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Wnt proteins form a family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis. Wnt genes and Wnt signaling are also implicated in cancer. Insights into the mechanisms of Wnt action have emerged from several systems: genetics in Drosophila and Caenorhabditis elegans; biochemistry in cell culture and ectopic gene expression in Xenopus embryos. Many Wnt genes in the mouse have been mutated, leading to very specific developmental defects. As currently understood, Wnt proteins bind to receptors of the Frizzled and LRP families on the cell surface. Through several cytoplasmic relay components, the signal is transduced to b-catenin, which then enters the nucleus and forms a complex with TCF to activate transcription of Wnt target genes. These pages contain some diagrams of the pathway. Wnt signaling has been discussed in many reviews, listed here.

High-sensitivity glycoprotein analyses are of particular interest in modern biomedical and clinical research, as well as in the development of recombinant protein products. The evolution of new hyphenated methodologies in high-sensitivity glycoprotein analysis is highlighted in this thematic review. These methodologies include, in particular, capillary LC/MALDI/TOF/TOF MS in conjunction with online permethylation platform, and silica-based lectin microcolumns interfaced to MS. The potential of these methodologies in glycomic and glycoproteomic analysis is demonstrated for model glycoproteins as well as total glycomes and glycoproteomes derived from biological samples. Additionally, the applications of CE-MS, CEC, and nanoLC with graphitized carbon in the areas of glycomics and glycoproteomics are described. Keywords: Glycomics, Glycoproteomics, LC/MALDI/TOF/TOF MS, LC/MS, Lectin, Oligosaccharides, Online permethylation, Review, Tandem mass spectrometry

Binding of beta-arrestins to seven-membrane-spanning receptors (7MSRs) not only leads to receptor desensitization and endocytosis but also elicits additional signaling processes. We recently proposed that stimulation of the angiotensin type 1A (AT(1A)) receptor results in independent beta-arrestin 2- and G protein-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation. Here we utilize two AT(1A) mutant receptors to study these independent pathways, one truncated at residue 324, thus removing all potential carboxyl-terminal phosphorylation sites, and the other bearing four mutations in the serine/threonine-rich clusters in the carboxyl terminus. As assessed by confocal microscopy, the two mutant receptors interacted with beta-arrestin 2-green fluorescent protein with much lower affinity than did the wild-type receptor. In addition, the mutant receptors more robustly stimulated G protein-mediated inositol phosphate production. Approximately one-half of the wild-type AT(1A) receptor-stimulated ERK1/2 activation was via a beta-arrestin 2-dependent pathway (suppressed by beta-arrestin 2 small interfering RNA), whereas the rest was mediated by a G protein-dependent pathway (suppressed by protein kinase C inhibitor). ERK1/2 activation by the mutant receptors was insensitive to beta-arrestin 2 small interfering RNA but was reduced more than 80% by a protein kinase C inhibitor. The biochemical consequences of ERK activation by the G protein and beta-arrestin 2-dependent pathways were also distinct. G-protein-mediated ERK activation enhanced the transcription of early growth response 1, whereas beta-arrestin 2-dependent ERK activation did not. In addition, stimulation of the truncated AT(1A) mutant receptor caused significantly greater early growth response 1 transcription than did the wild-type receptor. These findings demonstrate how the ability of receptors to interact with beta-arrestins determines both the mechanism of ERK activation as well as the physiological consequences of this activation.

A growing body of data supports the conclusion that G protein-coupled receptors can regulate cellular growth and differentiation by controlling the activity of MAP kinases. The activation of heterotrimeric G protein pools initiates a complex network of signals leading to MAP kinase activation that frequently involves cross-talk between G protein-coupled receptors and receptor tyrosine kinases or focal adhesions. The dominant mechanism of MAP kinase activation varies significantly between receptor and cell type. Moreover, the mechanism of MAP kinase activation has a substantial impact on MAP kinase function. Some signals lead to the targeting of activated MAP kinase to specific extranuclear locations, while others activate a MAP kinase pool that is free to translocate to the nucleus and contribute to a mitogenic response.

