directly to late each other (a phenomenon called cross-talk) through numerous specific effector proteins, which leads to a the regulation of phosphodiesterases. For example, oocyte change in their activities. Additionally, Ca2+ regulates arrest is maintained by high levels of cAMP. The LH surge several effector proteins indirectly, through binding decreases cGMP in surrounding follicle cells by decreasing to the signaling protein, calmodulin. Several of the the local production of a natriuretic peptide. This results Ca2+/calmodulin targets are enzymes, which amplify in lowered oocyte cyclic GMP. Because cGMP inhibits the the initial signal of increased cytosolic Ca2+. The oocyte cAMP-specific phosphodiesterase, lowered cGMP Ca2+-dependent signal is terminated by the lowering leads to decreased cAMP, thereby allowing the oocyte to of cytosolic Ca2+ by cell membrane and endoplasmic complete the first meiotic division (seeChapter 10). reticular Ca2+ ATPases (i.e., Ca2+ pumps). 6. Generation of lipid informational molecules, which act as intracellular messengers. These include diacyl- Transmembrane Receptors Using G Proteins

glycerol (DAG) and inositol 1,4,5-triphosphate (IP3), The largest family of hormone receptors is the G-protein- which are cleaved from phosphatidylinositol 4,5-bis- coupled receptor (GPCR) family. These receptors span the

phosphate (PIP2) by membrane-bound phospholipase cell membrane seven times and are referred to as 7-helix C (PLC). DAG activates certain isoforms of protein transmembrane receptors. The G proteins that directly

kinase C (Fig. 1.12). IP3 binds to the IP3 receptor, which interact with GPCRs are termed heterotrimeric G proteins is a large complex forming a Ca2+ channel, on the endo- and are composed of an α subunit (Gα), and a β/γ subunit plasmic reticulum membrane, and promotes Ca2+ efflux dimer (Gβ/γ). The Gα subunit binds GTP and functions (see later) from the endoplasmic reticulum into the as the primary G-protein signal transducer. GPCRs are, in cytoplasm. Some isoforms of DAG-activated PKC are fact, ligand-activated GEFs (see earlier). This means that 2+ also Ca dependent, so the actions of IP3 converge on on hormone binding, the conformation of the receptor and reinforce those of DAG. The DAG signal is termi- shifts to the active state. Once active, the GPCR induces the

nated by , whereas IP3 is rapidly inactivated by exchange of GDP for GTP, thereby activating Gα. One hor- dephosphorylation. mone-bound receptor activates 100 or more G proteins. 7. Noncovalent Ca2+ binding (see Fig. 1.12). Cytosolic GTP-bound Gα then dissociates from Gβ/γ and binds to levels of Ca2+ are maintained at very low levels (i.e., and activates one or more effector proteins (Fig. 1.13). 10- 7 to 10–8 M), by either active transport of Ca2+ out How do G proteins link specific hormone receptor– of the cell, or into intracellular compartments (e.g., binding events with specific downstream effector proteins?

endoplasmic reticulum). As discussed earlier, IP3 There are at least 16 Gα proteins that show specificity with 2+ binding to the IP3 receptor increases the flow of Ca respect to cell-type expression, GPCR binding, and effec- into the cytoplasm from the endoplasmic reticulum. tor protein activation. A rather ubiquitous Gα protein is Ca2+ can also enter the cytoplasm through the regu- called Gs-α, which stimulates the membrane enzyme, ade- lated opening of Ca2+ channels in the cell membrane. nylyl cyclase, and increases the levels of another messenger, 14 CHAPTER 1 Introduction to the Endocrine System

Hormone

GPCR GPCRиHormone complex (inactive) (active)

Adenylyl cyclase GDP ␣ ␣ ␣ Phospholipase ␤ GTP GTP Effector proteins /␥ Others ␤/␥ GDP GTP Increased level of 2nd messengers

cAMP (Fig 1-13)

DAG (Fig 1-15)

IP3 Ca2ϩ (Fig 1-15)

Specific cellular response to specific hormone- Fig. 1.13 Signaling pathway for hor- GPCR signal mones that bind to GPCRs.

