After studying this chapter, the reader should be able to:

• Distinguish between water- and lipid-soluble signaling molecules.
• Trace the steps of gene activation by steroid hormones.
• Understand the importance of the kinase cascade in signal transduction.
• Know about signal amplification by intracellular messengers and other signal transducers.

1. General information
1. Cells can send signals both to neighboring and distant cells
2. Chemical signals from the sender cell (or signaling cell) are received and processed by the target cells (or recipient cells)
3. In general, the chemical signals may be either lipid or water soluble; the recipient cell makes different responses to the two types of signals
4. Signal transduction is the acquisition and processing of signaling information by the target cell, in order to generate a cellular response to the incoming signal
5. A chain of intercommunicating signal molecules are involved in the process of signal transduction
6. Changes in the target cell can be short term or long term
2. Signaling strategies
1. Signaling is important for organism-to-organism and cell-to-cell communication; strategies vary according to the distance the signal molecule needs to travel to reach the target
2. At the inter-organismal level, some insects produce signal molecules called pheromones during mating; an example is the bombykol produced by the mulberry silk moth Bombyx mori
3. Within the organism, endocrine signals are hormones that travel long distances in the circulation system
1. Different endocrine cells synthesize structurally different hormones
2. The different hormones are mixed and diluted in the blood during circulation
3. The target cells need to recognize and retrieve the specific hormonal signal from the hormone mixture in the blood
4. Within the organism, paracrine signals travel short distances and act as local chemical mediators
1. The prostaglandins are common local mediators
1. Prostaglandins, 20-carbon fatty acid derivatives made from arachidonic acid, are mediators of the inflammatory response
2. The inflammatory response is a local response of a tissue to injury or infection
3. Prostaglandins act as vasodilators in the vascular system, leading to increased blood flow and edema; they are also involved in acute pain and fever
4. Aspirin blocks an intermediate step of prostaglandin synthesis, inhibiting cyclooxygenase activity
2. For synaptic signaling, neurotransmitters at chemical synapses also act as paracrine signals
5. Within the organism, autocrine signals are synthesized and received by the same cell, e.g., some tumor cells may produce growth factors and stimulate their own growth
6. Several discrete steps can be discerned in cell-to-cell signaling
1. The chemical signal is synthesized by the signaling cell
2. The signal is released by the signaling cell
3. The signal is transported to the target cell
4. The signal is detected by the target cell, via receptor proteins
5. The signal is received by the target cell
6. The target cell generates a specific response to the signal
7. The nature of the response is determined by the state of the target cell
8. Signals are usually short lived; however, in some instances, the signal needs to be removed in order to terminate the cellular response
3. Chemical nature of signaling molecules
1. Different types of chemicals, including derivatives of amino acids, fatty acids, cholesterol, small peptides, and proteins, have been used as cellular signals
1. Even a gas, nitric oxide (NO), serves as signal in functions such as blood vessel relaxation
2. NO as endothelium-derived relaxing factor (EDRF) leading to the production of cGMP is the topic of the 1998 Nobel award to Robert Furchgott, Louis Ignarro and Ferid Murad
3. The gas carbon monoxide (CO) may be function in nervous system
2. Steroid hormones such as testosterone are cholesterol derivatives
3. Polypeptide hormones such as insulin are proteins
4. Other hormones, such as thyroxine and epinephrine, are amino acid derivatives
5. In general, chemical signals can be classified as either hydrophilic or hydrophobic
1. Steroid and thyroid hormones are lipid soluble; most other hormones, local chemical mediators, and neurotransmitters are water soluble
2. For hydrophobic signals, they can enter the target cell via the lipid bilayer of the plasma membrane and bind to intracellular receptors
3. For hydrophilic signals, they act by binding to cell-surface receptors, because they cannot pass the plasma membrane
4. Since the hydrophilic signals cannot enter the cell, these signals, known as primary messengers, must activate a system of intracellular messengers, known as secondary messengers, for the signal transduction process
