The signaling molecules that activate receptor tyrosine kinases are soluble or membrane-bound peptide or protein hormones, including many that were initially identified as growth factors for specific types of cells. Many of these RTK ligands, such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF), stimulate proliferation and differentiation of specific cell types. Others, such as insulin, regulate expression of multiple genes that control sugar and lipid metabolism in liver, muscle, and adipose (fat) cells. Many RTKs and their ligands were identified in studies of human cancers associated with mutant forms of growth- factor receptors that stimulate proliferation even in the absence of the growth factor. The mutation “tricks” the receptor into behaving as though the ligand is present at all times, so the receptor is constantly in an active state (constitutively active). Other RTKs have been uncovered during analysis of developmental mutations that lead to blocks in differentiation of specific cell types in C. elegans, Drosophila, and the mouse.
Binding of Ligand Promotes Dimerization of an RTK and Leads to Activation of Its Intrinsic Tyrosine Kinase
All RTKs have three essential components: an extracellular domain containing a ligand-binding site, a single hydro phobic transmembrane α helix, and a cytosolic segment that includes a domain with protein tyrosine kinase activity ( Figure 1). Most RTKs are monomeric, and ligand binding to the extracellular domain induces formation of receptor dimers. As with cytokine receptors, the formation of functional dimers is a necessary step in activation of all RTKs.
Fig1. General structure and activation of receptor tyrosine kinases (RTKs). The cytosolic domain of RTKs contains an intrinsic protein tyrosine kinase catalytic site. In the absence of ligand (step 1), RTKs generally exist as monomers with poorly active kinases. Binding of two ligands to the extracellular domains of two RTKs forms or stabilizes an activated dimeric receptor. This brings together two poorly active kinases such that each one phosphorylates the other on a tyrosine residue in the activation loop (step 2). Phosphorylation causes the loop to move out of the kinase catalytic site, thus increasing the ability of ATP and/or the protein substrate to bind. The activated kinase then phosphorylates several tyrosine residues in the receptor’s cytosolic domain (step 3). The resulting phosphotyrosines function as docking sites for SH2 and other binding domains on downstream signal-transducing proteins.
RTK activation can be summarized as follows: In the resting, unstimulated (no ligand bound) state, the intrinsic kinase activity of an RTK is very low (Figure 1, step 1). Like most other kinases, including the JAK kinases discussed earlier, RTKs contain a flexible domain termed the activation loop. In the resting state, the activation loop is nonphosphorylated and assumes a conformation that blocks kinase activity. In some receptors (e.g., the insulin receptor), it prevents binding of ATP. In others (e.g., the FGF receptor), it prevents binding of substrate. Binding of ligand causes a conformational change that promotes dimerization of the extracellular domains of RTKs, which brings their transmembrane segments—and therefore their cytosolic domains—close together. In a manner similar to the activation of the JAK kinases, the kinase in each subunit then phosphorylates a particular tyrosine residue in the activation loop of the other subunit (Figure 1, step 2). This phosphorylation leads to a conformational change in the activation loop that unblocks kinase activity by reducing the KD for ATP or the substrate to be phosphorylated. The resulting enhanced kinase activity can then phosphorylate additional tyrosine residues in the cytosolic domain of the receptor (Figure 1, step 3), which bind SH2 or other domains of signaling proteins, as well as phosphorylating other target proteins, leading to intracellular signaling.
Although dimerization is a necessary step in the activation of all RTKs, functional dimers can be formed in multiple ways. Many receptors are dimerized in a manner similar to the fibroblast growth factor (FGF) receptor (Figure 2), in which each of two FGF molecules binds simultaneously to the extracellular domains of two receptor subunits, stabilizing the dimer. FGF also binds tightly to heparan sulfate, a negatively charged polysaccharide component of some cell-surface proteins and of the extracellular matrix; this association enhances ligand binding and formation of a dimeric receptor-ligand complex (see Figure 2). The participation of the heparan sulfate is essential for efficient receptor activation.