cAMP (see earlier). Some GPCRs couple to Gi-α, which returns the G protein to an inactive state (bound to GDP). inhibits adenylyl cyclase. A third major hormonal signaling Another termination mechanism involves desensitiza- pathway is through Gq-α, which activates phospholipase tion and endocytosis of the GPCR (Fig. 1.15). Hormone C (PLC). As discussed previously, PLC generates two lipid binding to the receptor increases the ability of GPCR messengers, DAG and IP3, from PIP2. Defects in G-protein kinases (GRKs) to phosphorylate the intracellular domain structure and expression are linked to endocrine diseases of GPCRs. This phosphorylation recruits proteins called such as pseudohypoparathyroidism (loss of Gs activity) or β-arrestins. GRK-induced phosphorylation and β-arrestin pituitary tumors (loss of intrinsic GTPase activity in Gs, binding inactivate the receptor, and β-arrestin couples the thereby extending its time in the active state). receptor to clathrin-mediated endocytotic machinery. Some GPCR-dependent signaling pathways regulate a broad GPCRs are dephosphorylated and rapidly recycled back to range of cellular responses. For example, the pancreatic the cell membrane (without hormone), whereas others are hormone, glucagon, regulates numerous aspects of hepatic degraded in lysosomes. GRK/β-arrestin-dependent inacti- metabolism (see Chapter 3). The glucagon receptor is vation and endocytosis is an important mechanism for hor- linked to the Gs-cAMP-PKA pathway, which diverges to monal desensitization of a cell after exposure to excessive regulate enzyme activity at both posttranslational and hormone. Hormone receptor endocytosis (also called recep- transcriptional levels. PKA phosphorylates and thereby tor-mediated endocytosis) is also an important mechanism activates phosphorylase kinase. Phosphorylase kinase for clearing protein and peptide hormones from the blood. phosphorylates and activates glycogen phosphorylase, which catalyzes the release of glucose molecules from gly- Receptor Tyrosine Kinases cogen. Catalytic subunits of PKA also enter the nucleus, Receptor tyrosine kinases (RTKs) can be classified into two where they phosphorylate and activate the transcription groups: the first acting as receptors for several growth fac- factor, CREB protein. Phospho-CREB then increases the tors (e.g., epidermal growth factor, platelet-derived growth transcriptional rate of genes encoding specific enzymes factor), and the second group for insulin and IGFs. The (e.g., phosphoenolpyruvate carboxykinase). former group of RTKs comprises transmembrane glyco- In summary, signaling from one GPCR can regulate a proteins with an intracellular domain containing intrinsic number of targets in different cellular compartments with tyrosine kinase activity. Growth factor binding induces different kinetics (Fig. 1.14). dimerization of the RTK within the cell membrane, fol- As mentioned, G-protein signaling is terminated by lowed by transphosphorylation of tyrosine residues, gen- intrinsic GTPase activity, converting GTP to GDP. This erating phosphotyrosine (pY). The phosphotyrosines CHAPTER 1 Introduction to the Endocrine System 15

Glucagon GPCR Hormone • GPCR Hormone • GPCR • P

GDP • Gαβγ GRK Glucagon receptor GTP • Gα Recycling β-arrestin binding GTP • Gs Pi

Adenylyl cyclase Phosphatase Inactivation of GPCR and endocytosis of hormone-GPCR–β arrestin complex cAMP by lysosomal enzymes PKA Catalytic Fig. 1.15 GPCR inactivation and endocytosis to lysosomes (de- subunit sensitization) and/or recycling back to the cell membrane in a Nucleus Cytoplasm dephosphorylated form (resensitization).

