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Receptor Tyrosine Kinases and Activation of Ras

We return now to the receptor tyrosine kinases (RTKs), which have intrinsic protein tyrosine kinase activity in their cytosolic domains. The ligands for RTKs are soluble or membrane-bound peptide or protein hormones including nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin. Ligand-induced activation of an RTK stimulates its tyrosine kinase activity, which subsequently stimulates the Ras-MAP kinase pathway and several other signal-transduction pathways. RTK signaling pathways have a wide spectrum of functions including regulation of cell proliferation and differentiation, promotion of cell survival, and modulation of cellular metabolism.

Some RTKs have been identified in studies on human cancers associated with mutant forms of growth-factor receptors, which send a proliferative signal to cells even in the absence of growth factor. For example, a constitutively active mutant form of Her2, a receptor for EGF-like proteins, enables uncontrolled proliferation of cancer cells even in the absence of EGF, which is required for proliferation of normal cells (see Figure 23-14). Alternatively, overproduction of the wild-type receptor for EGF in certain human breast cancers results in proliferation at low EGF levels that do not stimulate normal cells; monoclonal antibodies targeted to the EGF receptor have proved thera-peutically useful in these patients. Other RTKs have been uncovered during analysis of developmental mutations that lead to blocks in differentiation of certain cell types in C. elegans, Drosophila, and the mouse. I

Here we discuss how ligand binding leads to activation of RTKs and how activated receptors transmit a signal to the

Ras protein, the GTPase switch protein that functions in transducing signals from many different RTKs. The trans-duction of signals downstream from Ras to a common cascade of serine/threonine kinases, leading ultimately to activation of MAP kinase and certain transcription factors, is covered in the following section.

Ligand Binding Leads to Transphosphorylation of Receptor Tyrosine Kinases

All RTKs constitute an extracellular domain containing a ligand-binding site, a single hydrophobic transmembrane a helix, and a cytosolic domain that includes a region with protein tyrosine kinase activity. Most RTKs are monomeric, and ligand binding to the extracellular domain induces formation of receptor dimers, as depicted in Figure 14-4 for the EGF receptor. Some monomeric ligands, including FGF, bind tightly to heparan sulfate, a negatively charged polysaccharide component of the extracellular matrix (Chapter 6); this association enhances ligand binding to the monomeric receptor and formation of a dimeric receptor-ligand complex (Figure 14-15). The ligands for some RTKs are dimeric; their binding brings two receptor monomers together directly. Yet other RTKs, such as the insulin receptor, form disulfide-linked dimers in the absence of hormone; binding of ligand to this type of RTK alters its conformation in such a way that the receptor becomes activated.

Regardless of the mechanism by which ligand binds and locks an RTK into a functional dimeric state, the next step is universal. In the resting, unstimulated state, the intrinsic kinase activity of an RTK is very low. In the dimeric receptor, however, the kinase in one subunit can phosphorylate one or more tyrosine residues in the activation lip near the catalytic site in the other subunit. This leads to a conforma-tional change that facilitates binding of ATP in some receptors (e.g., insulin receptor) and binding of protein substrates in other receptors (e.g., FGF receptor). The resulting enhanced kinase activity then phosphorylates other sites in the cytosolic domain of the receptor. This ligand-induced activation of RTK kinase activity is analogous to the activation of the JAK kinases associated with cytokine receptors (see Figure 14-5). The difference resides in the location of the kinase catalytic site, which is within the cytosolic domain of RTKs, but within a separate JAK kinase in the case of cytokine receptors.

As in signaling by cytokine receptors, phosphotyrosine residues in activated RTKs serve as docking sites for proteins involved in downstream signal transduction. Many phos-photyrosine residues in activated RTKs interact with adapter proteins, small proteins that contain SH2, PTB, or SH3 domains but have no intrinsic enzymatic or signaling activities (see Figure 14-6). These proteins couple activated RTKs to other components of signal-transduction pathways such as the one involving Ras activation.

▲ FIGURE 14-15 Structure of the dimerized ligand-bound receptor for fibroblast growth factor (FGF), which is stabilized by heparan sulfate. Shown here are side and top views of the complex comprising the extracellular domains of two FGF receptor (FGFR) monomers (green and blue), two bound FGF molecules (white), and two short heparan sulfate chains (purple), which bind tightly to FGF In the side view, the upper domain of one receptor (blue) is situated behind that of the other (green). In the top view, the heparan sulfate chains thread between and make numerous contacts with the upper domains of both receptor monomers. These interactions promote binding of the ligand to the receptor and receptor dimerization. [Adapted from J. Schlessinger et al., 2000, Mol. Cell 6:743.]

Ras, a GTPase Switch Protein, Cycles Between Active and Inactive States

Ras is a monomeric GTP-binding switch protein that, like the Ga subunits in trimeric G proteins, alternates between an active on state with a bound GTP and an inactive off state with a bound GDP. As discussed in Chapter 13, trimeric G proteins are directly linked to cell-surface receptors and transduce signals, via the Ga subunit, to various effectors such as adenylyl cyclase. In contrast, Ras is not directly linked to cell-surface receptors.

