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MOLECULAR CELL BIOLOGY SEVENTH EDITION

CHAPTER 15

Signal Transduction and G Protein–

Coupled Receptors

Copyright © 2013 by W. H. Freeman and Company

Figure 15.1 Overview of signaling by cell-surface receptors.

Signaling by cell-surface receptors 1. The overall process of converting extracellular signals into intracellular responses, as well as the individual steps in this process, is termed signal transduction. 2. Communication by extracellular signals usually involves the following steps (Figure 15-1) : ① synthesis of the signaling molecule by the signaling cell and its incorporation into small intracellular

vesicles (step 1), ② its release into the extracellular space by exocytosis (step 2), ③ and transport of the signal to the target cell (step 3). ④ Binding of the signaling molecule to a specific cell-surface receptor protein triggers a conformational

change in the receptor, thus activating it (step 4). ⑤ The activated receptor then activates one or more downstream signal transduction proteins or small-

molecule second messengers (step 5), ⑥ eventually leading to activation of one or more effector proteins (step 6). ⑦ The end result of a signaling cascade can be either a short-term change in cellular function, metabolism,

or movement (step 7a) or a long-term change in gene expression or development (step 7b). ⑧ Termination or down-modulation of the cellular response is caused by negative feedback from

intracellular signaling molecules (step 8) and ⑨ by removal of the extracellular signal (step 9).

1. Gap junction 2. Signal molecules & receptors

Two types of cell-to-cell communication

Molecular Biology of the Cell (4th ed)

Two types of cell-to-cell communication 1. Adjacent cells often communicate by direct cell-cell contact. Another way to coordinate the activities of

neighboring cells is through gap junction. These are specialized cell-cell junctions that can form between closely apposed plasma membranes and directly connect the cytoplasms of the joined cells via narrow water-filled channels. Thus, cells connected by gap junctions can communicate with each other directly, without having to surmount the barrier presented by the intervening plasma membranes.

2. Extracellular signaling molecules are synthesized and released by signaling cells and produce a specific response only in target cells that have receptors for the signaling molecules (Molecular Biology of the Cell).

1. Gap junction


1) Intestinal epithelial cell

2) Pancreatic acinar cell (膵臓腺胞細胞)


Chemical synapse Electric synapse

3) Nerve cell


Gap junction Gap junctions permit the rapid diffusion of small, water-soluble molecules between the cytoplasm of adjacent cells (Molecular Biology of the Cell). 1. In epithelia, gap junctions are distributed along the lateral surfaces of adjacent cells. Tight junctions

also control the flow of solutes between the cells forming an epithelial sheet. 2. For instance, binding of secretory hormones, such as secretin, to receptors on the basal plasma

membranes of pancreatic acinar cells leads to increase in the intracellular concentration of either cAMP or Ca2+ions, both of which trigger secretion of the contents of secretory vesicles. Because Ca2+and cAMP can pass through the gap junctions, hormonal stimulation of one cell triggers secretion by many.

3. In nervous tissue, some neurons are connected by gap junctions through which ions pass rapidly, thereby allowing very rapid transmission of electrical signals.

2. Receptor communication


Receptor communication

1. Most signal molecules are hydrophilic and are therefore unable to cross the plasma membrane directly; instead, they bind to cell-surface receptors, which in turn generate one or more signals inside the target cell.

2. Some small signal molecules, by contrast, diffuse across the plasma membrane and bind to receptors inside the target cell―either in the cytosol or in the nucleus. Many of these small signal molecules are hydrophobic and nearly insoluble in aqueous solutions; they are therefore transported in the bloodstream and other extracellular fluids after binding to carrier proteins, from which they dissociate before entering the target cell (Molecular Biology of the Cell).

1) Intracellular receptors Hydrophobic ligand


Intracellular receptors: transcription factors


Intracellular receptors 1. A number of small hydrophobic signal molecules diffuse directly across the plasma membrane of target cells and bind to intracellular receptor proteins. These signal molecules include steroid hormones, thyroid hormones, retinoids, and vitamin D. 2. When these signal molecules bind to their receptor proteins, they activate the receptors, which bind to DNA to regulate the transcription of specific genes. The receptors are all structurally related, being part of the nuclear receptor superfamily. The intracellular receptors for the steroid and thyroid hormones, retinoids, and vitamin D all bind to specific DNA sequences adjacent to the genes the ligand regulates. Some receptors, such as those for cortisol, are located primarily in the cytosol and enter the nucleus after ligand binding; others, such as the thyroid and retinoid receptors, are bound to DNA in the nucleus even in the absence of ligand. In either case, the inactive receptors are bound to inhibitory protein complexes, and ligand binding alters the conformation of the receptor protein, causing the inhibitory complex to dissociate. The ligand binding also causes the receptor to bind to coactivator proteins that induce gene transcription. The transcriptional response usually takes place in successive steps: ① the direct activation of a small number of specific genes occurs within about 30 minutes and constitutes

the primary response; ② the protein products of these genes in turn activate other genes to produce a delayed, secondary

response; and so on. In this way, a simple hormonal trigger can cause a very complex change in the pattern of gene expression (Molecular Biology of the Cell).

