Cell Signaling, Growth Factors and Their Receptors

Lewis C. Cantley

Chris L. Carpenter

William C. Hahn

Matthew Meyerson

SIGNAL TRANSDUCTION SYSTEMS

Signal transduction is the chemistry that allows communication at the cellular level. Cells sense signals from the extracellular and intracellular environments, as well as directly from other cells. Cells respond to these signals in a variety of ways, primarily by modifying protein levels, activities, and locations. Protein levels are controlled by rates of transcription, translation, and proteolysis, whereas protein activities are affected by covalent modifications and noncovalent interactions with other proteins and small molecules. Signal transduction pathways regulate differentiation, division, and death in the mature and developing organisms. Some pathways are common to all cells, but others are specific to specialized cells (e.g., synthesis and secretion of insulin by the pancreas, migration and phagocytosis by neutrophils). Disruption or alterations of signal transduction pathways plays a key causative role in disease. Indeed, mutations in nearly all of these signaling pathways are found in a wide range of cancers.

To emphasize the essentials of signal transduction, the focus in this chapter is on the variety of solutions to the two common problems faced by cells and organisms in signal transduction:

How is a signal sensed?
How are the levels, activities, and locations of proteins modified in response to the signal?

Most signals are initiated by ligands and are sensed by the receptors to which they bind. Binding of a ligand to a receptor stimulates the activities of proteins necessary to continue the transmission of the signal through the formation of multiprotein complexes and the generation of small-molecule second messengers. Integration of signals from multiple pathways determines the cell’s ultimate response to competing and complementary signals. In addition, cell signaling pathways are highly interconnected to permit dynamic regulation of the strength, duration, and timing of cell responses.

SENSORY MACHINERY: LIGANDS AND RECEPTORS

Signals

Signal transduction pathways have evolved to respond to an enormous variety of stimuli. Molecules that initiate signaling cascades include proteins, amino acids, lipids, nucleotides, gases, and light (Table 5.1). Most extracellular signals, such as growth factors, bind to receptors on the plasma membrane, but others such as androgens or estrogen, diffuse into the cell and bind to receptors in the cytoplasm and nucleus. Some signals are continuous, such as those sent by the extracellular matrix, whereas others are episodic, like the secretion of insulin by pancreatic β cells in response to increases in blood glucose. Signaling molecules originate from a variety of sources. Some, such as neurotransmitters, are stored in the cell and are released to provide communication with other cells under specific conditions. Other ligands are stored outside the cell (e.g., in the extracellular matrix) and become accessible in response to tissue damage or remodeling. Traditionally, signals have been divided based on the cell of origin into those that affect distant cells (endocrine), nearby cells (paracrine), or the same cell (autocrine). Cells also respond to signals that arise from within. Important examples include the checkpoint pathways that ensure the orderly progression of the cell cycle and the pathways that sense and repair damaged DNA.¹

Receptors

The plasma membrane of eukaryotic cells serves to insulate the cell from the outside environment, but this barrier must be breached to transmit signals of extracellular origin. This fundamental
problem of transmitting extracellular signals is solved in two ways. Signals cross the plasma membrane either by activating transmembrane receptors or by using ligands that are membrane permeable (Table 5.2). Cells are exquisitely sensitive to most ligands. The affinity of receptors for ligands generally is in the picomolar to nanomolar range, and very few receptors need to be occupied to transmit a signal. For example, it has been estimated that activation of ten T-cell receptors is sufficient to send a maximal signal. Cytokineresponsive cells may express only a few hundred receptors on the cell surface. Given the small number of receptors that are activated, amplification of most signals is necessary for cellular responses. A requirement for signal amplification also allows opposing or complementary pathways to affect signal strength more efficiently.² As a result of ligand binding, receptors undergo conformational changes or oligomerization, or both, and the intrinsic activity of the receptor or of associated proteins is stimulated. Receptors may bind and respond to more than one ligand. For example, the epidermal growth factor (EGF) receptor binds to transforming growth factor-alpha (TGF-α), EGF, heparin-binding EGF (HB-EGF), beta-cellulin, epiregulin, epigen, and amphiregulin. The stimulation of most receptors leads to the activation of several downstream pathways that either function cooperatively to activate a common target or stimulate distinct targets. Generally, some of the pathways activated are counter-regulatory and serve to attenuate the signal. Receptors may also activate other receptors. A well-studied example is the activation of the EGF receptor by G protein-coupled receptors (GPCR), which occurs as a result of protease cleavage and activation of HB-EGF.