signal into a cellular response involving a change in the CREB Phosphorylase kinase expression of genes encoding proteins involved in prolif- eration and survival. The insulin receptor (IR) differs from growth factor Gluconeogenic enzyme Phosphorylase RTKs in several respects. First, the latent IR is already gene transcription dimerized by Cys-Cys bonds, and insulin binding induces a conformational change that leads to transphosphorylation of the cytoplasmic domains (Fig. 1.16). A major recruited Gluconeogenesis Glycogenolysis protein to pY residues is the insulin receptor substrate (IRS), which is then phosphorylated on tyrosine residues by the IR. The pY residues on IRS recruit the Grb2-2/SOS complex, thereby activating growth responses to insulin Hepatic glucose output through the MAP kinase pathway (see Fig. 1.16). The pY residues on the IRS also recruit the lipid kinase, PI3K, acti- Fig. 1.14 Coordinated regulation of cytoplasmic and nuclear vating and concentrating the kinase near its substrate, PIP2, events by PKA to produce a general cellular response. in the cell membrane. As discussed earlier, this ultimately leads to activation of Akt/PKB, which is required for the function to recruit proteins. One recruited protein is metabolic responses to insulin (Fig. 1.17). The IR also acti- phospholipase C, which is then activated by phosphoryla- vates a pathway involving the small G protein, TC-10 (see tion and generates the messengers DAG and IP3 from PIP2 Fig. 1.17). The small G-protein-dependent pathway and (see earlier). A second critically important protein that the Akt/PKB pathway are both required for the actions of is recruited to pY residues is the adapter protein, Grb2, insulin on glucose uptake (see Chapter 3). which is complexed with a GEF named SOS. Recruitment RTKs are downregulated by ligand-induced endocy- of SOS to the membrane allows it to activate a small, mem- tosis. Additionally, the signaling pathways from RTKs, brane-bound monomeric G protein called Ras. Ras then including IR and IRS, are inhibited by serine/threonine binds to its effector protein, Raf. Raf is a serine-specific phosphorylation, tyrosine dephosphorylation, and the kinase that phosphorylates and activates the dual-function suppressor of signaling proteins (see next section). kinase, MEK. MEK then phosphorylates and activates a mitogen-activated protein kinase (MAP kinase, also called Receptors Associated with Cytoplasmic Tyrosine ERK). Activated MAP kinases then enter the nucleus and Kinases phosphorylate and activate several transcription factors. Another class of membrane receptor falls into the cyto- This signaling pathway is referred to as the MAP kinase cas- kine receptor family and includes receptors for GH, pro- cade, and it transduces and amplifies a growth factor–RTK lactin, erythropoietin, and leptin. These receptors, which 16 CHAPTER 1 Introduction to the Endocrine System

Insulin

IR

GDP pY pY pY Ras GDP Ras GTP SoS Grb Raf IRS

Mek MekиP

MAPK MAPKиP

Transfer to nucleus

Phosphorylation of transcription factors

Change in gene expression Fig. 1.16 Signaling from the insulin receptor (a ) Cellular response through the MAPK pathway. pY, (Primarily mitogenic actions Phosphorylated tyrosine residue in of insulin) protein.

Insulin

IR PIP2 PIP3 R pY C pY pY P PI3K PP Akt/ P PDK IRS PKB

Also recruitment of Active Akt/PKB activation of PKC isoforms

Activation of Protein phosphorylation small G protein TCIO

Cellular response GLUT 4 (primarily metabolic actions Fig. 1.17 Signaling from the insu- (in vesicle) of insulin) lin receptor through the phosphati- dylinositol-3-kinase (PI3K)/Akt/PKB Insertion of pathway. PIP , Phosphatidylinositol GLUT4 into 2 cell membrane 4,5-bisphosphate; PIP3, Phosphati- dylinositol 3,4,5 trisphosphate; PKC, Increased uptake ; pY, phosphorylated of glucose tyrosine residue in protein; R and C; regulatory and catalytic subunits, re- Glucose spectively, of PI3K. CHAPTER 1 Introduction to the Endocrine System 17

Hormone/cytokine Cytokine receptor Insulin receptor

Hormone/cytokine receptor

SOCS inhibits recruitment Cytoplasm Recruitment by Recruitment via pY residues pY residues JAK JAK

pY pY

STAT STAT pY pY ↑ SOCS expression

Cellular responses Cellular responses Fig. 1.19 Role of suppressor of cytokine signaling (SOCS) protein in terminating signals from cytokine family and insulin receptors. STAT dimer