Nitric oxide (NO)

Figure 15-12a Molecular Biology of the Cell (© Garland Science 2008)

Essentials of Physiology (4th ed)

Nitric oxide (NO) 1. An important and remarkable example is the gas nitric oxide (NO), which acts as a signal molecule in both animals and plants. In mammals, one of its functions is to regulate smooth muscle contraction. Acetylcholine, for example, is released by autonomic nerves in the walls of a blood vessel, and it causes smooth muscle cells in the vessel wall to relax. The acetylcholine acts indirectly by inducing the nearby endothelial cells to make and release NO, which then signals the underlying smooth muscle cells to relax. NO gas is made by the deamination of the amino acid arginine, catalyzed by the enzyme NO synthase. Because it passes readily across membranes, dissolved NO rapidly diffuses out of the cell where it is produced and into neighboring cells. It acts only locally because it has a short half life―about 5-10 seconds―in the extracellular space before it is converted to nitrates and nitrites by oxygen and water. 2. Acetylcholine released by nerve terminals in the blood vessel wall activates NO synthase in endothelial cells lining the blood vessel, causing the endothelial cells to produce NO. The NO diffuses out of the endothelial cells and into the underlying smooth muscle cells, where it binds to and activates guanylyl cyclase to produce cyclic GMP. The cyclic GMP triggers a response that causes the smooth muscle cells to relax, enhancing blood flow through the blood vessel. In many target cells, including endothelial cells, NO binds to iron in the active site of the enzyme guanylyl cyclase, stimulating this enzyme to produce the small intracellular mediator cyclic GMP, which we discuss later. The effects of NO can occur within seconds, because the normal rate of turnover of cyclic GMP is high; a rapid degradation to GMP by a phosphodiesterase constantly balances the production of cyclic GMP from GTP by guanylyl cyclase (Figure 15-12a Molecular Biology of the Cell).

Cell surface receptor

Ligand: hydrophilic 1) Polypeptides 2) Small peptides 3) Amino acids and derivatives 4) Nucleotides and derivates 5) Fatty acids and derivatives arachidonic acid and prostaglandin


Cell surface receptor

ion channel Molecular Biology of the Cell (4th ed)

Cell surface receptor As mentioned previously, all water-soluble signal molecules (including neurotransmitters and all signal proteins) bind to specific receptor proteins on the surface of the target cells that they influence. Most cell-surface receptor proteins belong to one of three classes, defined by the transduction mechanism they use (Molecular Biology of the Cell). 1. Ion-channel-linked receptors, also known as transmitter-gated ion channels or ionotropic receptors,

are involved in rapid synaptic signaling between electrically excitable cells. This type of signaling is mediated by a small number of neurotransmitters that transiently open or close an ion channel formed by the protein to which they bind, briefly changing the ion permeability of the plasma membrane and thereby the excitability of the postsynaptic cell.

2. G-protein-linked receptors act directly to regulate the activity of a separate plasma-membrane-bound target protein, which can be either an enzyme or an ion channel. The interaction between the receptor and this target protein is mediated by a third protein, called a trimeric GTP-binding protein (G protein).

3. Enzyme-linked receptors, when activated, either function directly as enzymes or are directly associated with enzymes that they activate. They are formed by single-pass transmembrane proteins that have their ligand-binding site outside the cell and their catalytic or enzyme-binding site inside. Enzyme-linked receptors are heterogeneous in structure compared with the other two classes. The great majority, however, are protein kinases, or are associated with protein kinases, and ligand binding to them causes the phosphorylation of specific sets of proteins in the target cell.

Figure 15.2 Types of extracellular signaling.

