CELL SIGNALING

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3 CELL SIGNALING

Cells respond to extracellular signals produced by other cells or by themselves. This mechanism, called cell signaling, allows cell-cell communication and is necessary for the functional regulation and integration of multicellular organisms. Our discussion in this chapter not only provides the basis for understanding normal cell function but serves also as an introduction to the role of abnormal cell signaling in human disease.

Signaling molecules are either secreted or expressed at the cell surface of one cell. Signaling molecules can bind to receptors on the surface of another cell or the same cell.

Different types of signaling molecules transmit information in multicellular organisms, and their mechanisms of action on their target cells can be diverse. Some signaling molecules can act on the cell surface after binding to cell surface receptors; others can cross the plasma membrane and bind to intracellular receptors in the cytoplasm and nucleus.

When a signaling molecule binds to its receptor, it initiates a cascade of intracellular reactions to regulate critical functions such as cell proliferation, differentiation, movement, metabolism, and behavior. Because of their critical role in the control of normal cell growth and differentiation, signaling molecules have acquired significant relevance in cancer research.

Cell signaling mechanisms

Five major types of cell-cell signaling are considered (Figure 3-1):

1. Endocrine cell signaling involves a signaling molecule, called a hormone, secreted by an endocrine cell and transported through the circulation to act on distant target cells. An example is the steroid hormone testosterone produced in the testes, which stimulates the development and maintenance of the male reproductive tract.

2. Paracrine cell signaling is mediated by a signaling molecule acting locally to regulate the behavior of a nearby cell. An example is the action of neurotransmitters produced by nerve cells and released at a synapse. See Box 3-A for a summary of the four major families of paracrine signaling molecules.

3. Autocrine cell signaling is defined by cells responding to signaling molecules that they themselves produce. A classic example is the response of cells of the immune system to foreign antigens or growth factors that trigger their own proliferation and differentiation. Abnormal autocrine signaling leads to the unregulated growth of cancer cells.

4. Neurotransmitter cell signaling, a specific form of paracrine signaling.

5. Neuroendocrine cell signaling, a specific form of endocrine signaling.

Figure 3-1 Mechanisms of hormone action

Box 3-A Paracrine cell signaling

• Paracrine signaling molecules include four major families of proteins: 1. The fibroblast growth factor (FGF) family. 2. The Hedgehog family. 3. The wingless (Wnt) family. 4. The transforming growth factor β (TGF-β) super-family.

• Each of these signaling proteins can bind to one or more receptors. Mutations of genes encoding these proteins may lead to abnormal cell-cell interaction.

• The first member of the Hedgehog family was isolated in a Drosophila mutant with bristles in a naked area in the normal fly. The most widely found hedgehog homolog in vertebrates is sonic hedgehog (Shh). Shh participates in the development of the neural plate and neural tube (see Chapter 8, Nervous Tissue). Shh binds to a transmembrane protein encoded by the patched gene and suppresses transcription of genes encoding members of the Wnt and TGF-β families and inhibits cell growth. Mutation of the patched homolog in humans (PTC) causes the Gorlin’s syndrome (rib abnormalities, cyst of the jaw, and basal cell carcinoma, a form of skin cancer).

• The Wnt family of genes is named after the Drosophila gene wingless. In vertebrates, Wnt genes encode secretory glycoproteins that specify the dorsal-ventral axis and formation of the brain, muscles, gonads, and kidneys.

• The TGF-β superfamily encodes protein forming homodimers and heterodimers. Members of this superfamily include the TGF-β family itself, the bone morphogenetic protein (BMP) family, the activin family, and the vitellogenin 1 (Vg1) family. Mutations in a member of the BMP family, cartilage-derived morphogenetic protein-1 (CDMP1), cause skeletal abnormalities. Vg1 is a signaling molecule determining the left-right axis in embryos.

Mechanisms of action of cell signaling molecules

Cell signaling molecules exert their action after binding to receptors expressed by their target cells. Target cells, in turn, can determine either a negative or positive feedback action to regulate the release of the targeting hormone (Figure 3-2).

Figure 3-2 Positive and negative feedback

Cell receptors can be expressed on the cell surface of the target cells. Some receptors are intracellular proteins in the cytosol or the nucleus of target cells. Intracellular receptors require that the signaling molecules diffuse across the plasma membrane (Figure 3-3).

