Hormone actions on target cells begin when the hormone binds to a membrane receptor, forming a hormone-receptor complex. In many hormonal systems, the hormone-receptor complex is coupled to effector proteins by guanosine triphosphate (GTP)–binding proteins (G proteins). The effector proteins usually are enzymes, either adenylyl cyclase or phospholipase C. When the effector proteins are activated, a second messenger, either cAMP or IP3 (inositol 1,4,5-triphosphate), is produced, which amplifies the original hormonal signal and orchestrates the physiologic actions.
The major mechanisms of hormone action on target cells are the adenylyl cyclase mechanism, in which cAMP is the second messenger; the phospholipase C mechanism, in which IP3/Ca2+ is the second messenger; and the steroid hormone mechanism. In addition, insulin and insulin-like growth factors (IGFs) act on their target cells through a tyrosine kinase mechanism. Finally, several hormones activateguanylate cyclase, in which cyclic guanosine monophosphate (cyclic GMP, or cGMP) is the second messenger. The mechanisms of action of the major hormones are summarized in Table 9-3.
Table 9–3 Mechanisms of Hormone Action
G proteins are discussed in Chapter 2 in the context of autonomic receptors. Briefly, G proteins are a family of membrane-bound proteins that couple hormone receptors to effector enzymes (e.g., adenylyl cyclase). Thus, G proteins serve as “molecular switches” that decide whether the hormone action can proceed.
At the molecular level, G proteins are heterotrimeric (i.e., they have three subunits) proteins. The three subunits are designated alpha (α), beta (β), and gamma (γ). The α subunit can bind either guanosine diphosphate (GDP) or GTP, and it contains GTPase activity. When GDP is bound to the α subunit, the G protein is inactive; when GTP is bound, the G protein is active and can perform its coupling function. Guanosine nucleotide releasing factors (GRFs) facilitate dissociation of GDP so that GTP binds more rapidly, whereas GTPase activating factors (GAPs) facilitate hydrolysis of GTP. Thus, the relative activity of GRFs and GAPs influences the overall rate of G protein activation.
G proteins can be either stimulatory or inhibitory and are called, accordingly, Gs or Gi. Stimulatory or inhibitory activity resides in the α subunit (αs or αi). Thus, when GTP is bound to the αs subunit of a Gsprotein, the Gs protein stimulates the effector enzyme (e.g., adenylyl cyclase). When GTP is bound to the αi subunit of a Gi protein, the Gi protein inhibits the effector enzyme.
Adenylyl Cyclase Mechanism
The adenylyl cyclase/cAMP mechanism is utilized by many hormonal systems (see Table 9-3). This mechanism involves binding of a hormone to a receptor, coupling by a Gs or Gi protein, and then activation or inhibition of adenylyl cyclase, leading to increases or decreases in intracellular cAMP. cAMP, the second messenger, then amplifies the hormonal signal to produce the final physiologic actions.
The steps in the adenylyl cyclase/cAMP mechanism are shown in Figure 9-4. In this example, the hormone utilizes a Gs protein (rather than a Gi protein). The receptor–Gs–adenylyl cyclase complex is embedded in the cell membrane. When no hormone is bound to the receptor, the αs subunit of the Gs protein binds GDP. In this configuration, the Gs protein is inactive. When hormone binds to its receptor, the following steps (see Fig. 9-4) occur:
Figure 9–4 Steps involved in the adenylyl cyclase (cAMP) mechanism of action. See the text for an explanation of the circled numbers. AMP, Adenosine monophosphate; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; GDP, guanosine diphosphate; GTP, guanosine triphosphate.
1. Hormone binds to its receptor in the cell membrane, producing a conformational change in the αs subunit (Step 1), which produces two changes: GDP is released from the αs subunit and is replaced by GTP, and the αs subunit detaches from the Gs protein (Step 2).
2. The αs-GTP complex migrates within the cell membrane and binds to and activates adenylyl cyclase (Step 3). Activated adenylyl cyclase catalyzes the conversion of ATP to cAMP, which serves as the second messenger (Step 4). Although not shown, intrinsic GTPase activity in the G protein converts GTP back to GDP, and the αs subunit returns to its inactive state.
3. cAMP, via a series of steps involving activation of protein kinase A, phosphorylates intracellular proteins (Steps 5 and 6). These phosphorylated proteins then execute the final physiologic actions (Step 7).
4. Intracellular cAMP is degraded to an inactive metabolite, 5′ AMP, by the enzyme phosphodiesterase, thereby turning off the action of the second messenger.