Types of extracellular signaling In animals, signaling by extracellular molecules can be classified into three types―endocrine, paracrine, or autocrine―based on the distance over which the signal acts (Figure 15-2). 1. In addition, certain membrane-bound proteins on one cell can directly signal an adjacent cell. 2. In endocrine signaling, the signaling molecules are synthesized and secreted by signaling cells (for

example, those found in endocrine glands), transported through the circulatory system of the organism, and finally act on target cells distant from their site of synthesis.

3. In autocrine signaling, cells respond to substances that they themselves release. Some growth factors act in this fashion, and cultured cells often secrete growth factors that stimulate their own growth and proliferation.

4. Integral membrane proteins located on the cell surface also play an important role in signaling. In some cases, such membrane-bound signals on one cell bind receptors on the surface of an adjacent target cell, triggering its differentiation.

Figure 15.8 Four common intracellular second messengers.

Intracellular signal transduction The binding of ligands (“first messengers”) to many cellsurface receptors leads to a short-lived increase (or decrease) in the concentration of certain low-molecular-weight intracellular signaling molecules termed second messengers. These molecules include 3,5-cyclic AMP (cAMP), 3,5-cyclic GMP (cGMP), 1,2-diacylglycerol (DAG), and inositol 1,4,5-trisphosphate (IP3), whose structures are shown in Figure 15-8. Other important second messengers are Ca2+ and various inositol phospholipids, also called phosphoinositides, which are embedded in cellular membranes. The elevated intracellular concentration of one or more second messengers following binding of an external signaling molecule triggers a rapid alteration in the activity of one or more enzymes or nonenzymatic proteins (Figure 15-8).

Figure 15.4 Regulation of protein activity by a kinase/phosphatase switch.

- Tyr kinase and Ser/Thr kinase

Figure 15.6 GTPase switch proteins cycle between active and inactive forms.

Intracellular signal transduction 1. Activation of virtually all cell-surface receptors leads directly or indirectly to changes in protein phosphorylation through the activation of protein kinases, which add phosphate groups to specific residues of specific target proteins. Some receptors activate protein phosphatases, which remove phosphate groups from specific residues on target proteins. Phosphatases act in concert with kinases to switch the function of various proteins on or off (Figure 15-4). 2. Many signal transduction pathways utilize intracellular “switch” proteins that turn downstream proteins on or off. The most important group of intracellular switch proteins is the GTPase superfamily. All the GTPase switch proteins exist in two forms: (1) an active (“on”) form with bound GTP (guanosine triphosphate) that modulates the activity of specific target proteins and (2) an inactive (“off”) form with bound GDP (guanosine diphosphate). Conversion of the inactivate to active state is triggered by a signal (e.g., a hormone binding to a receptor) and is mediated by a guanine nucleotide exchange factor (GEF), which causes release of GDP from the switch protein. Subsequent binding of GTP, favored by its high intracellular concentration relative to its binding affinity, induces a conformational change to the active form. The rate of GTP hydrolysis regulates the length of time the switch protein remains in the active conformation and is able to signal its downstream target proteins: the slower the rate of GTP hydrolysis, the longer the protein remains in the active state. The rate of GTP hydrolysis is often modulated by other proteins. For instance, both GTPase-activating proteins (GAP) and regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis. Many regulators of G protein activity are themselves controlled by extracellular signals (Figure 15-6).

1. A signal molecule elicits different responses depending on the cell types.


1) Glucagon: A peptide hormone secreted by the pancreas glycogenolysis 2) Epinephrine: Produced in the chromaffin cells of the adrenal medulla. The fight-or-flight response (the acute stress response) Increases heart rate and blood pressure glycogenolysis

2. Different signal molecules could elicit the same cell response.

Signal molecules and cell responses 1. Different cell types are specialized to respond to acetylcholine in different ways.

The neurotransmitteracetylcholine, for example, stimulates the contraction of skeletal muscle cells, but it decreases the rate and force of contraction in heart muscle cells. This is because the acetylcholine receptor proteins on skeletal muscle cells are different from those on heart muscle cells. But receptor differences are not always the explanation for the different effects. In many cases, the same signal molecule binds to identical receptor proteins yet produces very different responses in different types of target cells, reflecting differences in the internal machinery to which the receptors are coupled. For instance, acetylcholine receptors are found on the surface of striated muscle cells, heart muscle cells, and pancreatic acinar cells. Release of acetylcholine from a neuron adjacent to a striated muscle cell triggers contraction, whereas release adjacent to a heart muscle slows the rate of contraction. Release adjacent to a pancreatic acinar cell triggers exocytosis of secretory granules that contain digestive enzymes.