5. An example of a primary messenger is insulin; an example of a secondary messenger is cAMP

1. General information
1. Hydrophobic or lipophilic signals such as steroid hormones interact with cytosolic and nuclear receptors and affect gene expression
2. Steroid hormones induce a long duration response, in terms of hours and days (in comparison to polypeptide hormones, with a short response, within minutes and hours)
3. The steroid effects are long term because these hormones are involved in changing the patterns of gene activity of target cells
2. Steroid hormone action
1. In the bloodstream, carrier proteins bind the steroid hormones and ship them to the target cell
2. The steroid hormone diffuses through the plasma membrane and binds to the intracellular steroid hormone receptor
1. A typical receptor has 3 domains: a DNA-binding domain, a gene-activating domain, and a hormone-binding site
2. In the inactive state, the receptor has a second protein, the inhibitor protein, that bound to it
3. The binding of the steroid hormone displaces the inhibitor protein and induces a conformational change in the receptor
4. The hormone-receptor complex leaves the cytoplasm and enters the nucleus
3. In the nucleus, the activated receptor binds to and activates specific genes
1. Hormone-receptor complex acts as a transcriptional enhancer
2. Specific regions, about 15 bases in size, known as response elements, are involved; an example is the estrogen response element (ERE) with the consensus palindromic sequence AGGTCANNNTGACCT
3. Binding of active receptor induces gene transcription
4. In addition to transcriptional control, steroid hormones may prolong mRNA stability; e.g., mRNA of casein, the milk protein, has its half life increased 30-50 fold in the presence of the hormone prolactin
5. Steroid hormones induce both a primary and a secondary response in the target cell
1. This can be experimentally demonstrated by the induction of chromosomal puffs of the polytene chromosomes of salivary glands of fruit flies
1. A chromosomal puff is an expansion of a band of polytene chromosome associated with the synthesis of RNA at some locus in the band, i.e., a site of active transcription
2. After treatment with the steroid hormone ecdysone, a few puffs (regions of active gene transcription) are seen within minutes
3. These are followed later by hundreds of additional secondary puffs
2. This finding suggests that gene products of primary response in turn activate additional genes to be transcribed, resulting in a secondary response to the original hormone signal
6. Steroid hormones regulate different genes in different target cells, as seen in humans with testicular feminization syndrome
1. In these patients, despite normal testosterone production, an abnormal testosterone receptor cannot recognize the hormone signal
2. Hence, there is an absence of male secondary sexual characteristics

1. General information
1. Hydrophilic signals induce a fast response (in terms of minutes and hours) by target cells
2. Intracellular messengers are needed to transduce the extracellular chemical signals that cannot enter the target cell
3. G proteins, protein kinases, and phosphatases are important in signal transduction
4. Several intracellular messengers, including cAMP, cGMP, DAG, IP₃ and Ca⁺⁺, have been identified
5. Signal amplification and reversible phosphorylation are involved in signal transduction
2. Discovery of cAMP as a second messenger
1. The second messenger concept was proposed by Earl Sutherland
1. Sutherland and colleagues were interested in the controls of glucose metabolism
2. The blood sugar level is controlled by three hormones: epinephrine, glucagon, and insulin
3. Insulin stimulates glucose polymerization to glycogen, whereas epinephrine or glucagon stimulates glycogen breakdown to glucose
4. Sutherland found that incubation of either epinephrine or glucagon with liver slices could stimulate the activity of glycogen phosphorylase, the enzyme responsible for the breakdown of glycogen to glucose-1-phosphate
5. The same result was noted if liver homogenate instead of liver slices was used
6. However, if the liver homogenate had been separated into soluble and particulate fractions, the glycogen phosphorylase, known to be in the soluble fraction, could no longer be activated by epinephrine or glucagon
7. Addition of either epinephrine or glucagon to the particulate fraction resulted in the appearance of a new substance, which could activate glycogen phosphorylase in the soluble fraction
8. This new substance was identified as 3',5'-cyclic adenosine monophosphate (cAMP) in 1956
2. Sutherland provided additional evidence for cAMP as the second messenger
1. Addition of epinephrine or glucagon to the liver elevates the level of cAMP