Seven membrane-spanning G protein-coupled receptors (GPCRs) function as ligand-activated guanine nucleotide exchange factors for heterotrimeric guanine nucleotide-binding (G) proteins that relay extracellular stimuli by activating intracellular effector enzymes or ion channels. Recent work, however, has shown that GPCRs also participate in numerous other protein-protein interactions that generate intracellular signals in conjunction with, or even independent of, G-protein activation. Nowhere has the importance of protein complex assembly in GPCR signaling been demonstrated more clearly than in the control of the spatial and temporal activity of the extracellular signal-regulated kinase (ERK1/2) mitogen-activated protein (MAP) kinase cascade. ERK1/2 activation by GPCRs often involves cross talk with classical receptor tyrosine kinases or focal adhesion complexes, which scaffold the assembly of a Ras activation complex. Even more surprising is the phenomenon of G protein-independent signaling using beta-arrestins, proteins originally characterized for their role in homologous GPCR desensitization, as scaffolds for the assembly of a multiprotein signalsome directly upon the GPCR. Although both forms of signaling lead to MAP kinase activation, the pathways appear to be functionally, as well as mechanistically, distinct. Transactivated receptor tyrosine kinases mediate rapid and transient MAP kinase activation that favors nuclear translocation of the kinases and transcriptional activation. In contrast, beta-arrestin-dependent signaling produces a slower and more sustained increase in MAP kinase activity that is often restricted to the cytosol. Together, these highly organized signaling complexes dictate the location, duration, and ultimate function of GPCR-stimulated MAP kinase activity.

Figure 20-22. Cycling of the Ras protein between the inactive form with bound GDP and the active form with bound GTP occurs in four steps. By mechanisms discussed later, binding of certain growth factors to their receptors induces formation of the active Ras · GTP complex. Step 1: Guanine nucleotide – exchange factor (GEF) facilitates dissociation of GDP from Ras. Step 2: GTP then binds spontaneously, and GEF dissociates yielding the active Ras · GTP form. Steps 3and 4: Hydrolysis of the bound GTP to regenerate the inactive Ras · GDP form is accelerated a hundredfold by GTPase-activating protein (GAP). Unlike Gα, cycling of Ras thus requires two proteins, GEF and GAP; otherwise, Gα and Ras exhibit many common features.

20.4. Receptor Tyrosine Kinases and Ras The receptor tyrosine kinases (RTKs) are the second major type of cell-surface receptors that we discuss in detail in this chapter (see Figure 20-3d, right). The ligands for RTKs are soluble or membrane-bound peptide/protein hormones including nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin. Binding of a ligand to this type of receptor stimulates the receptor's intrinsic protein-tyrosine kinase activity, which subsequently stimulates a signal-transduction cascade leading to changes in cellular physiology and/or patterns of gene expression (see Figure 20-6). RTK signaling pathways have a wide spectrum of functions including regulation of cell proliferation and differentiation, promotion of cell survival (Section 23.8), 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. One such mutant receptor, encoded at the neu locus, contributes to the uncontrolled proliferation of certain human breast cancers (Section 24.3). 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. In this section we discuss activation of RTKs and how they transmit a hormone signal to Ras, the GTPase switch protein that functions in transducing signals from many different RTKs. The second part of RTK-Ras signaling pathways, the transduction of signals downstream from Ras to a common cascade of serine/threonine kinases, is covered in the next section

Figure 20-23. Activation of Ras following binding of a hormone (e.g., EGF) to an RTK. The adapter protein GRB2 binds to a specific phosphotyrosine on the activated RTK and to Sos, which in turn interacts with the inactive Ras · GDP. The guanine nucleotide – exchange factor (GEF) activity of Sos then promotes formation of the active Ras · GTP. Note that Ras is tethered to the membrane by a farnesyl anchor (see Figure 3-36b). [See L. Buday and J. Downward, 1993, Cell 73:611; J. P. Olivier et al., 1993, Cell 73:179; S. E. Egan et al., 1993, Nature 363:45; E. J. Lowenstein et al., 1992, Cell 70:431; M. A. Simon et al., 1993, Cell 73:169.]