Phospholipase C Mechanism
Hormones that utilize the phospholipase C (IP3/Ca2+) mechanism also are listed in Table 9-3. The mechanism involves binding of hormone to a receptor and coupling via a Gq protein to phospholipase C. Intracellular levels of IP3 and Ca2+ are increased, producing the final physiologic actions. The steps in the phospholipase C (IP3/Ca2+) mechanism are shown in Figure 9-5.
Figure 9–5 Steps involved in the phospholipase C (IP3/Ca2+) mechanism of action. See the text for an explanation of the circled numbers. ER, Endoplasmic reticulum; GDP, guanosine diphosphate; GTP, guanosine triphosphate; IP3, inositol 1,4,5-triphosphate; PIP2, phosphatidylinositol 4,5-diphosphate; SR, sarcoplasmic reticulum.
The receptor–Gq–phospholipase C complex is embedded in the cell membrane. With no hormone bound to the receptor, the αq subunit binds GDP. In this configuration, the Gq protein is inactive. When the hormone binds to the receptor, Gq is activated, which activates phospholipase C, in the following steps (see Fig. 9-5):
1. Hormone binds to its receptor in the cell membrane, producing a conformational change in the αq subunit (Step 1). GDP is released from the αq subunit, is replaced by GTP, and the αq subunit detaches from the Gq protein (Step 2).
2. The αq-GTP complex migrates within the cell membrane and binds to and activates phospholipase C (Step 3). Activated phospholipase C catalyzes the liberation of diacylglycerol and IP3 from phosphatidylinositol 4,5-diphosphate (PIP2), a membrane phospholipid (Step 4). The IP3 generated causes the release of Ca2+ from intracellular stores in the endoplasmic or sarcoplasmic reticulum, resulting in an increase in intracellular Ca2+concentration (Step 5).
3. Together, Ca2+ and diacylglycerol activate protein kinase C (Step 6), which phosphorylates proteins and produces the final physiologic actions (Step 7).
Catalytic Receptor Mechanisms
Some hormones bind to cell surface receptors that have, or are associated with, enzymatic activity on the intracellular side of the cell membrane. These so-called catalytic receptors include guanylyl cyclase, serine/threonine kinases, tyrosine kinases, and tyrosine kinase-associated receptors. Guanylyl cyclase catalyzes the generation of cyclic GMP from GTP. The kinases phosphorylate serine, threonine, or tyrosine on proteins and thus add negative charge in the form of the phosphate group; phosphorylation of target proteins results in conformational changes that are responsible for the hormone’s physiologic actions.
Hormones acting through the guanylyl cyclase mechanism are also listed in Table 9-3. Atrial natriuretic peptide (ANP) and related natriuretic peptides act through a receptor guanylyl cyclase mechanism as follows (see Chapters 4and 6). The extracellular domain of the receptor has a binding site for ANP, while the intracellular domain of the receptor has guanylyl cyclase activity. Binding of ANP causes activation of guanylyl cyclase and conversion of GTP to cyclic GMP. Cyclic GMP then activates cyclic GMP-dependent kinase, which phosphorylates the proteins responsible for ANP’s physiologic actions.
Nitric oxide (NO) acts through a cytosolic guanylyl cyclase as follows (see Chapter 4). NO synthase in vascular endothelial cells cleaves arginine into citrulline and NO. The just-synthesized NO diffuses out of the endothelial cells into nearby vascular smooth muscle cells, where it binds to and activates soluble, or cytosolic, guanylyl cyclase. GTP is converted to cyclic GMP, which relaxes vascular smooth muscle.
As previously discussed, numerous hormones utilize G-protein-linked receptors as part of the adenylyl cyclase and phospholipase C mechanisms (see Table 9-3). In these mechanisms, the cascade of events ultimately activates protein kinase A or protein kinase C, respectively. The activated kinases then phosphorylate serine and threonine moieties on proteins that execute the hormone’s physiologic actions. In addition, Ca2+-calmodulin-dependent protein kinase (CaMK) and mitogen-activated protein kinases (MAPKs) phosphorylate serine and threonine in the cascade of events leading to their biologic actions.
Tyrosine kinases phosphorylate tyrosine moieties on proteins and fall in two major categories. Receptor tyrosine kinases have intrinsic tyrosine kinase activity within the receptor molecule. Tyrosine kinase-associated receptors do not have intrinsic tyrosine kinase activity but associate noncovalently with proteins that do (Fig. 9-6).
Figure 9–6 Tyrosine kinase receptors. Nerve growth factor (A) and insulin (B) utilize receptor tyrosine kinases that have intrinsic tyrosine kinase activity. Growth hormone (C) utilizes a tyrosine kinase–associated receptor. NGF, nerve growth factor; JAK, Janus family of receptor-associated tyrosine kinase.