TABLE 5.1 LIGANDS THAT STIMULATE SIGNAL TRANSDUCTION PATHWAYS

Types of Ligands	Examples
PROTEIN
Soluble	Insulin
Matrix	Fibronectin
Bound to other cells	Ephrines
AMINO ACIDS
*Nucleotides*
Soluble	Adenosine triphosphate
DNA	Double-strand breaks
LIPIDS	Prostaglandins
GASES	Nitric oxide
LIGHT	Rhodopsin, visual system

TABLE 5.2 RECEPTORS IN SIGNAL TRANSDUCTION

Types of Receptors	Examples	Types of Ligands
Tyrosine kinase	PDGF, EGF, FGF, and insulin receptors	Peptide growth factors
Serine kinase	TGF-β receptor	Activin
Heterotrimeric G protein	Thrombin, smell receptors	Thrombin
Receptors bound to tyrosine kinases	IL-2, interferon receptors	IL-2
TNF family	Fas receptor	Fas
Notch	Notch	Delta-Serrate-LAG-2
Guanylate cyclase	Atrial naturic factor receptor	Atrial natriuretic factor
Tyrosine phosphatase	CD45, LAR	Contactin
Nuclear receptors	Estrogen, androgen receptors	Estrogen
Adhesion receptors	Integrins, CD44	Fibronectin, hyaluronic acid
PDGF, platelet-derived growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; TGF-β, transforming growth factor-β; IL-2, interleukin-2; TNF, tumor necrosis factor.

There are a number of transmembrane receptor families. This chapter will discuss several of them to illustrate distinct signaling mechanisms.

Receptor Tyrosine Kinases

Receptor tyrosine kinases are transmembrane proteins that have an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain.³ The ligands for these receptors are proteins or peptides. Most receptor tyrosine kinases are monomeric, but members of the insulin-receptor family are heterotetramers in which the subunits are linked by disulfide bonds. Receptor tyrosine kinases have been divided into six classes, primarily on the basis of the sequence of the extracytoplasmic domain. Examples of tyrosine kinase receptors include the insulin receptor, the platelet-derived growth factor (PDGF)
receptor, the EGF receptor family, and the fibroblast growth factor (FGF) receptor family.

FIGURE 5.1 Dimerization of tyrosine kinase receptors. Most tyrosine kinase receptors are activated by ligand-induced dimerization. Some ligands, such as platelet-derived growth factor (PDGF), are dimeric and induce dimerization using the two receptor-binding domains. Other ligands, such as growth hormone, contain two receptor-binding domains in the same molecule. The fibroblast growth factors (FGFs) relay on proteoglycans to aid the formation of ligand dimmers. Some ligands, such as the ephrins (EPHs), are present on nearby cells and, when the cells come into contact, bind to the receptors and promote clustering.

Activation of receptor tyrosine kinases is generally believed to require tyrosine phosphorylation of the receptor. In the case of the insulin receptor, an insulin-stimulated conformational change activates the kinase. Most of the tyrosine kinases are activated by oligomerization, which brings the kinase domains of distinct molecules into close proximity so that they cross-phosphorylate. Autotransphosphorylation of tyrosine in the activation loop of the kinase domain locks the kinase into a high-activity conformation, stimulating phosphorylation of other sites on the receptor, as well as other substrates. However, cancerderived mutants of the EGF receptor may be activated without receptor autophosphorylation.⁴

Ligands stimulate receptor oligomerization in a variety of ways (Fig. 5.1). Some ligands, such as PDGF, are dimeric, so that the ligand is able to bind two receptors simultaneously.⁵ Other ligands, such as growth hormone, are monomeric but have two receptor-binding sites that allow them to induce receptor dimerization.⁶ FGFs are also monomeric but have only a single receptor-building site. FGF molecules bind to heparin sulfate proteoglycans, which concentrates FGF and facilitates dimerization of the FGF receptor.⁷ EGF is also monomeric, but binding of EGF to the receptor changes the receptor conformation and promotes interaction with a second ligand or receptor dimmer, leading to activation.⁸ Some ligand-receptor interactions result in signaling by the ligand, in addition to the receptor. Ephrins are ligands for EPH tyrosine kinase activity in the target cell, but they also stimulate signaling by ephrins in the ephrin-presenting cell.⁹

Studies of the EGF receptor-family illustrate some important concepts. The EGF-signaling pathways involve four receptors (EGF receptor, ERB2, ERB3, and ERB4) and many ligands.¹⁰ EGF stimulates homodimerization of the EGF receptor, but, under certain conditions, heterodimerization with other family members also occurs. Activation of EGFR proceeds via asymmetric dimerization of the receptor. Ligand causes extracellular dimerization, which then causes the kinase domains to form an intracellular head-totail dimer, which activates the receptor.¹¹,¹²