Receptor Serine/Threonine Kinase Receptors Nucleus One group of transmembrane receptors are bound and STAT regulation of activated by members of the transforming growth factor gene expression (TGF)-β family, which includes the hormones antimülle- rian hormone and inhibin. Unbound receptors exist as dissociated heterodimers, called RI and RII (Fig. 1.20). Fig. 1.18 Signaling from cytokine receptor family. Hormone binding to RII induces dimerization of RII with RI, and RII activates RI by phosphorylation. RI then acti- vates latent transcription factors called Smads. Activated exist as dimers, do not have intrinsic protein kinase activ- Smads heterodimerize with a Co-Smad, enter the nucleus, ity. Instead, the cytoplasmic domains are stably associ- and regulate specific gene expression. ated with members of the JAK kinase family (Fig. 1.18). Hormone binding induces a conformational change, Membrane Guanylyl Cyclase Receptors bringing the two JAKs associated with the dimerized As discussed previously, the membrane-bound forms of receptor closer together and causing their transphosphor- guanylyl cyclase constitute a family of a receptors for natri- ylation and activation. JAKs then phosphorylate tyrosine uretic peptides (see Fig. 1.11). The hormonal role of atrial residues on the cytoplasmic domains of the receptor. The natriuretic peptide (ANP) will be discussed in Chapter 7. pY residues recruit latent transcription factors called STAT (signal transducers and activators of transcription) Signaling from Intracellular Receptors proteins. STATs become phosphorylated by JAKs, which Steroid hormones, thyroid hormones, and 1,25-dihy- causes them to dissociate from the receptor, dimerize, droxyvitamin D act primarily through intracellular recep- and translocate into the nucleus, where they regulate gene tors. These receptors are structurally similar and are expression. members of the nuclear hormone receptor superfamily A loop has been identified for JAK/ that includes receptors for steroid hormones, thyroid STAT signaling. STATs stimulate expression of one or hormone, lipid-soluble vitamins, peroxisome prolifer- more suppressors of cytokine signaling (SOCS) proteins. ator–activated receptors (PPARs), and other metabolic SOCS proteins compete with STATs for binding to the pY receptors (liver X receptor, farnesyl X receptor). residues on cytokine receptors (Fig. 1.19). This terminates Nuclear hormone receptors act as transcriptional reg- the signaling pathway at the step of STAT activation. Recent ulators. This means that the signal of hormone receptor studies show that a SOCS protein is induced by insulin sig- binding is transduced ultimately into a change in the tran- naling. SOCS 3 protein plays a role in terminating the signal scriptional rate of a subset of the genes that are expressed from the IR, but also in reducing insulin sensitivity in hyper- within a differentiated cell type. One receptor binds to a spe- insulinemic patients. cific DNA sequence, called ahormone response element, 18 CHAPTER 1 Introduction to the Endocrine System

TGF-β–related RII/RI dimer often close to the promoter of one gene, and influences the hormones rate of transcription of that gene in a hormone-dependent­ RII manner (see later). However, multiple hormone receptor–­ binding events are collectively transduced into the regula- tion of several genes. Moreover, regulation by one hormone Cytoplasm usually includes activation and repression of the transcrip- P tion of many genes in a given cell type. Note that we have SMAD P already discussed examples of signaling to transcription factors by transmembrane receptors. Table 1.2 summarizes Co-SMAD the four general modes of hormonal regulation of gene transcription. P Nuclear hormone receptors have three major structural Active SMAD domains: an amino terminus domain (ATD), a middle Co-SMAD DNA-binding domain (DBD), and a carboxyl terminus ligand-binding domain (LBD) (Fig. 1.21). The amino terminus domain contains a hormone-independent tran- scriptional activation domain. The DNA-binding domain Nucleus contains two zinc finger motifs, which represent small Regulation of loops organized by Zn2+ binding to four cysteine residues at specific gene expression the base of each loop. The two zinc fingers and neighboring amino acids confer the ability to recognize and bind to spe- cific DNA sequences, which are calledhormone-response elements (HREs). The carboxyl terminal ligand-binding Fig. 1.20 Signaling from TGF- -related hormones. β domain contains several subdomains:

TABLE 1.2 Mechanisms by Which Hormones Regulate Gene Expression Catecholamines, Catecholamines, Hormone Type Steroid Hormones Thyroid Hormones Peptides, Proteins Peptides, Proteins Cell membrane Passes through cell Passes through cell Binds to extracellular Binds to extracellular membrane membrane, possibly domain of trans- domain of transmem- use transporter membrane receptor brane receptor Cytoplasm Binds to receptor, HRC Moves through Ultimately activates Activates a latent transcrip- translocates to nucleus cytoplasm directly cytoplasmic protein tion factor in cytoplasm, to nucleus to bind kinase, translocates TF translocates to the receptor to the nucleus nucleus Nucleus HRC binds to response Hormone binds to Phosphorylates TF, TF binds to DNA and elements (often as receptor already which binds to recruits coregulatory dimer), recruits coreg- bound to response DNA and recruits proteins, alters gene ulatory proteins and elements, HRC coregulatory pro- expression alters gene expression induces exchange teins, alters gene of coregulatory pro- expression teins, alters gene expression