Figure 3-3 Mechanism of action of steroid hormones

Steroid hormones (Box 3-B) belong to this class of signaling molecules. Steroid hormones are synthesized from cholesterol and include testosterone, estrogen, progesterone, and corticosteroids.

Box 3-B Steroid hormones

• They derive from cholesterol.

• They bind mainly to intracellular receptors in the cytosol and nucleus.

• They circulate in blood bound to a protein.

• They are nonpolar molecules.

• Steroid hormones are not stored in the producing endocrine cell.

• Steroid hormones can be administered orally and are readily absorbed in the gastrointestinal tract.

Testosterone, estrogen, and progesterone are sex steroids and are produced by the gonads. Corticosteroids are produced by the cortex of the adrenal gland and include two major classes: glucocorticoids, which stimulate the production of glucose, and mineralocorticoids, which act on the kidneys to regulate water and salt balance.

There are three cell signaling molecules that are structurally and functionally distinct from steroids but act on target cells by binding to intracellular receptors after entering the cell by diffusion across the plasma membrane. They include thyroid hormone (produced in the thyroid gland to regulate development and metabolism), vitamin D₃ (regulates calcium metabolism and bone growth), and retinoids (synthesized from vitamin A to regulate development).

Steroid receptors are members of the steroid receptor superfamily. They act as transcription factors through their DNA binding domains, which have transcription activation or repression functions. Steroid hormones and related molecules can therefore regulate gene expression.

In the androgen insensitivity syndrome (also known as the testicular feminization syndrome [Tfm]), there is a mutation in the gene expressing the testosterone receptor such that the receptor cannot bind the hormone, and hence the cells do not respond to the hormone. Although genetically male, the individual develops the secondary sexual characteristics of a female. We discuss the androgen insensitivity syndrome in Chapter 21, Sperm Transport and Maturation.

Nitric oxide

Nitric oxide is a signaling molecule. It is a simple gas synthesized from the amino acid arginine by the enzyme nitric oxide synthase. It acts as a paracrine signaling molecule in the nervous, immune, and circulatory systems. Like steroid hormones, nitric oxide can diffuse across the plasma membrane of its target cells. Unlike steroids, nitric oxide does not bind to an intracellular receptor to regulate transcription. Instead, it regulates the activity of intracellular target enzymes. The following are relevant characteristics of nitric oxide:

1. It is an unstable molecule with a limited half-life (seconds).

2. It has local effects.

3. A well-defined function of nitric oxide signaling is the dilation of blood vessels. For example, the release of the neurotransmitter acetylcholine from nerve cell endings in the blood vessel muscle cell wall stimulates the release of nitric oxide from endothelial cells.

Nitric oxide increases the activity of the second messenger cyclic guanosine monophosphate (cGMP; see later in this section) in smooth muscle cells, which then causes cell muscle relaxation and blood vessel dilation. Nitroglycerin, a pharmacologic agent used in the treatment of heart disease, is converted to nitric oxide, which increases heart blood flow by dilation of the coronary blood vessels.

Cell signaling molecules bind to cell surface receptors

A large variety of signaling molecules bind to cell surface receptors. Several groups are recognized

1. Peptides (Box 3-C): This group includes peptide hormones (insulin, glucagon, and hormones secreted by the hypophysis), neuropeptides, secreted by neurons (enkephalins and endorphins, which decrease pain responses in the central nervous system), and growth factors, which control cell growth and differentiation (nerve growth factor [NGF]; epidermal growth factor [EGF]; platelet-derived growth factor [PDGF]; and cytokines).

Box 3-C Peptide hormones

• They are synthesized as precursor molecules (prohormones).

• They are stored in membrane-bound secretory vesicles.

• They are generally water soluble (polar).

• They circulate in blood as unbound molecules.

• Peptide hormones cannot be administered orally.

• They usually bind to cell surface receptors.

NGF is a member of a family of peptides called neurotrophins, which regulate the development and viability of neurons. EGF stimulates cell proliferation and is essential during embryonic development and in the adult. PDGF is stored in blood platelets and released during clotting.

2. Neurotransmitters: These cell signaling molecules are released by neurons and act on cell surface receptors present in neurons or other type of target cells (such as muscle cells). This group includes acetylcholine, dopamine, epinephrine (adrenaline), serotonin, histamine, glutamate, and γ-aminobutyric acid (GABA). The release of neurotransmitters from neurons is triggered by an action potential. Released neurotransmitters diffuse across the synaptic cleft and bind to surface receptors on the target cells.