Figure 20-27. Structures of Ras · GDP-Sos complex and Ras · GTP determined by x-ray crystallography. (a) Sos (shown as a trace diagram) binds to two switch regions of Ras · GDP, leading to a massive conformational change in Ras. In effect, Sos pries open Ras by displacing the switch I region, thereby allowing GDP to diffuse out. (b) GTP is thought to bind to Ras-Sos first through its base; subsequent binding of the GTP phosphates complete the interaction. The resulting conformational change in Ras displaces Sos and promotes interaction of Ras · GTP with its effectors (discussed later). Ras is in a slightly different orientation in parts (a) and (b). GDP and GTP are shown as small stick models in the center of Ras; the adjacent sphere is a Mg2 ion. [From P. A. Boriack-Sjodin and J. Kuriyan, 1998, Nature 394:341; courtesy of John Kuriyan.]

Figure 20-6. Schematic overview of common signaling pathways downstream from G protein – coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). Hormone binding to the receptor initiates a series of events leading to phosphorylation of specific substrate proteins, which mediate the cellular responses such as changes in the activity of metabolic enzymes, gene expression, and cytoskeletal structures. The kinase cascade entails sequential activation of specific protein kinases induced by a signal from activated Ras protein. Second messengers (SM) play a role in some RTK signaling pathways, although not in the pathway depicted here. Likewise, some GPCR pathways do not involve second messengers; these lead to activation of MAP kinase. See text for discussion.

Receptors for Many Peptide Hormones Are Down-Regulated by Endocytosis The principal mechanism for down-regulating the receptors for many peptide hormones (e.g., insulin, glucagon, EGF, and PDGF) is ligand-dependent receptor-mediated endocytosis. In the absence of EGF ligand, for instance, the EGF receptor is internalized with bulk membrane flow. Binding to EGF induces a conformational change in the cytoplasmic tail of the receptor. This exposes a sorting motif that facilitates receptor recruitment into coated pits and subsequent internalization. After the receptor-hormone complex is internalized, the hormone is degraded in lysosomes — a fate similar to that of other endocytosed proteins, such as low-density lipoproteins (see Figure 17-64). Unlike the low-density lipoprotein (LDL) receptor, internalized receptors for many peptide hormones do not recycle efficiently to the cell surface. In the presence of EGF, for instance, the average half-life of an EGF receptor on a fibroblast cell is about 30 minutes; during its lifetime, each receptor mediates the binding, internalization, and degradation of only two EGF molecules. Each time an EGF receptor is internalized with bound EGF, it has a high probability (about 50 percent) of being degraded in an endosome or lysosome. Exposure of a fibroblast cell to high levels of EGF for 1 hour induces several rounds of endocytosis, resulting in degradation of most receptor molecules. If the concentration of extracellular EGF is then reduced, the number of EGF receptors on the cell surface recovers, but only after 12 – 24 hours. Synthesis of new receptors is needed to replace those degraded by endocytosis, which is a slow process that may take more than a day.

