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Proliferation Inflammation Response Differentiation Cell death Celt survival bind to promoters to initiate gene transcription

Figure 133 MAPK pathways consist of three protein kinases. The third kinase is a nuclear transcription factor. Reprinted from Geoffrey M.Cooper, The Cell: A Molecular Approach, 4th edn., © (2007), with permission from ASM Press, Washington DC.

activation of MAP kinase cascades. These scaffolds serve at least three functions in cells:

• To increase the efficiency of signalling between successive kinases in the phosphorylation cascade.

• To dampen cross talk between parallel MAP kinase cascades.

• To target MAP kinases to specific subcellular locations.

The ERK/MAP kinase pathway is well known for its predilection to be activated by growth factor receptors such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors. Auto phosphorylation of these receptor-tyrosine kinases recruits several adapter proteins, resulting in the activation of Ras (a small G protein that is attached to the plasma membrane by farnesyl and palmitoyl lipid groups). As for the GPCR-associated G proteins, inactive Ras contains tightly bound GDP and, during its activation, this nucleotide will dissociate and be replaced by GTP (Figure 134). Ras is a central player in signal transduction. Perhaps the best-characterized downstream effector of Ras is Raf, which leads to the ERK/MAP kinase pathway.

The link between GPCR-P-arrestin complexes and ERK/MAP kinase activation has been brought to light by data from yeast-2 hybrid screens (see Section 4.8) using P-arrestins, and from the biochemical characterization of receptor-P-arrestin complexes:

• AT1a receptor activation results in the formation of complexes between the receptor, P-arrestin 2 and the component kinases of the ERK cascade (Raf-1, MEK1 and ERK2). P-Arrestins appear to act as scaffolds for ERK/MAP kinase cascade as well as for the JNK3 MAP kinase cascade.

Figure 134 Three-dimensional structure (Reprinted with permission from Biochemistry, 33, Kraulis, P. J., Domaille, P. J., Campbell-Burk, S. L., Van Aken, T.and Laue, E.D., Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy, 3515-3531. Copyright (1994) American Chemical Society) and activation mechanism of the small G protein, Ras.

inactive Ras protein inactive Ras protein

Figure 134 Three-dimensional structure (Reprinted with permission from Biochemistry, 33, Kraulis, P. J., Domaille, P. J., Campbell-Burk, S. L., Van Aken, T.and Laue, E.D., Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy, 3515-3531. Copyright (1994) American Chemical Society) and activation mechanism of the small G protein, Ras.

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Figure 135 Fluorescence microscopy: GFP-P-arrestin 2 is in the cytosol of COS-7 cells and fluorescence of antibodies against active ERK (phospho-ERK1/2) is minimal. Angiotensin II redistributes its receptors along with GFP-P-arrestin 2 into large endosomal vesicles. Now, anti-phospho-ERK1/2 fluorescence partially co-localizes with GFP-P-arrestin 2. Note that there is only a little phospho-ERK1/2 in the nucleus (Tohgo et al., 2002, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

Figure 135 Fluorescence microscopy: GFP-P-arrestin 2 is in the cytosol of COS-7 cells and fluorescence of antibodies against active ERK (phospho-ERK1/2) is minimal. Angiotensin II redistributes its receptors along with GFP-P-arrestin 2 into large endosomal vesicles. Now, anti-phospho-ERK1/2 fluorescence partially co-localizes with GFP-P-arrestin 2. Note that there is only a little phospho-ERK1/2 in the nucleus (Tohgo et al., 2002, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

• Receptor-P-arrestin complexes are also associated with the activation and cytosolic retention of ERK (Figure 135, Figure 136). In this respect, activated ERK1/2 does not appear to undergo nuclear translocation. Instead bound ERK1/2 may phosphorylate plasma membrane, cytosolic or cytoskeletal substrates, or it may lead to transcriptional activation through the activation of other kinases.

• Certain receptor-P-arrestin complexes also induce cell mitogenesis by stimulating the ERK/ MAP kinase cascade. This signalling cascade is initiated by the interaction between P-arrestin and the SH3 domain of c-Src, a protein kinase that is capable of activating EGF receptors by phosphorylating some of their intracellular tyrosine residues (Figure 137). This 'cross talk' between GPCRs and EGF receptors results in a Ras-dependent activation of the ERK/MAP kinase pathway and, hence, to a mitogenic response.

Many GPCRs simultaneously employ multiple mechanisms to activate MAP kinases. For example, the AT1A receptor can activate ERK1/2 not only via P-arrestin-dependent pathways but also through G protein-dependent signals. G protein-dependent mechanisms (Figure 138) include:

• Protein kinase A-dependent phosphorylation of the small G protein, Rap1.

• Protein kinase C-dependent activation of Raf (a small G protein like Ras).

• 'Transactivation' of receptor tyrosine kinases, such as the EGF and PDGF receptors.

Figure 136 AT1A receptor stimulation triggers the consecutive binding of P-arrestin and ERK1/2. However, such activated ERK1/2 is unable to undergo nuclear translocation (i.e. to act as a nuclear transcription factor). Reproduced by permission of the Company of Biologists, Journal of Cell Science, 115, Luttrell, L. M. and Lefkowitz, R. J., The role of beta-arrestins in the termination and transduction of G protein-coupled receptor signals, 455-465, Copyright (2002).

Figure 136 AT1A receptor stimulation triggers the consecutive binding of P-arrestin and ERK1/2. However, such activated ERK1/2 is unable to undergo nuclear translocation (i.e. to act as a nuclear transcription factor). Reproduced by permission of the Company of Biologists, Journal of Cell Science, 115, Luttrell, L. M. and Lefkowitz, R. J., The role of beta-arrestins in the termination and transduction of G protein-coupled receptor signals, 455-465, Copyright (2002).

ERK activation by Gj -activating receptors is likely to result from the liberation of P-y subunits from Gi that can directly activate PI3-kinase, leading to the activation of Ras and the Raf/MEK/ERK cascade.

Figure 137 GPCR induces cell mitogenesis via EGF receptor activation. To start the signalling casade, GPCR-P-arrestin complexes bind to the SH3 domain of c-Src. c-Src is a protein kinase that is capable of phosphorylating EGF receptors. This triggers Ras-dependent activation of the ERK/MAP kinase pathway.

Figure 137 GPCR induces cell mitogenesis via EGF receptor activation. To start the signalling casade, GPCR-P-arrestin complexes bind to the SH3 domain of c-Src. c-Src is a protein kinase that is capable of phosphorylating EGF receptors. This triggers Ras-dependent activation of the ERK/MAP kinase pathway.

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Figure 138 G protein-dependent pathways for GPCR-mediated MAP kinase stimulation. Note that GPCRs can simultaneously employ multiple mechanisms.

Figure 138 G protein-dependent pathways for GPCR-mediated MAP kinase stimulation. Note that GPCRs can simultaneously employ multiple mechanisms.

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