There are differences that distinguish the mechanism of action of neurotransmitters. For example, acetylcholine is a ligand-gated ion channel. It induces a change in conformation of ion channels to control ion flow across the plasma membrane in target cells.

As we will see soon, neurotransmitter receptors can be associated to G proteins, a class of signaling molecules linking cell surface receptors to intracellular responses.

Some neurotransmitters have a dual function. For example, epinephrine (produced in the medulla of the adrenal gland) can act as a neurotransmitter and as a hormone to induce the breakdown of glycogen in muscle cells.

3. Eicosanoids and leukotrienes: These are lipid-containing cell-signaling molecules that, in contrast to steroids, bind to cell surface receptors (Box 3-D).

Box 3-D Eicosanoids

1. They derive from polyunsaturated fatty acids with 18, 20, and 22 carbons.

2. Arachidonic acid is the main precursor.

3. This group includes prostaglandins, leukotrienes, thromboxanes, and prostacyclin.

4. They have primary autocrine and paracrine actions.

5. The synthesis of eicosanoids is regulated by hormones.

6. They usually bind to cell surface receptors.

Prostaglandins, prostacyclin, thromboxanes, and leukotrienes are members of this group of molecules. They stimulate blood platelet aggregation, inflammatory responses, and smooth muscle contraction.

Eicosanoids are synthesized from arachidonic acid. During the synthesis of prostaglandins, arachidonic acid is converted to prostaglandin H₂ by the enzyme prostaglandin synthase. This enzyme is inhibited by aspirin and anti-inflammatory drugs. Inhibition of prostaglandin synthase by aspirin reduces pain, inflammation, platelet aggregation, and blood clotting (prevention of strokes).

Pathways of intracellular signaling by cell surface receptors

When a cell-signaling molecule binds to a specific receptor, it activates a series of intracellular targets located downstream of the receptor. Several molecules associated with receptors have been identified:

1. G protein–coupled receptors (guanine nucleotide–binding proteins): Members of a large family of G proteins (more than 1000 proteins) are present at the inner leaflet of the plasma membrane (Figure 3-4).

Figure 3-4 C protein-coupled receptors

When a signaling molecule or receptor ligand binds to the extracellular portion of a cell surface receptor, its cytosolic domain undergoes a conformational change that enables binding of the receptor to a G protein. This contact activates the G protein, which then dissociates from the receptor and triggers an intracellular signal to an enzyme or ion channel. We return to the G protein when we discuss the cyclic adenosine monophosphate (cAMP) pathway.

2. Tyrosine kinases as receptor proteins (Figure 3-5): These surface receptors are themselves enzymes that phosphorylate substrate proteins on tyrosine residues. EGF, NGF, PDGF, insulin, and several growth factors are receptor protein tyrosine kinases. Most of the receptor protein tyrosine kinases consist of single polypeptides, although the insulin receptor and other growth factors consist of a pair of polypeptide chains.

Figure 3-5 Tyrosine kinases

Binding of a ligand (a growth factor) to the extracellular domain of these receptors induces receptor dimerization that results in receptor autophosphorylation (the two polypeptide chains phosphorylate one another). The autophosphorylation of the receptors determines the binding of the tyrosine kinase domain to downstream signaling molecules. Downstream signaling molecules bind to phosphotyrosine residues through domains called SH2 domains (for Src homology 2). Src (for sarcoma) is a gene present in the tumor-producing Rous sarcoma virus and encodes a protein that functions as a protein tyrosine kinase.

3. Cytokine receptors: This family of receptors stimulates intracellular protein tyrosine kinases, which are not intrinsic components of the receptor. A growth factor ligand induces the dimerization and cross-phosphorylation of the associated tyrosine kinases. Activated kinases phosphorylate the receptors, providing binding sites for downstream signaling molecules that contain the SH2 domain.

The cytokine receptor–associated tyrosine kinases belong to two families: the Src family and the Janus kinase family (JAK).

4. Receptors linked to other enzymes (protein tyrosine phosphatases and protein serine and threonine kinases): Some receptors associate with protein tyrosine phosphatases to remove phosphate groups from phosphotyrosine residues. Therefore, they regulate the effect of protein tyrosine kinases by arresting signals initiated by protein tyrosine phosphorylation.