2. On the other hand, different receptor-ligand complexes can induce the same cellular response in some cell types. In liver cells, for example, the binding of either glucagon to its receptors or of epinephrine to its receptors can induce degradation of glycogen and release of glucose into the blood (Molecular Biology of the Cell).

A. Enzyme 1) Receptor Tyr kinase: GF receptor (insulin R, EGFR) 2) Receptor Ser/Thr kinase: TGF-β receptor 3) Protein phosphatase: CD45 4) Guanylyl cyclase: atrial natriuretic factor (ANF) receptor B. Tyrosine kinase-linked receptor

TGF-β

TGF-β In humans TGFβ consists of three protein isoforms, TGFβ-1, TGFβ-2, and TGFβ-3, each encoded by a unique gene and expressed in both a tissue-specific and developmentally regulated fashion. Each TGFβ isoform is synthesized as part of a larger precursor that contains a pro-domain. This domain is cleaved from but remains noncovalently associated with the mature domain after the protein is secreted. Most secreted TGFβ is stored in the extracellular matrix as a latent, inactive complex containing the cleaved TGFβ precursor and a covalently bound TGFβ-binding protein called Latent TGFβ Binding Protein, or LTBP. Binding of LTBP by the matrix protein thrombospondin or by certain cell-surface integrins triggers a conformational change in LTBP that causes release of the mature, active dimeric TGFβ. Alternatively, digestion of the binding proteins by matrix metalloproteases can result in activation of TGFβ. The monomeric form of TGFβ growth factors contains 110–140 amino acids and has a compact structure with four antiparallel strands and three conserved intramolecular disulfide linkages. An additional N-terminal cysteine in each monomer links TGFβ monomers into functional homodimers and heterodimers (Figure 16-3a 6th ed, Figure 16-27).

Figure 16.28 TGF-β/Smad signaling pathway.

TGF-β/Smad signaling pathway 1. Step 1a: In some cells, TGF-β binds to the type III TGF-β receptor (RIII), which increases the

concentration of TGF-β near the cell surface and also presents TGF-β to the type II receptor (RII). Step 1b: In other cells, TGF-β binds directly to RII, a constitutively phosphorylated and active kinase.

2. Step 2: Ligand-bound RII recruits and phosphorylates the juxtamembrane segment of the type I receptor (RI), which does not directly bind TGF-β. This releases the inhibition of RI kinase activity that otherwise is imposed by the segment of RI between the membrane and kinase domain.

3. Step 3: Activated RI then phosphorylates Smad2 or Smad3 (shown here as Smad 2/3), causing a conformational change that unmasks its nuclear-localization signal (NLS).

4. Step 4: Two phosphorylated molecules of Smad2/3 bind to a co-Smad (Smad4) molecule, which is not phosphorylated, and with an importin, forming a large cytosolic complex.

5. Steps 5 and 6: After the entire complex translocates into the nucleus, Ran·GTP causes dissociation of the importin as discussed in Chapter 13.

6. Step 7: A nuclear transcription factor (e.g., TFE3) then associates with the Smad2/3/Smad4 complex, forming an activation complex that cooperatively binds in a precise geometry to regulatory sequences of a target gene.

7. Step 8: This complex then recruits transcriptional co-activators and induces gene transcription. 8. Smad 2/3 is dephosphorylated by a nuclear phosphatase (step 9) and 9. recycles through a nuclear pore to the cytosol (step 10), where it can be reactivated by another TGF-β

receptor complex (Figure 16-28).

Figure 16.29 Model of Ski-mediated down-regulation of Smad transcription-activating function.

TGF-β/Smad signaling pathway Smad signaling is regulated by additional intracellular proteins, including two cytosolic proteins called SnoN and Ski (Ski stands for “Sloan-Kettering Cancer Institute”). These proteins were originally identified as oncoproteins because they cause abnormal cell proliferation when overexpressed in cultured fibroblasts. How they accomplish this was not understood until years later when SnoN and Ski were found to bind to the Smad2/Smad4 or Smad3/Smad4 complexes formed after TGF stimulation. SnoN and Ski do not affect the ability of the Smad complexes to bind to DNA control regions. Rather, they block transcription activation by the bound Smad complexes, thereby rendering cells resistant to the growth-inhibitory actions normally induced by TGF (Figure 16-29).

Nat Rev Cancer. (2003) 3(11):807-821

TGF-β Points of TGF-β action during cancer progression (Nat Rev Cancer. (2003) 3(11):807-821). 1. Transforming growth factor-β (TGF-β) limits the growth of normal epithelium and early-stage tumours. 2. Loss of growth-inhibitory responsiveness by loss of TGF-β receptors or SMAD proteins, or by

specific loss of cytostatic gene responses, selects for more aggressively growing tumours, facilitating the acquisition of additional oncogenic mutations.