2. Addition of the hormone activates plasma membrane adenylate cyclase, the enzyme that converts adenosine triphosphate into cAMP
3. The ability of epinephrine or glucagon to activate glycogen phosphorylase can be mimicked by exposing the cells directly to cAMP in the absence of the hormone
4. The activity of glycogen phosphorylase stimulation can be amplified by compounds, such as caffeine, that are inhibitors of the enzyme phosphodiesterase, an enzyme that breaks down cAMP to adenosine 5'-monophosphate (AMP)
3. Cyclic AMP was the first intracellular messenger to be discovered; others include cGMP, Ca⁺⁺, DAG, and IP₃
3. The cAMP pathway
1. From an extracellular hydrophilic signal (the first messenger, also called a ligand), via a cell-surface receptor, receptor-ligand binding leads to the activation of adenylate cyclase and cAMP production (the second messenger)
2. The major components of the plasma membrane that are involved in the cAMP pathway are the receptor, the G protein, and the adenylate cyclase
3. A collision-coupling model has been proposed for the activation of adenylate cyclase
1. The binding of ligand or signal to the receptor induces a conformational change in the receptor (a 7-pass transmembrane protein)
2. The activated receptor, in turn, activates the G protein
3. Then the activated G protein activates the cyclase
4. Ligand-triggered activation is a common mechanism of signal transduction by cell-surface receptors
5. The GTP-binding regulatory protein (G protein) is an important intermediate for signal transduction
1. G protein is a heterotrimer that is made up of 3 subunits known as a , b , and g subunits
2. The Gb g subunits act as an inhibitor of the Ga subunit
3. The inactive G protein has a GDP bound to the a subunit; upon activation by the ligand-receptor complex, there is an exchange of GTP for GDP
4. The activated Ga subunit dissociates from the Gb g inhibitor and activates the adenylate cyclase
5. Because of the intrinsic GTPase activity of the Ga subunit, hydrolysis of the bound GTP to GDP is thought to occur after 10-15 sec
6. The Ga subunit with bound GDP re-associates with the Gb g subunit, again becoming the inactive G protein
6. There are many different G proteins; G_sa activates or stimulates the adenylate cyclase and G_ia inhibits the cyclase
1. Cholera toxin, produced by the bacterium Vibrio cholera, acts on the G_sa subunit by the addition of ADP-ribose
1. Thus, the modified subunit cannot hydrolyze GTP and the G protein is always in the stimulated state
2. This leads to the release of salt and water by intestinal cells from the body to the intestinal tract and causes the patient to become dehydrated
2. In Bordetella pertussus, the causative organism of whooping cough, pertussis toxin acts on the G_ia subunit
3. Diseases involving G protein are known, such as the combined precocious puberty and pseudohypoparathyrodism
1. Two males patients have this rare combination of endocrine disorder, due to mutated G protein
2. At temperature below the body temperature, the protein is active even without ligand bounding
3. At body temperature, the protein is inactive even with ligand bounding
4. Normal testes produces testosterone in response to the pituitary hormone at puberty, the patient’s testes is active even without this signal (at 33^oC)
5. However, no parathyroid hormone is produced because the G protein is inactive
6. As this particular Ga isoform only affect this two conditions, other organs have normal functions
4. In addition to the cAMP pathway, G protein also participates in other signaling pathways (see below)
7. For the cAMP pathway, there is a rapid change in intracellular cAMP concentration during cell signaling, from 10^-6M to a 5-fold increase within seconds after hormone stimulation
8. Many hormone-induced cellular responses are mediated by cAMP; these include: epinephrine and glucose in liver, leading to glucose breakdown; thyroid stimulating hormone (TSH) in thyroid, leading to thyroxine synthesis and secretion; parathyroid hormone in bone cells, leading to bone resorption; and vassopressin in kidney, leading to water resorption
4. The inositol-phospholipid pathway
1. The inositol-phospholipid pathway is also known as the DAG/IP₃ (diacylglycerol/inositol trisphosphate) pathway, or the Ca⁺⁺ (calcium) pathway
2. The major components on the plasma membrane involved in this pathway are the receptor, the G protein, the phospholipase C, the protein kinase C, and the inositol phospholipids
1. Less than 10% of the membrane phospholipids are inositol phospholipids (also called phosphatidyl inositol or PI)
2. Phosphotidyl inositol (PI) is phosphorylated by the enzyme PI kinase to PIP (PI phosphate)
3. PIP is phosphorylated by the enzyme PIP kinase to PIP₂ (PI bisphosphate)