Receptors that Activate Tyrosine Kinases

A number of receptors do not have intrinsic enzymatic activity but stimulate associated tyrosine kinases. Important examples of this type of receptor include the cytokine and interferon receptors that associate constitutively with members of the Jak family of tyrosine kinases¹³ and the multichain immune recognition receptors that activate SKF and Syk family tyrosine kinases.¹⁴,¹⁵ The kinase appears to be inactive in the absence of ligand, but, as happens in receptors with intrinsic tyrosine kinase activity, signaling is initiated by ligand-stimulated heterodimerization and conformational changes of the receptors.

Serine-Threonine Kinase Receptors

The TGF-β family of receptors are transmembrane proteins with intrinsic serine-threonine kinase activity.¹⁶ TGF-β ligands are dimmers that bind to and oligomerize type I and type II receptors. The type I and type II receptors homologous but distinctly regulated. The type II receptors seem to be constitutively active but do not normally phosphorylate substrates, whereas the type I receptors are normally inactive. Ligand-mediated dimerization of the type I and type II receptors causes the type II receptor to phosphorylate the type I receptor, converting it to an active kinase. Subsequent signal propagation is dependent on the kinase activity of the type I receptor and the phosphorylation of downstream substrates.

Receptor Phosphotyrosine Phosphatases

Receptor protein tyrosine phosphatases (RPTPs) have an extracellular domain, a single transmembrane-spanning domain, and cytoplasmic catalytic domains.¹⁷ The extracellular domains of some receptor tyrosine phosphatases contain fibronectin and immunoglobulin repeats, suggesting that these receptors may recognize adhesion molecules as ligands. Several RPTPs are capable of homotypic interaction, but no true ligands are yet known for RPTPs. Most receptor tyrosine phosphatases have two catalytic domains, and both are active in at least some receptors. Functional and structural evidence suggests that the phosphatase activity of some of these receptors is inhibited by dimerization. Ligand-dependent dimerization could cause constitutively active tyrosine phosphatases to lose
activity, enhancing signals emanating from tyrosine kinases. RPTPs do not always function in strict opposition to tyrosine kinases, however. For example, CD45 is necessary for signaling by the B-cell receptor, which also requires tyrosine kinase activity.¹⁸ Since some Tyr-phosphorylation events, such as phosphorylation of a Tyr near the C-terminus of src-family protein-Tyr kinases, can be inhibitory to the Tyr kinase activity, activation of certain phospho-Tyr phosphatases can paradoxically cause an increase in global tyrosine phosphorylation (discussed in more detail below).

G Protein-Coupled Receptors

GPCRs are by far the most numerous receptors.¹⁹ Almost 700 GPCRs are present in the human genome.²⁰ The number of GPCRs is so high because they encode the light, smell, and taste receptors, all of which require great diversity. These receptors have seven membrane-spanning domains: The N-terminus and three of the loops are extracellular, whereas the other three loops and the C-terminus are cytoplasmic. A wide variety of ligands bind GPCRs, including proteins and peptides, lipids, amino acids, and nucleotides. No common binding domain exists for all ligands, and interactions of ligands with GPCRs are fairly distinct.²¹ In the case of the thrombin receptor, thrombin cleaves the N-terminus of the receptor, freeing a new N-terminus that self-associates with the ligand pocket, leading to activation. Amines and eicosanioids bind to the transmembrane domains of their GPCRs, whereas peptide ligands bind to the transmembrane domains of their GPCRs, and peptide ligands bind to the transmembrane domains and the extracellular loops of their GPCRs. Neurotransmitters and some peptide hormones require the N-terminus for binding and activation.

Intramolecular bonds that involve residues in the transmembrane or juxtamembrane regions keep GPCRs in an inactive conformation.²² In the inactive state, the receptor is bound to a heterotrimeric G protein, which is also inactive. Agonist binding causes a conformational change that stimulates the guanine nucleotide exchange activity of the receptor. Exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on the α-subunit of the heterotrimeric G proteins initiates signaling. Ultimately, GPCRs stimulate the same downstream pathways as other receptor types, including ion channels, cytosolic protein tyrosine and serine kinases, and enzymes that phosphorylate or hydrolyze membrane lipids.¹⁹ Certain GPCRs also activate receptor tyrosine kinases. As mentioned earlier, GPCR-dependent cleavage of HB-EGF stimulates the EGF receptor, which is necessary for the GPCR to activate the mitogenactivated protein kinase (MAP kinase) pathway.