3. Tumour cells that have the lost cytostatic response but retained TGF-β signalling components can undergo epithelial–mesenchymal transdifferentiation in response to TGF-β, becoming more invasive.

Cell (2004) 118(3): 277–279

Epithelial-to-mesenchymal transition

Semin Cancer Biol. 2008;18(1):12-22

Cell Res. 2009 Feb;19(2):156-72

Epithelial–mesenchymal transdifferentiation The migratory ability of epithelial cells relies on loss of cell–cell contacts and acquisition of fibroblastic characteristics, a process that is commonly referred to as the epithelial–mesenchymal transition (EMT). Such transitions occur frequently during development and in certain cases — such as embryonic cardiac development or palate formation — are influenced by members of the TGF-β family. One of the crucial targets for inactivation or transcriptional repression during EMT is the calcium-dependent cell–cell adhesion receptor, E-cadherin. E-cadherin is commonly downregulated in many cancers, and its overexpression can suppress invasion by tumour cells; indeed, TGF-β-induced EMT coincides with loss of E-cadherin expression. TGF-β induces expression of both Snail and SIP1, which are zinc-finger transcription factors that are known to repress the E-cadherin gene (Nat Rev Cancer. (2003) 3(11):807-821).

Molecular events in junctional complexes during EMT

Nat Rev Mol Cell Biol. (2006) 7(2):131-42

Barrallo-Glmeno A et al. (2005) Development

Epithelial–mesenchymal transdifferentiation EMT is controlled by various extracellular triggers. The intracellular pathways that are activated by these triggers exhibit extensive crosstalk and have many common endpoints (Nat Rev Mol Cell Biol. (2006) 7(2):131-142).

A. Enzyme 1) Receptor Tyr kinase: GF receptor 2) Receptor Ser/Thr kinase: TGF-β receptor 3) Protein phosphatase: CD45 4) Guanylyl cyclase: atrial natriuretic factor (ANF) receptor B. Tyrosine kinase-linked receptor

Figure 16.10 General structure and activation of cytokine receptors.

Cytokine receptor Cytokine receptors do not possess intrinsic enzymatic activity. Rather, a JAK kinase is tightly bound to the cytosolic domain of all cytokine receptors. The four members of the JAK family of kinases contain an N-terminal receptor-binding domain, a C-terminal kinase domain that is normally poorly active catalytically, and a middle domain that regulates kinase activity by an unknown mechanism. 1. As in RTKs, this kinase becomes activated after ligand binding and receptor dimerization (Figure 16-10,

step 1). 2. As a result of receptor dimerization, the associated JAKs are brought close enough together so that

one can phosphorylate the other on a critical tyrosine in the activation lip (Figure 16-10, step 2). 3. As with many other kinases, phosphorylation of the activation lip leads to a conformational change that

enhances the affinity for ATP or the substrate to be phosphorylated, thereby increasing kinase activity (Figure 16-10, step 3).

Figure 16.13 (a) Activation and structure of STAT proteins.

Cytokine receptor All STAT proteins contain an N-terminal DNA-binding domain, an SH2 domain that binds to one or more specific phophotyrosines in a cytokine receptor’s cytosolic domain, and a C-terminal domain with a critical tyrosine residue. Once a monomeric STAT is bound to the receptor via its SH2 domain, the C-terminal tyrosine is phosphorylated by an associated JAK kinase (Figure 16-13a). This arrangement ensures that in a particular cell, only those STAT proteins with an SH2 domain that can bind to a particular receptor protein will be activated and only when that receptor is activated. A phosphorylated STAT dissociates spontaneously from the receptor, and two phosphorylated STAT proteins form a dimer in which the SH2 domain on each binds to the phosphotyrosine in the other. Because dimerization involves conformational changes that expose the nuclear-localization signal (NLS), STAT dimers move into the nucleus, where they bind to specific enhancers (DNA regulatory sequences) controlling target genes (Figure 16-13b) and thus alter gene expression.

All cytokines evolved from a common ancestral protein and have a similar tertiary structure consisting of four long conserved α helices folded together. The interaction of one erythropoietin molecule with two identical erythropoietin receptor (EpoR) proteins, depicted in Figure 16-9, exemplifies the binding of a cytokine to its receptor.