Fig2. Structure of the extracellular domains of the active dimeric fibroblast growth factor (FGF) receptor, stabilized by binding of two ligands and by heparan sulfate. Shown here are side and top-down views of the complex comprising the extracellular domains of two FGF receptor (FGFR) monomers (purple and violet), two bound FGF molecules (red), and two short heparan sulfate chains, which bind tightly to FGF. (a) In the side view, the upper domain of one receptor monomer (purple) is seen situated behind that of the other (violet); the plane of the plasma membrane is at the bottom. A small segment of the extracellular domain whose structure is not known connects to the membrane-spanning α-helical segment of each of the two receptor monomers (not shown) that protrude downward into the membrane. (b) In the top view, the heparan sulfate chains are seen threading between and making numerous contacts with the upper domains of both receptor monomers. These interactions promote binding of the ligand to the receptor and receptor dimerization. [Data from J. Schlessinger et al., 2000, Mol. Cell 6:743, PDB ID 1fq9.]
Yet other RTKs, such as the insulin receptor, form disulfide-linked dimers even in the absence of hormone; binding of ligand to this type of RTK alters its conformation in such a way that the receptor kinase becomes activated. This last example highlights the fact that simply having two receptor monomers in close contact is not sufficient for receptor activation—the proper conformational changes must accompany receptor dimerization to lead to tyrosine kinase activation. Once an RTK is locked into a functional dimeric state, its associated tyrosine kinase becomes activated.
Homo- and Hetero-oligomers of Epidermal Growth Factor Receptors Bind Members of the Epidermal Growth Factor Family
Four RTKs participate in signaling by the many members of the epidermal growth factor (EGF) family of signaling molecules; these receptors are termed Erb-B1, 2, 3, and 4. In humans these receptors are also called HER (human epidermal growth factor receptor) 1, 2, 3, and 4, respectively. Epi dermal growth factors and their receptors have been studied intensively because of their involvement in many human diseases; drugs targeting receptors that are overproduced in tumors or have activating mutations are used to treat many cancers.
In the resting state, the majority of EGF receptor molecules are monomeric. HER1 (Erb-B1) directly binds EGF as well as six other members of the EGF family: heparin-binding EGF (HB-EGF), transforming growth fac tor α (TGF-α), amphiregulin (AREG), epiregulin (EREG), epigen (EPGN), and betacellulin (BTC). Binding of any of these ligands leads to homodimerization of the HER1 extra cellular domain (Figure 3). In the resting, monomeric, state, the β-hairpin in the segment of receptor domain II, termed the dimerization arm, forms a tight intramolecular interaction with domain IV. Binding of EGF induces a dramatic conformational change to the extracellular domain of HER1 in which the receptor “clamps down” on the hormone such that the dimerization arm becomes exposed. The dimerization arms from two activated receptors then bind tightly together, forming a stable receptor homodimer. In contrast to the FGF receptors (see Figure2), the ligand does not directly stabilize the active homodimer.
Fig3. Ligand-induced dimerization of HER1, a human receptor for epidermal growth factor (EGF). (a) The extracellular region of all EGF receptors contains four domains: domains I (blue) and III (yellow) are closely related in sequence, as are domains II (green) and IV (red). In the absence of bound EGF, the receptor is mostly monomeric and the intracellular kinase is inactive. The extracellular region adopts a configuration in which the β-hairpin from domain II that forms the “dimerization arm” binds to domain IV of the same receptor molecule. (b) EGF binds simultaneously to domains I and III; binding induces a major conformational change in the extracellular domain such that the dimerization arm of domain II is now exposed. (c) Dimerization of two identical ligand-bound receptor monomers in the plane of the membrane occurs primarily through interactions between the dimerization arms of the two receptors. [Data from H. Ogiso et al., 2002, Cell 110:775, PDB ID 1ivo.]
HER3 and HER4 also bind members of the EGF family. Neuregulins 1 and 2 (NRG1 and NRG2) bind to both HER3 and HER4; HB-EGF, NRG3, and NRG4 bind only to HER4. HER4, like HER1, can form stable homodimers after binding a growth factor, but HER3 cannot. So how can HER3 signal? The answer involves HER2, which does not bind a ligand. Rather, HER2 exists on the plasma mem brane in a conformation that is very similar to that of HER1 with a bound EGF; the HER2 dimerization arm protrudes outward and is able to bind to and form heterodimers with ligand-bound HER3 as well as with ligand-bound HER1 and HER4 (Figure4). HER2 can signal only by forming heterocomplexes with ligand-bound HER1, HER3, or HER4; thus it facilitates signaling by all EGF family members (see Figure4). Even though HER3 lacks a functional kinase domain, it can still participate in signaling; after binding a ligand, it heterodimerizes with HER2 and becomes phosphorylated by the HER2 kinase, activating downstream signal transduction pathways.