4. The product PIP₂ is less than 1% of total phospholipids
3. A model, similar to that for the cAMP pathway, has been proposed
1. The signal or ligand binds and activates the receptor by a conformational change
2. The receptor activates the G protein (of the PI pathway)
3. Activated G protein in turn activates phospholipase C
4. The phospholipase C cleaves PIP₂ to 2 compounds, DAG (diacylglycerol) and IP₃ (inositol trisphosphate)
5. DAG, located on the inner surface of the plasma membrane, activates protein kinase C (PKC, or C-kinase)
6. IP₃ is soluble in the cytosol; when it reaches the ER it acts as a ligand for a ligand-gated Ca⁺⁺ channel, leading to the release of Ca⁺⁺ to the cytosol from the ER compartment
7. The effect of DAG can be mimicked by plant phorbol esters such as TPA (tetradecanoylphorbol acetate, a tumor promoter); the effect of IP₃ can be mimicked by Ca⁺⁺ ionophores, such as A28317 or ionomycin
4. Although total cellular Ca⁺⁺ is high (10^-3 M), free cytosolic Ca⁺⁺ is low (10^-7 M); the free cytosolic Ca⁺⁺ concentration reaches 5X10^-6M after signal stimulation
5. Many hormone-induced cellular responses are mediated by the PI pathway; these include: acetylcholine to pancreatic b cells to secrete insulin, acetylcholine to pancreatic acinar cells to secrete digestive enzymes, antigen to mast cells to secrete histamine, platelet-derived growth factor (PDGF) to fibroblasts to initiate cell division
1. For PDGF action, the receptor is a protein-tyrosine kinase (receptor protein-tyrosine kinase); growth factor binding leads to receptor auto-phosphorylation at target Tyr residues
1. The PDGF ligand works as a dimer; the dimer cross-links two adjacent PDGF receptors on the plasma membrane and thus activates the receptor (receptor dimerization)
2. The two catalytic domains cross-phosphorylates each other on multiple Tyr residues
3. This step is called autophosphorylation (phosphorylation occurs within the receptor dimer)
2. One important recent discovery is the SH2 (src-homology 2) domain, a region of about 100 amino acids that recognizes and binds to the phospho-Tyr of other proteins only in the phosphorylated state
1. Phospholipase c has an SH2 domain
2. The SH2 domain is important for protein-protein interactions leading to the transduction of signals for growth
5. Mode of action of cAMP and Ca⁺⁺
1. The intracellular messengers cAMP and Ca⁺⁺ act by activating target protein kinases
2. Cyclic AMP activates a cAMP-dependent protein kinase (protein kinase A, PKA or A-kinase)
1. The inactive kinase is a tetramer, with 2 identical regulatory subunits inhibiting 2 catalytic subunits
2. Binding of cAMP to the regulatory subunits leads to their dissociation from the catalytic subunits
3. PKA adds phosphate groups to Ser and Thr residues of substrate proteins
4. The cAMP-dependent transcriptional factors are targets of PKA; activated factors bind to cAMP response elements of the genes and modulate transcription
3. Cyclic AMP also inactivates protein phosphatases
1. Activated PKA can phosphorylate a phosphatase inhibitor protein
2. The phosphorylated inhibitor is activated and inhibits protein phosphatase
4. Ca⁺⁺ activates calmodulin and the Ca⁺⁺/calmodulin-dependent protein kinases (Ca kinases)
1. Calmodulin is a protein of 148 amino acids, with 4 binding sites for Ca⁺⁺
2. Calmodulin is activated if its binding sites are filled with Ca⁺⁺
3. Ca⁺⁺/calmodulin in turn activates many target proteins, including Ca kinases, which are serine and threonine protein kinases
5. There are cross-links between the cAMP and PI pathways; e.g., cAMP is degraded by cAMP phosphodiesterase, an enzyme that is activated by binding to Ca⁺⁺/calmodulin
6. Another intracellular messenger is cyclic GMP (cGMP)
1. cGMP is produced by guanylate cyclase
2. It activates cGMP-dependent protein kinase (G-kinase), which is also a serine and threonine protein kinase
7. Extracellular signals are amplified by the use of second messengers during signal transduction
1. Via the enzyme cascade, there is signal amplification from 10^-9 M to 10^-10 M of extracellular signal to 10^-6 M intracellular messengers of cAMP or Ca⁺⁺
2. Cyclic AMP and Ca⁺⁺ activate protein kinases that activate many target proteins; thus, further signal amplification is achieved
8. Reversible phosphorylation reactions are important for signal transduction
1. Kinases phosphorylate and phosphatases de-phosphorylate target proteins
2. In the statement by the Nobel Academy concerning the 1992 award in medicine to Edmond Fischer and Edwin Krebs, it is observed that: "We now estimate that perhaps 1% of the genes in the entire genome encode protein kinases. These kinases regulate the function of a large proportion of the thousands of proteins in a cell."