Examples Cortisol T3 Glucagon Growth hormone HRC, Hormone-receptor complex; TF, transcription factor. CHAPTER 1 Introduction to the Endocrine System 19

progesterone-response element (PRE), glucocorticoid- ATD DBD LBD response element (GRE), mineralocorticoid-response element (MRE), and androgen-response element ATD (Amino Terminus Domain) (ARE). Once bound to their respective HREs, these • Ligand-independent association with coregulatory proteins receptors recruit other proteins, called coregulatory • Ligand-independent phosphorylation sites proteins, which are either coactivators or corepressors. Coactivators act to recruit other components of the tran- DBD (DNA Binding Domain) scriptional machinery and probably activate some of these • DNA binding via zinc finger domains components. Coactivators also possess intrinsic histone • Dimerization acetyltransferase (HAT) activity, which acetylates histones LBD (Ligand Binding Domain) in the region of the promoter. Histone acetylation relaxes • Ligand-binding chromatin coiling, making that region more accessible • Ligand-dependent association with coregulatory proteins to transcriptional machinery. Although the mechanistic • Dimerization details are beyond the scope of this chapter, the student • Nuclear translocation • Association with chaperone proteins should appreciate that steroid receptors can also repress gene transcription through recruitment of corepressors Fig. 1.21 Domains of nuclear hormone receptor. that possess histone deacetylase (HDAC) activity and that transcriptional activation and repression pathways are 1. Site of hormone recognition and binding induced concomitantly in the same cell. HDAC inhibitors 2. Hormone-dependent transcriptional activation are being studied in the context of treating some cancers domain because they restart the expression of silenced tumor sup- 3. Nuclear translocation signal pressor genes. 4. Binding domain for heat-shock proteins Pathway 2: Receptor is always in nucleus and exchanges 5. Dimerization subdomain corepressors with coactivators on hormone binding. This There are numerous variations in the details of nuclear pathway is used by the thyroid hormone receptors (THRs), receptor mechanisms of action. Two generalized pathways VDRs, PPARs, and retinoic acid receptors. For example, by which nuclear hormone receptors increase gene tran- the THR is bound, usually as a heterodimer, with the retinoic scription are the following (Fig. 1.22): acid X receptor (RXR). In the absence of thyroid hormone, Pathway 1: Unactivated receptor is cytoplasmic or the THR/RXR recruits corepressors. As stated earlier, core- nuclear and binds DNA and recruits coactivator proteins pressors recruit proteins with histone deacetylase (HDAC) on hormone binding. This mode is observed for the ER, activity. In contrast to histone acetylation, histone deacetyl- PR, GR, MR, and AR (i.e., steroid hormone receptors). ation allows tighter coiling of chromatin, which makes pro- In the absence of hormone, some of these receptors are moters in that region less accessible to the transcriptional held in the cytoplasm through an interaction with chap- machinery. Thus THR/RXR heterodimers are bound to thy- erone proteins (so-called heat-shock proteins because roid hormone response elements (TREs) in the absence of their levels increase in response to elevated temperatures hormone and maintain the expression of neighboring genes and other stresses). Chaperone proteins maintain the at a “repressed” level. Thyroid hormone (and other ligands stability of the nuclear receptor in an inactive configura- of this class) readily move into the nucleus and bind to their tion. Hormone binding induces a conformational change receptors. Thyroid hormone binding induces dissociation of in the receptor, causing its dissociation from heat-shock corepressor proteins, thereby increasing gene expression to proteins. This exposes the nuclear localization signal a basal level. The hormone receptor complex subsequently and dimerization domains, so receptors dimerize and recruits coactivator proteins, which further increase tran- enter the nucleus. Once in the nucleus, these receptors scriptional activity to the “stimulated” level. bind to their respective HREs. The HREs for the PR, GR, Termination of steroid hormone receptor signaling MR, and AR are inverted repeats with the recognition is poorly understood but appears to involve phosphor- sequence, AGAACANNNTGTTCT. Specificity is con- ylation, ubiquitination, and proteasomal degradation. ferred by neighboring base sequences and possibly by Circulating steroid and thyroid hormones are cleared as receptor interaction with other transcriptional factors in described previously. the context of a specific gene promoter. The ER usually In summary, hormones signal to cells through mem- binds to an inverted repeat with the recognition sequence, brane or intracellular receptors. Membrane receptors have AGGTCANNNTGACCT. The specific HREs are also rapid effects on cellular processes (e.g., enzyme activity, referred to as an estrogen-response element (ERE), cytoskeletal arrangement) that are independent of new Pathway 1 (Steroid hormones) (–) Hormone

GTFs

HRE Gene

Basal transcription

(+) Hormone Recruitment of coactivators Recruitment and activation of Coact general transcription factor

HR HR GTFs

HRE Gene Chromatin structure Stimulated transcription

Pathway 2 (Thyroid hormones, vitamin D, PPARs) (–) Hormone

Corepress Blocking general transcription factor

RXR HR

HRE Gene Chromatin structure Repressed transcription

(+) Hormone Dissociation of corepressors

RXR HR GTFs HRE Gene

Basal transcription

(+) Hormone Recruitment of coactivators Recruitment of activation of Coact general transcription factors