Receptor tyrosine kinases have an extracellular binding domain that binds the hormone or ligand, a hydrophobic transmembrane domain, and an intracellular domain that contains tyrosine kinase activity. When activated by hormone or ligand, the intrinsic tyrosine kinase phosphorylates itself and other proteins.
One type of receptor tyrosine kinase is a monomer (e.g., nerve growth factor [NGF] and epidermal growth factor receptors, see Fig. 9-6A). In this monomeric type, binding of ligand to the extracellular domain results in dimerization of the receptor, activation of intrinsic tyrosine kinase, and phosphorylation of tyrosine moieties on itself and other proteins, leading to its physiologic actions.
Another type of receptor tyrosine kinase is already a dimer (e.g., insulin and insulin-like growth factor [IGF] receptors, see Fig. 9-6B). In this dimeric type, binding of the ligand (e.g., insulin) activates intrinsic tyrosine kinase and leads to phosphorylation of itself and other proteins and ultimately the hormone’s physiologic actions. The mechanism of the insulin receptor is also discussed later in the chapter.
Tyrosine kinase-associated receptors (e.g., growth hormone receptors, see Fig. 9-6C) also have an extracellular domain, a hydrophobic transmembrane domain, and an intracellular domain. However, unlike the receptor tyrosine kinases, the intracellular domain does not have tyrosine kinase activity but is noncovalently “associated” with tyrosine kinase such as those in the Janus kinase family (JAK, Janus family of receptor-associated tyrosine kinase, or “just another kinase”). Hormone binds to the extracellular domain, leading to receptor dimerization and activation of tyrosine kinase in the associated protein (e.g., JAK). The associated tyrosine kinase phosphorylates tyrosine moieties on itself, the hormone receptor, and other proteins. Downstream targets of JAK include members of the STAT (signal transducers and activators of transcription) family, which cause transcription of mRNAs and ultimately new proteins involved in the hormone’s physiologic actions.
Steroid and Thyroid Hormone Mechanism
Steroid hormones and thyroid hormones have the same mechanism of action. In contrast to the adenylyl cyclase and phospholipase C mechanisms utilized by peptide hormones and involving cell membrane receptors and generation of intracellular second messengers, the steroid hormone mechanism involves binding to cytosolic (or nuclear) receptors (Fig. 9-7) that initiate DNA transcription and synthesis of new proteins. In further contrast to peptide hormones, which act quickly on their target cells (within minutes), steroid hormones act slowly (taking hours).
Figure 9–7 Structure of cytosolic (or nuclear) steroid hormone receptor. Letters A–F represent the six domains of the receptor. DNA, Deoxyribonucleic acid.
The steps in the steroid hormone mechanism (shown in Fig. 9-8) are described as follows:
Figure 9–8 Steps involved in the steroid hormone mechanism of action. See the text for an explanation of the circled numbers. DNA, Deoxyribonucleic acid; mRNA, messenger ribonucleic acid; SREs, steroid-responsive elements.
1. The steroid hormone diffuses across the cell membrane and enters its target cell (Step 1), where it binds to a specific receptor protein (Step 2) that is located in either the cytosol (as shown in Fig. 9-8) or nucleus. Steroid hormone receptors are monomeric phosphoproteins that are part of a gene superfamily of intracellular receptors. Each receptor has six domains (see Fig. 9-7). The steroid hormone binds in theE domain located near the C-terminus. The central C domain is highly conserved among different steroid hormone receptors, has two zinc fingers, and is responsible for DNA-binding. With hormone bound, the receptor undergoes a conformational change and the activated hormone-receptor complex enters the nucleus of the target cell.
2. The hormone-receptor complex dimerizes and binds (at its C domain) to specific DNA sequences, called steroid-responsive elements (SREs) located in the 5′ region of target genes (Step 3).
3. The hormone-receptor complex has now become a transcription factor that regulates the rate of transcription of that gene (Step 4). New messenger RNA (mRNA) is transcribed (Step 5), leaves the nucleus (Step 6), and is translated to new proteins (Step 7) that have specific physiologic actions (Step 8). The nature of the new proteins is specific to the hormone and accounts for the specificity of the hormone’s actions. For example, 1,25-dihydroxycholecalciferol induces the synthesis of a Ca2+-binding protein that promotes Ca2+ absorption from the intestine; aldosterone induces synthesis of Na+ channels (ENaC) in the renal principal cells that promote Na+reabsorption in the kidney; and testosterone induces synthesis of skeletal muscle proteins.