ignals Pass from Activated Ras to a Cascade of Protein Kinases A remarkable convergence of biochemical and genetic studies in yeast, C. elegans, Drosophila, and mammals has revealed a highly conserved cascade of protein kinases that operate in sequential fashion downstream from activated Ras as follows (Figure 20-28): 1. Activated Ras binds to the N-terminal domain of Raf, a serine/threonine kinase. 2. Raf binds to and phosphorylates MEK, a dual-specificity protein kinase that phosphorylates both tyrosine and serine residues. 3. MEK phosphorylates and activates MAP kinase, another serine/threonine kinase. 4. MAP kinase phosphorylates many different proteins, including nuclear transcription factors, that mediate cellular responses. Several types of experiments have demonstrated that Raf, MEK, and MAP kinase lie downstream of Ras and their sequential order in the pathway. For example, constitutively active mutant Raf proteins induce quiescent cultured cells to proliferate in the absence of hormone stimulation. These mutant Raf proteins, which initially were identified in tumor cells, are encoded by oncogenes and stimulate uncontrolled cell proliferation. Conversely, cultured mammalian cells that express a mutant, defective Raf protein cannot be stimulated to proliferate uncontrollably by a mutant, constitutively active RasD protein. This finding establishes a link between the Raf and Ras proteins. In vitro binding studies have shown that purified Ras · GTP protein binds directly to Raf. An interaction between the mammalian Ras and Raf proteins also has been demonstrated in the yeast two-hybrid system, a genetic system in yeast used to select cDNAs encoding proteins that bind to target, or “bait” proteins (Figure 20-29). The binding of Ras and Raf to each other does not induce the Raf kinase activity. The location of MAP kinase downstream of Ras was evidenced by the finding that in quiescent cultured cells expressing a constitutively active RasD, activated MAP kinase is generated in the absence of hormone stimulation. More importantly, in Drosophila mutants that lack a functional Ras or Raf but express a constitutively active MAP kinase specifically in the developing eye, R7 photoreceptors were found to develop normally. This finding indicates that activation of MAP kinase is sufficient to transmit a proliferation or differentiation signal normally initiated by ligand binding to an RTK. Biochemical studies showed that Raf does not activate MAP kinase directly. The signaling pathway thus appears to be a linear one: activated RTK → Ras → Raf → (?) → MAP kinase. Finally, fractionation of cultured cells that had been stimulated with growth factors led to identification of MEK, a kinase that specifically phosphorylates threonine and tyrosine residues on MAP kinase, thereby activating its catalytic activity. (The acronym MEK comes from MAP and ERK kinase, where ERK is another acronym for MAP.) Later studies showed that MEK binds to the C-terminal catalytic domain of Raf and is phosphorylated by the Raf serine/ threonine kinase activity; this phosphorylation activates the catalytic activity of MEK. Hence, activation of Ras induces a kinase cascade that includes Raf, MEK, and MAP kinase.top link

Molecular Cell Biology 20. Cell-to-Cell Signaling: Hormones and Receptors 20.5. MAP Kinase Pathways Figure 20-28. Kinase cascade that transmits signals downstream from activated Ras protein. In unstimulated cells, most Ras is in the inactive form with bound GDP (top); binding of a growth factor to its RTK leads to formation of the active Ras · GTP (see Figure 20-23). A signaling complex then is assembled downstream of Ras, leading to activation of MAP kinase by phosphorylation of threonine and tyrosine residues separated by a single amino acid. Phosphorylation at both sites is necessary for activation of MAP kinase. Several details not shown in the diagram are discussed in the text. [See A. B. Vojtek et al., 1993, Cell 74:205; L. Van Aelst et al., 1993, Proc. Nat'l. Acad. Sci. USA 90:6213; S. A. Moodie et al., 1993, Science 260:1658; H. Koide et al., 1993, Proc. Natl. Acad. Sci. USA 90:8683; P. W. Warne et al., 1993, Nature 364:352.]

Figure 20-45. Activation of protein kinase B by the Ras-independent insulin signaling pathway. The insulin receptor is a dimeric RTK. Step 1: Insulin binding to the receptor leads to a conformational change that induces autophosphorylation, similar to activation of other RTKs (see Figure 20-21). After IRS1 binds to a phosphotyrosine residue through a PTB domain, the activated kinase in the receptor's cytosolic domain phosphorylates IRS1. One subunit of PI-3 kinase binds to the receptor-bound IRS1 via its SH2 domain, and the other subunit then phosphorylates PI 4,5-bisphosphate and PI 4-phosphate to PI 3,4,5- trisphosphate and PI 3,4-biphosphate, respectively. Step 2 : The phosphoinositides bind the PH domain of protein kinase B (PKB), thereby recruiting it to the membrane. Two membrane-bound kinases, in turn, phosphorylate membrane-associated PKB and activate it. Step 3: Activated PKB is released from the membrane and promotes glucose uptake by the GLUT4 transporter and glycogen synthesis. The former effect results from translocation of the GLUT4 glucose transporter from intracellular vesicles to the plasma membrane. The latter effect occurs by PKB-catalyzed phosphorylation of glycogen synthase kinase 3 (GSK3), converting it from its active to inactive form. As a result, GSK3-mediated inhibition of glycogen synthase is relieved, promoting glycogen synthesis. [See from J. Downward, 1998, Curr. Opin. Cell Biol. 10:262.]