Fig4. The HER family of receptors and their ligands. Humans and mice express four receptor tyrosine kinases, denoted HER1, 2, 3, and 4 in humans and Erb-B1, 2, 3, and 4 in mice and other animals. Only the extracellular domains of these receptors are depicted here. These receptors bind epidermal growth factor (EGF) and the other EGF family members: heparin-binding EGF (HB-EGF), transforming growth factor α (TGF-α), amphiregulin (AREG), epiregulin (EREG), epigen (EPGN), betacellulin (BTC), and the four neuregulins. Note that Erb-B2 (HER2), which does not directly bind a ligand, exists in a conformation that is very similar to that of the activated Erb-B1 with a bound EGF. Erb-B2 can form a heterodimer with ligand-activated Erb-B1, 3, or 4; thus Erb-B2 facilitates signaling by all EGF family members. Erb-B3 (HER3) has a very poorly active kinase domain and can signal only when complexed with Erb-B2. [Erb-B1 data from K. M. Ferguson et al., 2003, Mol. Cell 11:507, PDB ID 1nql. Erb-B2 data from H.-S. Cho et al., 2003, Nature 421:756, PDB ID 1n8z. Erb-B3 data from H. S. Cho and D. J. Leahy, 2002, Science 297:1330, PDB ID 1m6b. Erb-B4 data from S. Bouyain et al., 2005, Proc. Natl. Acad. Sci. USA 102:15024, PDB ID 1ahx. EFG and Erb-B1 data from H. Ogiso et al., 2002, Cell 110:775, PDB ID 1ivo.]
Thus an increase in HER2 on the cell surface makes a cell more sensitive to signaling by many EGF family members because the rate at which the signaling heterodimers are formed after ligand binding will be enhanced. As we learn in Chapter 24, understanding the HERs has helped explain why a common form of breast cancer, in which the HER2 gene is amplified and HER2 is overexpressed, is so dangerous and has led to important drug therapies. Overexpression of HER2 makes the tumor cells sensitive to growth stimulation by low levels of any member of the EGF family of growth factors—levels that would not stimulate proliferation of cells with normal HER2 levels.
Activation of the EGF Receptor Results in the Formation of an Asymmetric Active Kinase Dimer
As is the case with most receptor tyrosine kinases, the kinase domain in the FGF receptor is activated by phosphorylation of a tyrosine in the activation loop. In contrast, activation of the EGF receptor kinases does not involve loop phosphorylation; their mechanism of activation was uncovered only recently through structural studies of the receptor cytosolic domain in both the active and inactive states. The kinase domains are separated from the transmembrane segment by a so-called juxtamembrane segment, colored blue in Figure 5. In the inactive, monomeric state, the activation loop is localized to the active site of the kinase, blocking its activity; in this way the kinase is maintained in the “off” state (Figure5, left). Receptor dimerization generates an asymmetric kinase dimer (Figure 5, right) in which one kinase domain—termed the donor—binds the second kinase domain—the acceptor. This binding changes the conformation of the top lobe of the receiver, causing the activation loop to move out of the kinase active site and activating the kinase.
Fig5. Activation of the EGF receptor by EGF results in the formation of an asymmetric kinase domain dimer. In the inactive, monomeric state, the activation loop is localized to the kinase active site and thus Juxtamembrane segment Kinase domain C-terminal tail N lobe Activation loop C lobe Acceptor kinase P P P P P P P Autophosphorylation sites Asymmetric kinase dimer Donor kinase P P P inhibits kinase activation. Receptor dimerization generates an asymmetric kinase dimer such that the C-terminal C-lobe of the donor kinase binds to the N-terminal N-lobe of the acceptor kinase in the opposite receptor; the dimer is stabilized by interactions between the juxtamembrane segments of the two receptors. These inter actions cause a conformational change that removes the activation loop from the kinase site of the acceptor kinase, activating its kinase activity. The active kinase then phosphorylates tyrosine residues in the C-terminal segments of the receptor cytosolic domain. [EGF receptor data from H. Ogiso et al., 2002, Cell 110:775, PDB ID 1ivo. Asymmetric kinase dimer data from K. M. Ferguson et al., 2003, Mol. Cell. 11:507, PDB ID 1nql.]
Thus evolution has produced many variations on the theme of this simple ligand-RTK mechanism: RTKs are activated by dimerization, but different receptors use different mechanisms to accomplish this. Similarly, kinases become activated by movement of the activation loop away from the kinase catalytic site, but different receptors use different mechanisms to accomplish this task. Down-regulation of signaling from RTKs is also common, and different mechanisms have evolved to accomplish this task as well.