Members of the transforming growth factor-β (TGF-β) family are protein kinases that phosphorylate serine and threonine residues (rather than tyrosine). TGF-β inhibits the proliferation of their target cells. Like tyrosine kinase and cytokine receptors, binding of ligand to the TGF-β receptor induces receptor dimerization and the cytosolic protein serine or threonine kinase domain cross-phosphorylates the polypeptide chains of the receptor.

Clinical significance: Tyrosine kinases, targets for therapeutic agents

There are two main classes of tyrosine kinases: (1) receptor tyrosine kinases are transmembrane proteins with a ligand-binding extracellular domain and a catalyic intracellular kinase domain (see Figure 3-5), and (2) nonreceptor tyrosine kinases found in the cytosol, nucleus, and inner side of the plasma membrane.

The transmembrane receptor kinase subfamily belongs to the PDGF family, which includes c-kit. The subfamily of nonreceptor tyrosine kinases includes the Src family, the Fujinami poultry sarcoma/feline sarcoma (Fps/Fes), and Fes-related (Fer) subfamily.

In the absence of a ligand, receptor tyrosine kinases are unphosphorylated and monomeric. The nonreceptor tyrosine kinase is maintained in an inactive state by cellular inhibitor proteins. Activation occurs when the inhibitors are dissociated or by recruitment to transmembrane receptors that trigger autophosphorylation. Tyrosine kinase activity terminates when tyrosine phosphatases hydrolyze tyrosyl phosphates and by induction of inhibitory molecules.

The activity of tyrosine kinases in cancer cells can be disrupted by a protein that determines unregulated autophosphorylation in the absence of a ligand, by disrupting au to regulation of the tyrosine kinase, or by overexpression of receptor tyrosine kinase and/or its ligand. Abnormal activation of tyrosine kinases can stimulate the proliferation and anticancer drug resistance of malignant cells.

Tyrosine kinase activity can be inhibited by imatinib mesylate, a molecule that binds to the adenosine triphosphate (ATP)–binding domain of the tyrosine kinase catalytic domain. Imatinib can induce hematologic remission in patients with chronic myeloid leukemia and in tumors caused by activated receptor tyrosine kinase PDGF receptor (chronic myelomonocytic leukemia) and c-kit (systemic mastocytosis and mast cell leukemias). Imatinib has been successfully used in the treatment of gastrointestinal solid tumors.

Major pathways of intracellular cell signaling

Upon ligand binding, most cell surface receptors stimulate intracellular target enzymes to transmit and amplify a signal. An amplified signal can be propagated to the nucleus to regulate gene expression in response to an external cell stimulus.

The major intracellular signaling pathways include the cAMP and cGMP pathways, the phospholipase C–Ca²⁺ pathway, the NF-κB (for nuclear factor involved in the transcription of the κ light chain gene in B lymphocytes) transcription factor pathway, the Ca²⁺-calmodulin pathway, the MAP (for mitogen-activated protein) kinase pathway, and the JAK-STAT (for signal transducers and activators of transcription) pathway.

The cAMP pathway

The intracellular signaling pathway mediated by cAMP was discovered in 1958 by Earl Sutherland while studying the action of epinephrine, a hormone that breaks down glycogen into glucose before muscle contraction.

When epinephrine binds to its receptor, there is an increase in the intracellular concentration of cAMP. cAMP is formed from adenosine triphosphate (ATP) by the action of the enzyme adenylyl cyclase and degraded to adenosine monophosphate (AMP) by the enzyme cAMP phosphodiesterase. This mechanism led to the concept of a first messenger (epinephrine) mediating a cell-signaling effect by a second messenger, cAMP. The epinephrine receptor is linked to adenylyl cyclase by G protein, which stimulates cyclase activity upon epinephrine binding.

The intracellular signaling effects of cAMP (Figure 3-6) are mediated by the enzyme cAMP-dependent protein kinase (or protein kinase A). In its inactive form, protein kinase A is a tetramer composed of two regulatory subunits (to which cAMP binds) and two catalytic subunits. Binding of cAMP results in the dissociation of the catalytic subunits. Free catalytic subunits can phosphorylate serine residues on target proteins.