Notch Family of Receptors

The Notch receptor has a large extracellular domain, a single transmembrane domain, and a cytoplasmic domain.²³ Ligands for the Notch receptor are proteins expressed on the surface of adjacent cells, and activation results in two proteolytic cleavages of Notch. Initial cleavage by ADAM family proteases removes the extracellular domain and causes endocytosis. Subsequent proteolysis by the preselinin protease family releases the cytoplasmic region of Notch as a soluble signal. This fragment moves to the nucleus, where it complexes with the transcriptional repressor CBFI, relieving its inhibitory effects and stimulating transcription.

Guanylate Cyclases

Guanylate cyclases (GCs) convert guanosine triphosphate to cyclic guanosine monophosphate (cGMP) upon activation.²⁴ There are both transmembrane and soluble forms of GCs. The membrane GCs are receptors for atrial natriuretic hormone, peptides that regulate intestinal secretion and are necessary for regulating cGMP levels for vision. In addition to the catalytic domain, the cytoplasmic tail includes a protein kinase homology domain that lacks kinase activity. Soluble GCs are activated by nitrous oxide. These receptors are widely expressed and regulate vascular tone and neuron function. They are heterodimers and each subunit has catalytic activity.

Tumor Necrosis Factor Receptor Family

The tumor necrosis factor family of receptors has a conserved cysteine-rich region in the extracellular domain, a transmembrane domain, and a domain called the death domain in the cytoplasmic tail.²⁵ The receptors undergo oligomerization after ligand binding, which is necessary for signaling. These receptors are distinct in several respects. Stimulation of the receptor leads to recruitment of cytoplasmic proteins that bind to each other and the receptor through death domains, thereby activating a protease, caspase 8, that initiates apoptosis. Under some conditions, however, tumor necrosis factor receptors (TNFRs) stimulate antiapoptotic signals. This family of receptors also includes “decoys” or receptors that are missing all or part of the cytoplasmic tail and thus cannot transmit a signal. This feature provides a unique mechanism for inhibiting and further regulating signaling. A second class of TNFRs lack death domains but bind to TNFR-associated factors.

WNT Receptors

The Wnt family of growth and differentiation factors are small proteins that bind to cell surface receptors of the Frizzled family.²⁶ These receptors resemble GPCRs but utilize a unique mechanism
of signal transduction (Fig. 5.2). Binding of Wnt to the receptor suppresses a kinase cascade involving the protein Ser/Thr kinases casein kinase I (CK I) and glycogen synthase kinase 3 (GSK3) and the low-density lipoprotein-related protein (LRP). Active Wnt signaling requires inactivation of Axin and the adenomatous polyposis coli (APC) protein. This complex mediates phosphorylation and ultimately proteosome-dependent degradation of β-catenin. Suppression of β-catenin degradation in response to Wnt allows β-catenin to accumulate to higher levels in the cell and to migrate into the nucleus where it regulates genes involved in cell growth regulation, acting as a heterodimer with the T-cell factor (TCF) transcription factors.

FIGURE 5.2 Wingless (Wnt)/β catenin signaling. Wnt extracellular ligands bind Frizzled receptors and regulate the phosphorylation status of axin. Axin functions as part of the destruction complex that regulates the stability of β-catenin, a transcriptional regulator.

Nuclear Receptors

Ligands for nuclear hormone receptors diffuse into the cell and bind their receptors either in the cytoplasm or the nucleus. The ligands include steroids, eicosanoids, retinoids, and thyroid hormone. The sex steroids, androgens such as testosterone and estrogen and progesterone, are ligands for the androgen receptor, estrogen receptor, and progesterone receptor, respectively. Inhibition of androgen receptor is central to treatment of prostate cancer, while inhibition of estrogen receptor is central to treatment of estrogen receptor-positive breast cancer. The receptors are transcription factors that have both DNA- and ligand-binding domains. The unliganded receptor is bound to heat shock proteins that are dissociated after ligand binding. Release from the chaperone complex and ligand association lead to binding of the receptor to cofactors and DNA to regulate transcription.

Adhesion Receptors

Cell adherence, either to the extracellular matrix or to other cells, is mediated by receptors that function mechanically and stimulate intracellular signaling pathways, primarily through tyrosine kinases.²⁷ Integrins mediate adherence to extracellular matrix and are composed of heterodimers of α and β

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