Figure 16.2 Overview of signal transduction pathways triggered by receptors that activate protein tyrosine kinases.

Cytokine receptor The phosphorylated target proteins can then activate one or more signaling pathways. These pathways are noteworthy because they regulate most aspects of cell proliferation, differentiation, survival, and metabolism. There are two broad categories of receptors that activate tyrosine kinases (Figure 16-2) : 1. those in which the tyrosine kinase enzyme is an intrinsic part of the receptor’s polypeptide chain

(encoded by the same gene), called the receptor tyrosine kinases (RTKs), and 2. those, such as cytokine receptors, in which the receptor and kinase are encoded by different genes

yet bound tightly together. For cytokine receptors, the tightly bound kinase is known as JAK kinase. Both classes of receptors activate similar intracellular signal transduction pathways, and we therefore consider them together in this section..

Figure 16.8 Erythropoietin and formation of red blood cells (erythrocytes).

Cytokine receptor Erythroid progenitor cells, called colony-forming units erythroid (CFU-E), are derived from hematopoietic stem cells, which also give rise to progenitors of other blood cell types. In the absence of erythropoietin (Epo), CFU-E cells undergo apoptosis. Binding of Epo to its receptors on a CFU-E induces transcription of several genes whose encoded proteins prevent programmed cell death (apoptosis), allowing the cell to survive and undergo a program of three to five terminal cell divisions (Figure 16-8).

Figure 16.14 Two mechanisms for terminating signal transduction from the erythropoietin receptor (EpoR).

Cytokine receptor Signal-induced transcription of target genes for too long a period can be as dangerous for the cell as too little induction. Thus cells must be able to turn off a signaling pathway quickly unless the extracellular signal remains continuously present. In various progenitor cells, two classes of proteins serve to dampen signaling from cytokine receptors, one over the short term (minutes) and the other over longer periods of time (Figure 16-14). 1. Short-term regulation: SHP1, a phosphotyrosine phosphatase, is present in an inactive form in

unstimulated cells. Binding of an SH2 domain in SHP1 to a particular phosphotyrosine in the activated receptor unmasks its to particular phophotyrosine in the activated receptor unmasks its phophatase catalytic site and positions it near the phosphorylated tyrosine in the lip region of JAK2. Removal of the phosphate from this tyrosine inactivates the JAK kinase.

2. Long-term regulation: SOCS proteins, whose expression is induced by STAT proteins in erythropoietin-stimulated erythroid cells, inhibit or permanently terminate signaling over longer time periods. Binding of SOCS to phosphotyrosine residues on the EpoR or JAK2 blocks binding of other signaling proteins (left). The SOCS box can also target proteins such as JAK2 for degradation by the ubiquitin-proteasome pathway (right). Similar mechanisms regulate signaling from other cytokine receptors.

A. Enzyme 1) Receptor Tyr kinase: GF receptor (insulin R, EGFR) 2) Receptor Ser/Thr kinase: TGF-β receptor 3) Protein phosphatase: CD45 4) Guanylyl cyclase: atrial natriuretic factor (ANF) receptor B. Tyrosine kinase-linked receptor

Protein phosphatase: CD45 1. There are a large number of transmembrane protein tyrosine phosphatases, but the functions of most of them are unknown. At least some are thought to function as receptors; as this has not been directly demonstrated, however, they are referred to as receptor like tyrosine phosphatases. They all have a single transmembrane segment and usually possess two tyrosine phosphatase domains on the cytosolic side of theplasma membrane. An important example is the CD45 protein, which is found on the surface of all white blood cells and has an essential role in the activation of both T and B lymphocytes by foreign antigens. The ligand that is presumed to bind to the extracellular domain of the CD45 protein has not been identified (http://www.ncbi.nlm.nih.gov/books/NBK26822/). 2. For simplicity, signal transduction pathways can be grouped into several basic types, based on the sequence of intracellular events. In one very common type of signal transduction pathway (Figure 16-1a), ligand binding to a receptor triggers activation of a receptor-associated kinase. This kinase may be an intrinsic part of the receptor protein or be tightly bound to the receptor. These kinases often directly phosphorylate and activate a variety of signal transduction proteins, including transcription factors located in the cytosol (Figure 16-1a).

Wnt signaling

Figure 16.30 Wnt signaling pathway.