RXR HR GTFs

HRE Gene Chromatin structure Stimulated transcription Fig. 1.22 Two general mechanisms by which nuclear receptor and hormone complexes increase gene tran- scription. Coact, Coactivator proteins; corepress, corepressor proteins; GTFs, general transcription factors; HR, hormone receptor; HRE, hormone response element; RXR, retinoid X receptor. CHAPTER 1 Introduction to the Endocrine System 21 protein synthesis. Membrane receptors can also rapidly there exists a wide array of intracellular mechanisms regulate gene expression through either mobile kinases that terminate the signaling pathway within the target (e.g., PKA, MAPKs) or mobile transcription factors cells. Some of these are listed in Table 1.3. Note that (e.g., STATs, Smads). Steroid hormones have slower, overactivity of terminating mechanisms can lead to hor- longer-term effects that involve chromatin remodeling monal resistance. and changes in gene expression. Increasing evidence points to rapid, nongenomic effects of steroid hormones TABLE 1.3 Some Modes of Signal as well, but these pathways are still being elucidated. The presence of a functional receptor is an absolute Transduction Termination requirement for hormone action, and loss of a receptor Mechanism of Signal produces essentially the same symptoms as loss of hor- Transduction Termination Example mone. In addition to the receptor, there are fairly complex Receptor-mediated Many transmembrane pathways involving numerous intracellular messengers endocytosis linked to receptors and effector proteins. Accordingly, endocrine diseases can lysosomal degradation arise from abnormal expression or activity of any of these Phosphorylation/dephos- Serine phosphorylation signal transduction pathway components. phorylation of receptor of insulin receptor Overview of the Termination Signals or “downstream” and insulin receptor components of signaling ­substrate by other Most of what has been discussed in this chapter pathway signaling pathways describes the stimulatory arm of signal transduction. As Ubiquitination/proteasomal Steroid hormone noted earlier, all signal transduction of hormonal sig- degradation ­receptors nals must have termination mechanisms to avoid sus- Binding of an inhibitory Regulatory subunit of tained and uncontrolled stimulation of target cells. Part regulatory factor PKA of this stems from the cessation of the original stimulus Intrinsic terminating GTPase activity of for increasing a hormone’s level, and mechanisms to enzymatic activity G proteins clear the hormone (i.e., removal of signal). However,

SUMMARY 1. The endocrine system is composed of: ­membrane-bound secretory vesicles. The release of • Dedicated hormone-producing glands (pituitary, these vesicles represents a regulated mode of exocy- thyroid, parathyroid, and adrenal) tosis. Each hormone is first made as a prehormone, • Hypothalamic neuroendocrine neurons containing a signal peptide that guides the elongat- • Scattered endocrine cells that exist as clusters of ing polypeptide into the cisternae of the endoplas- endocrine-only cells (islets of Langerhans) or as cells mic reticulum. within organs that are have a nonendocrine primary • Are frequently synthesized as preprohormones. function (pancreas, GI tract, kidney) After removal of the signal peptide, the prohor- • Testes and ovaries, whose intrinsic endocrine func- mone is processed by prohormone convertases. tion is absolutely necessary for gametogenesis • Typically do not cross cell membranes and act 2. Endocrine signaling involves the secretion of a through transmembrane receptors (see later). chemical messenger, called a hormone, that cir- • Mostly circulate as free hormones, and are excreted culates in the blood and reaches an equilibrium in the urine or cleared by receptor-mediated endo- with the extracellular fluid. Hormones alter many cytosis and lysosomal degradation. functions of their target cells, tissues, and organs 4. Catecholamine hormones: through specific, high-affinity interactions with • Include the hormones, epinephrine (Epi) and nor- their receptors. epinephrine (Norepi). Epi and Norepi are deriva- 3. Protein/peptide hormones: tives of tyrosine, which is enzymatically modified • Are produced on ribosomes, become inserted into by several reactions. Ultimately, Epi and Norepi are the cisternae of the endoplasmic reticulum, tran- stored in a secretory vesicle and are released through sit the Golgi apparatus, and finally are stored in regulated exocytosis. 22 CHAPTER 1 Introduction to the Endocrine System