Multiple Mechanisms Down-Regulate Signaling from RTKs
Earlier in this chapter we discussed two mechanisms by which signaling from cytokine receptors is down-modulated—those involving phosphotyrosine phosphatases and SOCS proteins. These proteins are also used to depress signaling by some RTKs, but two other mechanisms— receptor-mediated endocytosis and degradation in lysosomes—are more common.
Receptor-Mediated Endocytosis Prolonged treatment of cells with ligand often reduces the number of available cell- surface receptors, so that the cells have a less robust response to exposure to a given concentration of ligand than they did before ligand addition. This desensitization response helps prevent inappropriately prolonged receptor activity. In the absence of EGF, for instance, cell-surface HER1 receptors are relatively long-lived, with an average half-life of 10–15 hours. Unbound receptors are internalized via clathrin-coated pits into endosomes at a relatively slow rate, on average once every 30 minutes, and are often returned rapidly to the plasma membrane so that there is little reduction in total sur face receptor numbers. Following binding of an EGF ligand, the rate of endocytosis of HER1 is increased about tenfold, and only a fraction of the internalized receptors return to the plasma membrane; the rest are degraded in lysosomes. Each time a HER1–EGF complex is internalized via the process of receptor-mediated endocytosis, the receptor has about a 20 to 80 percent chance of being degraded, depending on the cell type. Exposure of a fibroblast cell to high levels of EGF for several hours induces several rounds of endocytosis, resulting in degradation of most cell-surface EGF receptor molecules and thus a reduction in the cell’s sensitivity to EGF. In this way, prolonged treatment with a given concentration of EGF desensitizes the cell to that level of hormone, though the cell may respond if the level of EGF is increased.
HER1 mutants that lack kinase activity do not undergo accelerated endocytosis in the presence of ligand. It is likely that ligand-induced activation of the kinase activity in nor mal HER1 induces a conformational change in the cytosolic tail, exposing a sorting motif that facilitates receptor recruitment into clathrin-coated pits and subsequent internalization of the receptor-ligand complex. Despite extensive study of mutant HER1 cytosolic domains, the identity of these “sorting motifs” is controversial, and most likely multiple motifs function to enhance endocytosis. Interestingly, internalized receptors can continue to signal from endosomes or other intracellular compartments before their degradation, as evidenced by their binding to signaling proteins such as GRB2 and Sos, which are discussed in the next section.
Lysosomal Degradation Several processes influence recycling of surface receptors to the plasma membrane versus lysosomal degradation; one is covalent modification by the small protein ubiquitin. There is a strong correlation between monoubiquitinylation (addition of a single ubiquitin to a given lysine of a protein) of the HER1 cytosolic domain and HER1 degradation. The monoubiquitinylation, which also occurs in other ligand-activated RTKs, is mediated by the enzyme c-Cbl. This enzyme, which is an E3 ubiquitin ligase, contains an EGF receptor–binding domain, which binds directly to phosphorylated EGF receptors, and a RING finger domain, which recruits ubiquitin-conjugating enzymes and mediates transfer of ubiquitin to the receptor. The ubiquitin functions as a “tag” on the receptor that stimulates its incorporation from endosomes into multivesicular bodies that are ultimately degraded in lysosomes. A role for c-Cbl in EGF receptor trafficking emerged from genetic studies in C. elegans, which established that c-Cbl negatively regulates the function of the nematode EGF receptor (Let-23), prob ably by inducing its degradation. Similarly, knockout mice lacking c-Cbl show hyperproliferation of mammary gland epithelia, consistent with a role of c-Cbl as a negative regulator of EGF signaling.
Experiments with mutant cell lines have demonstrated that internalization of RTKs plays an important role in regulating cellular responses to EGF and other growth fac tors. For instance, a mutation in the HER1 EGF receptor that prevents it from being incorporated into coated pits makes it resistant to ligand-induced endocytosis. As a result, this mutation leads to substantially elevated numbers of EGF receptors on cells and thus increased sensitivity of cells to EGF as a mitogenic signal. Such mutant cells are prone to EGF-induced transformation into tumor cells. Interestingly, the other EGF family receptors—HER2, HER3, and HER4—do not undergo ligand-induced internalization, an observation that emphasizes how each receptor evolved to be regulated in its own appropriate manner.