Wnt signaling 1. A current model of the Wnt pathway is shown in Figure 16-30. The central player in intracellular Wnt signal transduction is called β-catenin in vertebrates and Armadillo in Drosophila. This remarkable protein functions both as a transcriptional activator and as a membrane–cytoskeleton linker protein. ① In the absence of a Wnt signal, β-catenin is phosphorylated by a complex containing GSK3β, a

protein kinase; the adenomatosis polyposis coli (APC) protein, an important human tumor suppressor; and Axin, a scaffolding protein. Phosphorylated β-catenin is ubiquitinated and then degraded in proteasomes.

② In the presence of Wnt, β-catenin is stabilized and translocates to the nucleus. There, it is believed to associate with the TCF transcription factor to activate expression of particular target genes (e.g.,wg, cyclin D1, myc, and metalloprotease genes), depending on cell type. Findings from genetic studies have shown that Wnt induced stabilization of β-catenin depends on Dishevelled (Dsh) protein. In the presence of Wnt, Dsh and the Lrp membrane protein appear to interact with components of the phosphorylation complex, thereby inhibiting the phosphorylation and subsequent degradation of β-catenin. The importance of β-catenin stability and location means that Wnt signals affect a critical balance between the three pools of β-catenin in the cytoskeleton, cytosol, and nucleus.

2. Wnt signals help control numerous critical developmental events, such as gastrulation, brain development, limb patterning, and organogenesis. Disturbances in signal transduction through the Wnt pathway and many other developmentally important signaling pathways are associated with various human cancers. Several extracellular activators can trigger EMT, that extensive crosstalk exists between the signalling pathways that activate and repress EMT, and that EMT-inducing signalling pathways have many common endpoints, including downregulation of E-cadherin expression and expression of EMT-associated genes. he activity of GSK3β is inhibited by the AKT/PKB (protein kinase B), Wnt and Hedgehog pathways, each of which regulate EMT (Nat Rev Mol Cell Biol. (2006) 7(2):131-142).

Hedgehog signaling

Figure 16.31 Processing of Hedgehog (Hh) precursor protein.

Hedgehog signaling The Hedgehog signal is secreted from cells as a 45-kDa precursor protein. Cleavage of this secreted precursor produces a 20-kDa N-terminal fragment, which is associated with the plasma membrane and contains the inductive activity, and a 25-kDa C-terminal fragment. As depicted in Figure 16-31, this process includes adding cholesterol to a glycine residue, splitting the molecule into two fragments, and leaving the N-terminal signaling fragment with an attached hydrophobic cholesterol moiety. A second modification to Hedgehog, the addition of a palmitoyl group to the N-terminus, makes the protein even more hydrophobic. Together, the two modifications may tether Hedgehog to cells, thereby affecting its range of action in tissue (Figure 16-31).

Figure 16.32 Hedgehog signaling in flies.

Figure 16.33 Hedgehog signaling in vertebrates.

Hedgehog signaling 1. Figure 16-32 depicts a current model of the Hedgehog pathway. Although the signal-transduction mechanisms are only partly understood, the pathway includes a cytoplasmic complex of proteins consisting of Fused (Fu), a serinethreonine kinase; Costal-2 (Cos-2), a microtubule-associated kinesin-like protein; and Cubitis interruptus (Ci), a transcription factor (Figure 16-32). ① In the absence of Hedgehog, when Patched inhibits Smoothened, these three proteins form a

complex that binds to microtubules in the cytoplasm. Proteolytic cleavage of Ci in this complex generates a Ci fragment that translocates to the nucleus and represses target-gene expression.

② In the presence of Hedgehog, which relieves the inhibition of Smoothened, the complex of Fu, Cos-2, and Ci is not associated with microtubules, cleavage of Ci is blocked, and an alternatively modified form of Ci is generated. After translocating to the nucleus, this Ci form binds to the transcriptional coactivator CREB-binding protein (CBP), promoting the expression of target genes.

2. In vertebrates, Hedgehog signaling occurs in primary cilia, but otherwise the overall process is similar to that in flies. Hh signaling pathway in vertebrates shares many features with the Drosophila pathway, but there are also some striking differences. First, mammalian genomes contain three hh genes and two ptc genes, which are expressed differentially among various tissues. Second, mammals express three Gli transcription factors that divide up the roles of the single Ci protein in Drosophila. All other components of the Hh pathway also are conserved. At the same time degradation of Gli to a repressor fragment is blocked, and the motor protein Kif7 moves Gli to the tip of the cilium. There it becomes activated by Smo by a mechanism as yet unknown, and then another motor protein, a dynein, moves the activated Gli to the base of the cilium. As in flies, this active transcription factor them moves into the nucleus, where it can activate expression of multiple target genes (Figure 16-33).