Figure 20-30. Structures of MAP kinase in its inactive, unphosphorylated form (a) and active, phosphorylated form (b). Phosphorylation of MAP kinase by MEK at tyrosine 185 (pY185) and threonine 183 (pT183) leads to a marked conformational change in the phosphorylation lip (red). This change promotes dimerization of MAP kinase and binding of its substrates, ATP and certain proteins. [From B. J. Canagarajah et al., 1997, Cell 90:859; courtesy of Bertram Canagarajah and Elizabeth Goldsmith.]

Figure 20-38. Several second messengers are derived from phosphatidylinositol (PI). (a) Pathway for synthesis of DAG and IP3, two important second messengers. Each membrane-bound PI kinase places a phosphate on a specific hydroxyl group on the inositol ring, producing the phosphoinositides PIP and PIP2. Cleavage of PIP2 by phospholipase C (PLC) yields DAG and IP3. (b) Formation of other phosphoinositides (yellow) and inositol phosphates (blue). These reactions are catalyzed by various kinases and PLC. The pathway shown in (a) is highlighted in red. See text for discussion. [See A. Toker and L. C. Cantley, 1997, Nature 387:673-676 and C. L. Carpenter and L. C. Cantley, 1996, Curr. Opin. Cell Biol. 8:153-158.]

Substrate Binding by Protein Kinases The small domain of the kinase core binds ATP, while the large domain binds the target peptide (Figure 3-23). The structure of the ATP-binding site complements the structure of the nucleotide substrate. The adenine ring of ATP sits snugly at the base of the cleft, which is characterized by a highly conserved sequence, Gly-X-Gly-X-X-Gly. This triad of glycine residues, the “glycine lid,” is part of a strand-loop-strand motif that closes over the adenine of ATP and holds it in position. The adenine ring sits in a hydrophobic pocket and is positioned by hydrogen bonds and van der Waals attractions with the glycine residues and backbone amide groups. Two invariant residues, lysine at position 72 and aspartic acid at position 184, stabilize the phosphate groups, which protrude from the nucleotide-binding cleft (step 1 in Figure 3-24). Lys-72 bridges to the α and β phosphates of ATP, while the γ-phosphate group is chelated by a Mg2 ion bound to Asp-184. ATP is a common substrate for all protein kinases, but the sequence of the target peptide varies among different kinases. The peptide sequence recognized by cAPK is Arg-Arg-X-Ser-Y, where X is any amino acid and Y is a hydrophobic amino acid. The portion of the polypeptide chain containing the target serine, threonine, or tyrosine is bound to a shallow groove in the large domain of the kinase core. The peptide specificity of cAPK is conferred by several glutamic acid residues in the large domain, which bind the two arginine residues in the target peptide. Different residues determine the specificity of other protein kinases.top link

A Glimpse into the Dark Side of src Tyrosine Kinase: by Deanne Greene Department of Biology Lycoming College

How Src works The Src protein has three major domains, SH2 (for Src homology 2), SH3, and the kinase catalytic domain (or SH1), as shown above. SH2 and SH3 both play a part in protein-protein interactions, while the kinase catalytic domain contains the kinase active site. Src can be switched from an inactive to an active state through control of its phosphorylation state, or through protein interactions. There are two major phosphorylation sites on Src: one is at Tyr416 (or Y416), the other at Tyr527, as marked in the drawing above for chicken Src. Tyr416 can be auto-phosphorylated, which activates Src by displacing the P-Tyr416 from the binding pocket, allowing the substrate to gain access. A more critical site is Tyr527, which can be phosphorylated and dephosphorylated by various proteins, such as CSK kinase (phosphorylates), or SHP-1 phosphorylase (dephosphorylates). Phosphorylation of Tyr527 inactivates Src through the interaction of P-Tyr527 with the SH2 domain, which effectively folds Src up into a closed, inaccessible bundle. Dephosphorylation of Tyr527 releases this bond, opening up the molecule to an active state. Protein interactions also act to regulate Src by either directly activating Src, or by moving Src to sites of action. Both platelet-derived growth factor and focal adhesion kinase are able to bind to the SH2 domain, causing Src to open up into the active form. Many of the substrates that Src can phosphorylate with its kinase domain form part of signalling cascades. These include Fak and Cas, which are important for integrin signalling, as well as Shc and Stat3, which are involved in growth regulation. Signalling systems often involve a cascade mechanism of sequential phosphorylation and dephosphorylation of proteins in the cascade, as occurs here.