• Act through transmembrane GPCRs receptors • Phosphorylation and dephosphorylation, using called adrenergic receptors. kinases and phosphatases, respectively. The phos- 5. Steroid hormones: phorylation state of a protein affects activity, sta- • Include cortisol (glucocorticoid), aldosterone bility, subcellular localization, and recruitment (mineralocorticoid), testosterone, and dihydrotes- binding of other proteins. Note that phosphorylated

tosterone (androgens), estradiol (estrogen), proges- lipids such as PIP3 also play a role in signaling. terone (progestin), and 1,25 dihydroxyvitamin D3 8. Transmembrane receptor families: (secosteroid). • G-protein-coupled receptors (GPCRs) act as gua- • Are derivatives of cholesterol, which is modified by nine nucleotide exchange factors (GEFs) to acti- a series of cell-specific enzymatic reactions. vate the Gα subunit of the heterotrimeric α/β/γ • Are lipophilic and cross membranes readily. Thus G-protein complex. Depending on the type of Gα steroid hormones cannot be stored in secretory ves- subunit that is activated, this will increase cAMP icles. Steroid production is regulated at the level of levels, decrease cAMP levels, or increase protein synthesis. Several steroid hormones are produced kinase C activity and Ca2+ levels. All catecholamine to a significant extent by peripheral conversion of receptors (adrenergic receptors) are GPCRs. GPCRs precursors. are internalized by a receptor-mediated endocyto- • Circulate bound to transport proteins. Steroid hor- sis that involves GRK and β-arrestin. Endocytosis mones are cleared by enzymatic modifications that results in the lysosomal clearance of the hormone. increase their solubility in blood and decrease their The receptor may be digested in the lysosome or affinity for transport proteins. Steroid hormones may be recycled to the cell membrane. and their inactive metabolites are excreted in the • The insulin receptor is a tyrosine kinase receptor urine. that activates the Akt/PKB pathway, the G-protein • Act through intracellular receptors, which are mem- TC10-related pathway, and the MAPK pathway. bers of the nuclear hormone receptor family. Most The insulin receptor uses the scaffolding protein steroid hormone receptors reside in the cytoplasm insulin receptor substrate (IRS; four isoforms) as and are translocated to the nucleus after ligand (hor- part of its signaling to these three pathways. mone) binding. Each steroid hormone regulates the • Some protein hormones (e.g., growth hormone, expression of numerous genes in their target cells. prolactin) bind to transmembrane receptors that 6. Thyroid hormones are: belong to the cytokine receptor family. These are • Iodinated derivatives of thyronine. The term thyroid constitutively dimerized receptors that are bound by hormone typically refers to 3,5,3′,5′-tetraiodothy- janus kinases (JAKs). Hormone binding interacts ronine (T4 or thyroxine) and 3,5,3′-triiodothy- with both extracellular domains and induces JAK- ronine (T3). T4 is an inactive precursor of T3, which JAK cross-phosphorylation, followed by recruitment is produced by 5′-deiodination of T4. and binding of STAT proteins. Phosphorylation of • Synthesized and released by the thyroid STATs activates them and induces their transloca- (see Chapter 6 for more detail) tion to the nucleus, where they act as transcription • Circulate tightly bound to transport proteins factors.

• Lipophilic and cross cell membranes. T3 binds to one • Hormones that are related to transforming growth of several isoforms of thyroid hormone receptors factor-β (TGF-β), such as antimüllerian hormone, (THRs), which form heterodimers with retinoid X signal through a coreceptor (receptor I and recep- receptor (RXR) and reside bound to their response tor II) complex that ultimately signals to the nucleus elements in the nucleus in the absence of hormone. through activated Smad proteins. Hormone binding induces an exchange in the coreg- • Atrial natriuretic peptide (and related peptides) ulatory proteins that interact with the THRs. bind to a transmembrane receptor that contains 7. Protein, peptide, and catecholamine hormones signal a guanylyl cyclase domain within the cytoso- through transmembrane receptors and use several lic domain. These receptors signal by increasing common forms of informational transfer: cGMP, which activates protein kinase G (PKG) and • Conformational change cyclic nucleotide-gated channels. cGMP also regu- • Binding by activated G proteins lates selective phosphodiesterases. 2+ 2+ • Binding by Ca or Ca -calmodulin. IP3 is a major 9. Intracellular Receptors lipid messenger that increases cytosolic Ca2+ levels • Steroid hormones bind to members of the nuclear

through binding to the IP3 receptor. hormone transcription factor family. Steroid CHAPTER 1 Introduction to the Endocrine System 23

hormone receptors usually reside in the cytoplasm. 10. Thyroid hormone (T3) receptors (THRs) are related to Hormone binding induces nuclear translocation, steroid hormone receptor, but they constitutively remain dimerization, and DNA binding. Steroid hormone in the nucleus bound to thyroid hormone response DNA

receptor complexes regulate many genes in a target elements. T3 binding typically induces an exchange of cell. coregulatory proteins and altered gene expression.