Ras activation is accelerated by a guanine nucleotide-exchange factor (GEF), which binds to the Ras-GDP complex, causing dissociation of the bound GDP (see Figure 3-29). Because GTP is present in cells at a higher concentration than GDP, GTP binds spontaneously to "empty" Ras molecules, with release of GEF and formation of the active Ras-GTP. Subsequent hydrolysis of the bound GTP to GDP deactivates Ras. Unlike the deactivation of Ga-GTP, deactivation of Ras-GTP requires the assistance of another protein, a GTPase-activating protein (GAP) that binds to Ras-GTP and accelerates its intrinsic GTPase activity by more than a hundredfold. Thus the average lifetime of a GTP bound to Ras is about 1 minute, which is much longer than the average lifetime of Ga-GTP. In cells, GAP binds to specific phosphotyrosines in activated RTKs, bringing it close enough to membrane-bound Ras-GTP to exert its accelerating effect on GTP hydrolysis. The actual hydrolysis of GTP is catalyzed by amino acids from both Ras and GAP. In particular, insertion of an arginine side chain on GAP into the Ras active site stabilizes an intermediate in the hydrolysis reaction.

The differences in the cycling mechanisms of Ras and Ga are reflected in their structures. Ras («170 amino acids) is smaller than Ga proteins («300 amino acids), but its three-dimensional structure is similar to that of the GTPase domain of Ga (see Figure 13-8). Recent structural and biochemical studies show that Ga also contains another domain that apparently functions like GAP to increase the rate of GTP hydrolysis by Ga. In addition, the direct interaction between an activated receptor and inactive G protein promotes release of GDP and binding of GTP, so that a separate nucleotide exchange factor is not required.

Both the trimeric G proteins and Ras are members of a family of intracellular GTP-binding switch proteins collectively referred to as the GTPase superfamily, which we introduced in Chapter 3. The many similarities between the structure and function of Ras and Ga and the identification of both proteins in all eukaryotic cells indicate that a single type of signal-transducing GTPase originated very early in evolution. In fact, their structures are similar to those of the GTP-binding factors involved in protein synthesis, which are found in all prokaryotic and eukaryotic cells. The gene encoding this ancestral protein subsequently duplicated and evolved to the extent that the human genome encodes a su-perfamily of such GTPases, comprising perhaps a hundred different intracellular switch proteins. These related proteins control many aspects of cellular growth and metabolism.

Mammalian Ras proteins have been studied in great detail because mutant Ras proteins are associated with many types of human cancer. These mutant proteins, which bind but cannot hydrolyze GTP, are permanently in the "on" state and contribute to neoplastic transformation (Chapter 23). Determination of the three-dimensional structure of the Ras-GAP complex explained the puzzling observation that most oncogenic, constitu-tively active Ras proteins (RasD) contain a mutation at position 12. Replacement of the normal glycine-12 with any other amino acid (except proline) blocks the functional binding of GAP, and in essence "locks" Ras in the active GTP-bound state. I

An Adapter Protein and Guanine Nucleotide-Exchange Factor Link Most Activated Receptor Tyrosine Kinases to Ras

The first indication that Ras functions downstream from RTKs in a common signaling pathway came from experiments in which cultured fibroblast cells were induced to proliferate by treatment with a mixture of PDGF and EGF. Microinjection of anti-Ras antibodies into these cells blocked cell proliferation. Conversely, injection of RasD, a constitu-tively active mutant Ras protein that hydrolyzes GTP very inefficiently and thus persists in the active state, caused the cells to proliferate in the absence of the growth factors. These findings are consistent with studies showing that addition of FGF to fibroblasts leads to a rapid increase in the proportion of Ras present in the GTP-bound active form.

How does binding of a growth factor (e.g., EGF) to an RTK (e.g., the EGF receptor) lead to activation of Ras? Two cytosolic proteins—GRB2 and Sos—provide the key links (Figure 14-16). An SH2 domain in GRB2 binds to a specific phosphotyrosine residue in the activated receptor. GRB2 also contains two ,SH3 domains, which bind to and activate Sos. GRB2 thus functions as an adapter protein for the EGF receptor. Sos is a guanine nucleotide-exchange protein (GEF), which catalyzes conversion of inactive GDP-bound Ras to the active GTP-bound form. Genetic analyses of mutants in the worm C. elegans and in the fly Drosophila blocked at particular stages of differentiation were critical in elucidating the roles of these two proteins in linking an RTK to Ras activation. To illustrate the power of this experimental approach, we consider development of a particular type of cell in the compound eye of Drosophila.

► FIGURE 14-16 Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs). The receptors for epidermal growth factor (EGF) and many other growth factors are RTKs. The cytosolic adapter protein GRB2 binds to a specific phosphotyrosine on an activated, ligand-bound receptor and to the cytosolic Sos protein, bringing it near its substrate, the inactive Ras-GDR The guanine nucleotide-exchange factor (GEF) activity of Sos then promotes formation of active Ras-GTR Note that Ras is tethered to the membrane by a hydrophobic farnesyl anchor (see Figure 5-15). [See J. Schlessinger, 2000, Cell 103:211, and M. A. Simon, 2000, Cell 103:13.]

Binding of hormone causes dimer- |1 ization and phosphorylation of cytosolic receptor tyrosine residues

Sos promotes dissociation of GDP from Ras; GTP binds and active Ras dissociates from Sos

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