Figure 20-3. Four classes of ligand-triggered cell-surface receptors. Common ligands for each receptor type are listed in parentheses. (a) G protein – linked receptors. Binding of ligand (maroon) triggers activation of a G protein, which then binds to and activates an enzyme that catalyzes synthesis of a specific second messenger. (b) Ion-channel receptors. A conformational change triggered by ligand binding opens the channel for ion flow. (c) Tyrosine kinase – linked receptors. Ligand binding causes formation of a homodimer or heterodimer, triggering the binding and activation of a cytosolic protein-tyrosine kinase. The activated kinase phosphorylates tyrosines in the receptor; substrate proteins then bind to these phosphotyrosine residues and are phosphorylated. (d) Receptors with intrinsic ligand-triggered enzymatic activity in the cytosolic domain. Some activated receptors are monomers with guanine cyclase activity and can generate the second messenger cGMP (left). The receptors for many growth factors have intrinsic protein-tyrosine kinase activity (right). Ligand binding to most such receptor tyrosine kinase (RTKs) causes formation of an activated homodimer, which phosphorylates several residues in its own cytosolic domain as well as certain substrate proteins. [Part (c) see J. E. Darnell et al., 1994, Science 264:1415. Part (d) see S. Schulz et al., 1989, FASEB J. 3:2026; D. Garbers, 1989, J. Biol. Chem. 264:9103; and W. J. Fantl et al., 1993, Annu. Rev. Biochem. 62:453.] Navigation About this book 20. Cell-to-Cell Signaling: Hormones and Receptors 20.1. Overview of Extracellular Signaling 20.2. Identification and Purification of Cell-Surface Receptors 20.3. G Protein –Coupled Receptors and Their Effectors 20.4. Receptor Tyrosine Kinases and Ras 20.5. MAP Kinase Pathways 20.6. Second Messengers 20.7. Interaction and Regulation of Signaling Pathways 20.8. From Plasma Membrane to Nucleus PERSPECTIVES for the Future PERSPECTIVES in the Literature Testing Yourself on the Concepts MCAT/GRE-Style Questions References Search This book All books PubMed

20.4. Receptor Tyrosine Kinases and Ras The receptor tyrosine kinases (RTKs) are the second major type of cell-surface receptors that we discuss in detail in this chapter (see Figure 20-3d, right). The ligands for RTKs are soluble or membrane-bound peptide/protein hormones including nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin. Binding of a ligand to this type of receptor stimulates the receptor's intrinsic protein-tyrosine kinase activity, which subsequently stimulates a signal-transduction cascade leading to changes in cellular physiology and/or patterns of gene expression (see Figure 20-6). RTK signaling pathways have a wide spectrum of functions including regulation of cell proliferation and differentiation, promotion of cell survival (Section 23.8), and modulation of cellular metabolism.

Figure 20-21. General structure and activation of receptor tyrosine kinases (RTKs). The ligands for some RTKs, such as the receptor for EGF depicted here, are monomeric; ligand binding induces a conformational change in receptor monomers that promotes their dimerization. The ligands for other RTKs are dimeric; their binding brings two receptor monomers together directly (see Figure 20-4d). In either case, the kinase activity of each subunit of the dimeric receptor initially phosphorylates tyrosine residues near the catalytic site in the other subunit. Subsequently, tyrosine residues in other parts of the cytosolic domain are autophosphorylated. See text for discussion. [See G. Panayotou and W. D. Waterfield, 1993, Bioessays 15:171; M. Mohammadi et al., 1996, Cell 86:577.]

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