3. To date, about half of the oncogenes are known to encode tyrosine protein kinases
6. The ras pathway
1. To date, the best characterized pathway for signal transduction is the ras pathway
2. The central mechanism is a kinase cascade
1. At least 3 kinases have been identified
2. The mitogen-activated protein kinase (MAPK) is activated by phosphorylation of both Thr and Tyr residues
3. The MAPK kinase (MAPKK) is a novel protein kinase that has the dual properties of Tyr and Ser/Thr phosphorylation
4. The MAPKK is activated by Ser and Thr phosphorylation via the action of MAPKK kinase (MAPKKK), a kinase that is similar to raf (a proto-oncogene) in mammalian cells
3. In the ras pathway, a growth factor such as the epidermal growth factor (EGF) binds and activates its own receptor, a tyrosine protein kinase, by auto-phosphorylation
4. The activated receptor turns the inactive GDP-ras into the active GTP-ras
1. Other proteins, such as ras exchange factors, aid in the release of GDP and the binding of GTP to the ras protein
2. The ras protein is a specific G protein because it is a single 21 Kd protein; thus, it is different from the heterotrimer of other G proteins
3. Mutated ras protein has been noted in human cancer
5. GTP-ras then passes the signal to raf or MAPKKK, by recruiting raf to the plasma membrane, thus initiating the kinase cascade
6. The ras pathway has been seen in yeast, fruit flies, frogs, and mammals; such findings suggest a common signal transduction among organisms
7. JAK-STAT pathway
1. This is a fast pathway for cytokine and cytokine receptors
2. The first characterized is the interferon gamma
1. A dimer of interferon leads to receptor dimerization (a heterodimer)
2. Dimerized receptor leads to the activation of JAK (Janus family of protein kinases, non-receptor protein-tyrosine kinases)
3. Activated JAK phosphorylates STAT (signal transducer and activator of transcription)
4. Monomeric STAT dimerizes at the SH2 domain (phospho-Tyr recognition)
5. Activated STAT dimer enters the nucleus and acts as trancription factor
3. This is a fast response: transcription of interferon responsive genes is up 20-fold in 15-30 min
8. Target cell adaptation
1. Organisms and cells are adaptable; if they are bombarded by a prolonged stimulus signal, target cell adaptation (or desensitization) occurs
2. Adaptation requires that the target cell becomes insensitive to the current level of signaling molecule
3. The target cells have different methods of adaptation or desensitization
1. The cell-surface receptor number can be down-regulated by endocytosis; the endocytozed receptors can then be degraded in the lysosome or they can be hidden in the endosome
2. The receptors can be made inactive by phosphorylation; alternatively, G proteins can be made inactive by phosphorylation

1. Differentiate among the different signaling strategies, with an emphasis on the distance the signal molecule needs to travel to reach its target cell.
2. Describe steroid hormone action.
3. Discuss the role of G proteins in the cAMP, PI, and ras signaling pathways.
4. Discuss the kinase cascade and the signal amplification of the signal transduction process.
5. In terms of cellular economy, compare the relative merits of reversible phosphorylation and receptor degradation for target cell adaptation.