Notch signaling

Figure 16.35 Notch/Delta signaling pathway.

Notch signaling Both Notch and its ligand Delta are transmembrane proteins with numerous EGF-like repeats in their extracellular domains. They participate in a highly conserved and important type of cell differentiation in both invertebrates and vertebrates, called lateral inhibition, in which adjacent and developmentally equivalent cells assume completely different fates (Figure 16-35). 1. Notch protein is synthesized as a monomeric membrane protein in the endoplasmic reticulum, where it

binds presenilin 1, a multispanning membrane protein; the complex travels first to the Golgi and then on to the plasma membrane. In the Golgi, Notch undergoes a proteolytic cleavage that generates an extracellular subunit and a transmembrane cytosolic subunit; the two subunits remain noncovalently associated with each other in the absence of interaction with Delta residing on another cell.

2. Binding of Notch to Delta triggers two proteolytic cleavages in the responding cell. The second cleavage, within the hydrophobic membrane-spanning region of Notch, is catalyzed by presenilin 1 and releases the Notch cytosolic segment, which immediately translocates to the nucleus. Such signal-induced regulated intramembrane proteolysis (RIP) also occurs in the response of cells to high cholesterol and to the presence of unfolded proteins in the endoplasmic reticulum

Figure 16.36 Proteolytic cleavage of APP and Alzheimer’s disease.

Intramembrane proteolysis Proteolytic cleavage of APP, a neuronal plasma membrane protein : Presenilin 1 (PS1) was first identified as the product of a gene that commonly is mutated in patients with an early-onset autosomal dominant form of Alzheimer’s disease. A major pathologic change associated with Alzheimer’s disease is accumulation in the brain of amyloid plaques containing aggregates of a small peptide containing 42 residues termed Aβ42. This peptide is derived by proteolytic cleavage of APP (amyloid precursor protein), a cell-surface protein of unknown function expressed by neurons. APP actually undergoes cleavage by two pathways (Figure 16-36). In each pathway the initial cleavage occurs within the extracellular domain, catalyzed by α- or β-secretase; γ-secretase then catalyzes a second cleavage at the same intramembrane site in both pathways. The pathway initiated by α-secretase, which involves the same membrane-bound metalloprotease TACE that cleaves Notch, generates a 26-residue peptide that apparently does no harm. The pathway initiated by β-secretase generates the pathologic A42. The missense mutations in presenilin 1 involved in Alzheimer’s disease enhance the formation of the A42 peptide, leading to plaque formation and eventually to the death of neurons (Figure 16-36).

Figure 16.34 (a) Activation of the NF-κB signaling pathway.

Figure 16.34 (b) Activation of the NF-κB signaling pathway.

NF-kB 1. Biochemical studies in mammalian cells and genetic studies in flies have provided important insights into the operation of the NF-κB pathway (Figure 16-34a). The two subunits of heterodimeric NF-κB (p65 and p50) share a region of homology at their N-termini that is required for their dimerization and binding to DNA. ① In resting cells, NF-κB is sequestered in an inactive state in the cytosol by direct binding to an inhibitor

called I-κB. A single molecule of I-κB binds to the N-terminal domains of each subunit in the p50/p65 heterodimer, thereby masking the nuclear-localization signals. A protein kinase complex termed I-κB kinase is the point of convergence of all of the extracellular signals that activate NF-κB.

② Within minutes of stimulation, I-κB kinase becomes activated and phosphorylates two N-terminal serine residues on I-κB. An E3 ubiquitin ligase then binds to these phosphoserines and polyubiquitinates I-κB, triggering its immediate degradation by a proteasome. In cells expressing mutant forms of I-κB in which these two serines have been changed to alanine, and thus cannot be phosphorylated, NF-κB is permanently repressed, demonstrating that phosphorylation of I-κB is essential for pathway activation. The degradation of I-κB exposes the nuclear-localization signals on NF-κB, which then translocates into the nucleus and activates transcription of a multitude of target genes. Despite its activation by proteolysis, NF-κB signaling eventually is turned off by a negative feedback loop, since one of the genes whose transcription is immediately induced by NF-κB encodes I-κB. The resulting increased levels of the I-κB protein bind active NF-κB in the nucleus and return it to the cytosol.

2. Polyubiquitin chains linked to the activated IL-1 receptor form a scaffold that brings the TAK1 kinase near its substrate, a subunit of the I-κB kinase, and thus allows isgnals to be transmitted from the receptor to downstream components of the NF-κB pathway (Figure 16-34b)

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