SELF-STUDY PROBLEMS 1. How do protein hormones differ from steroid hormones 5. Name an example of a transmembrane receptor– in terms of their storage within an endocrine cell? associated transcription factor that translocates to the 2. How does binding to serum transport proteins influ- nucleus. ence hormone metabolism and hormone action? 6. Explain the mechanism of receptor-mediated endocy- 3. How would a large increase in the GTPase activity of tosis of a hormone that binds to a GPCR. Gs-α affect signaling through GPCRs linked to Gs-α? 7. What is the importance of the GEF activity of a GPCR 4. What role does the IRS protein play in transducing to its ability to signal? insulin receptor signaling into a growth response? 8. Explain how PLC generates two second messengers. A metabolic response?

KEY WORDS AND CONCEPTS 7-Helix transmembrane receptors Adenylyl cyclase Endocrine system Adrenal cortex Epinephrine Agonist Estrogen Androgen Estrogen receptor Androgen receptor Estrogen response element (ERE) Androgen response element (ARE) Antagonist Exocytosis β-Arrestins G-protein exchange factor (GEF) Ca2+ Ga Ca2+ ATPases Gi-α Ca2+ channels Glucocorticoid Calmodulin Glucocorticoid receptor cAMP phosphodiesterase Glucocorticoid response element (GRE) cAMP response element–binding protein (CREB) Glucuronide conjugation Catecholamine GPCR kinase (GRK) Cellular response G-protein-coupled receptor (GPCR) cGMP phosphodiesterase Gq-α Circadian (diurnal) rhythms Grb2/SOS Coactivator proteins Gs-α Corepressors GTP-binding proteins (G proteins) Corticosteroid-binding globulin Guanylyl cyclase Covalent phosphorylation of proteins and lipids Gβ/γ Cyclic AMP Heterotrimeric G proteins Cyclic GMP High-affinity receptor Cyclic nucleotide monophosphates Histone acetyltransferase (HAT) Cycloperhydrophenanthrene ring Histone deacetylase (HDAC) Cytokine receptor family Hormonal desensitization Diacylglycerol (DAG) Hormonal resistance Docking protein Hormone Effector proteins Hormone response elements (HREs)

Eicosanoids Inositol 1,4,5-triphosphate (IP3) 24 CHAPTER 1 Introduction to the Endocrine System

Insulin receptor (IR) Protein kinase G (PKG) Insulin receptor substrate (IRS) Protein/ Intracellular messengers Raf Intrinsic GTPase activity Ras Iodothyronine Receptor JAK kinase family Receptor serine/threonine kinases Receptor tyrosine kinases (RTKs) Ligand Regulated secretory pathway Ligand-activated GEF Regulators of G-protein signaling (RGS proteins) Ligand-induced endocytosis Second messenger hypothesis MEK Serine/threonine-specific kinases and phosphatases Mineralocorticoid Set-point Mineralocorticoid receptor Sex hormone–binding globulin Mineralocorticoid response element (MRE) Signal peptidase Mitogen-activated protein kinase (MAPK) Signal peptide Mixed-function kinases and phosphatases Signal recognition complex Nitric oxide (NO) Signal transduction pathway Norepinephrine Smads Nuclear receptor superfamily STAT Ovary Steroid hormone Peripheral conversion Steroidogenic cells

Phosphatidylinositol 3,4,5-triphosphate (PIP3) Stimulus-secretion coupling Phosphatidylinositol-3-kinase (PI3K) Sulfate conjugation Phospholipase C Suppressors of cytokine signaling (SOCS) proteins Phosphotyrosine (pY) Target cell PKA catalytic subunit Target organ PKA regulatory subunit Testis Placenta Thromboxanes Prehormone Thyroid hormone receptor Preprohormone Thyroid hormone–binding globulin Progesterone receptor Thyroid hormone–response element (TRE) Progesterone response element (PRE) Transforming growth factor (TGF)-β family Progestin Transport proteins Prohormone convertase Tyrosine kinases and phosphatases Prostacyclin Ultradian rhythms Vitamin D Protein kinase A (PKA) Vitamin D receptor Protein kinase B (PKB/Akt) Vitamin D response element (VRE)