Activation of many GPCRs stimulates cell growth and differentiation in a variety of physiological and experimental systems (Gutkind 1998; Pierce et al. 2001). In many cases, these actions are correlated with receptor capacity to stimulate mitogen-activated protein kinase (MAPK) signalling pathways. Although mechanisms responsible for GPCR-directed stimulation of MAPK pathways are poorly understood, recent studies demonstrate that ß-arrestins serve as adaptor/scaffolding proteins which form stable complexes with certain GPCRs and various intracellular non-receptor kinases (Miller and Lefkowitz 2001; Pierce et al. 2001). ß-arrestins recruit members of the c-Src tyrosine kinases to ß-adrenergic receptors (Luttrell etal. 1999; Miller et al. 2000) and neurokinin-1 receptors (DeFea etal. 2000a). Similarly,

ß-arrestins recruit the Src family kinases c-Fgr and Hck to the interleukin CXCR-1 receptors (Barlic et al. 2000). In each of these cases, ß-arrestin-mediated complex formation confers kinase activity, albeit indirectly, to associated receptors. Thus, GPCRs can signal similarly to other non-G protein-coupled growth factor receptors with intrinsic tyrosine kinase catalytic activity.

ß-arrestins also complex directly with kinases that comprise MAPK signalling cascades, in particular those of the extracellular regulated kinases (ERKs) and c-jun N terminal kinases (JNKs) (Fig. 7.1a). Diverse upstream signals activate raf-1 kinase that phosphorylates and activates MEK1/2 kinases. Activated MEK1/2 then phosphorylate and activate ERK1/2 kinases leading to transcriptional regulation of target genes involved in cell growth and differentiation (Pearson etal. 2001). In a separate but parallel MAPK cascade, upstream activation of the serine/threonine kinase ASK1 leads to phosphorylation and activation of MKK4/7 kinases which, in turn, phosphorylate and activate JNK kinases (JNK1-3). Recent studies indicate that certain GPCR/ß-arrestin complexes bind cognate proteins of both the ERK and JNK pathways. Agonist-occupied protease-activated receptors (PAR-2) and angiotensin II type 1a receptors (ATlaR) each form stable complexes with ß-arrestins, raf-1 and Erk2 (DeFea et al. 2000b). In the case of AT1aR, MEK1 also complexes with ß-arrestin. Similarly, AT1a1 receptors can complex with ß-arrestin, ASK1 and JNK3, and stimulation of AT1aR leads to activation of JNK3 (McDonald et al. 2000). These findings demonstrate that ß-arrestins are hormone and GPCR-regulated scaffolding proteins for certain branches of the ERK and JNK signalling cascades.

Collectively, these observations indicate that ß-arrestins serve as adaptor proteins that physically and functionally link GPCRs to components of both the endocytotic pathways (clathrin, AP-2, NSF) and MAPK signalling cascades. Emerging models propose that following agonist activation GRKs are recruited to the plasma membrane to phosphorylate receptors. In turn, ß-arrestins are recruited to bind GRK-phosphorylated GPCRs to form a stable complex that is recognized by clathrin and AP-2 for targeted internalization. This stable GPCR/ß-arrestin complex can also serve as a platform to assemble raf-1, MEK1, and ERK2 or, alternatively, ASK1, MKK4, and JNK3 (Fig. 7.1a). As such, ß-arrestins apparently act in a manner analogous to the cytosolic scaffolding proteins MP-1 and JIP that assemble kinases involved with ERK and JNK signalling, respectively (Schaeffer et al. 1998; Yasuda et al. 1999). Such assemblies provide a cellular mechanism for promoting and localizing highly efficient MAPK signalling events. GPCR/ß-arrestin complexes allow a novel mechanism, whereby MAPK may phosphorylate substrates at cellular sites such as the plasma membrane or internalized vesicles at various temporal and spatial stages of sorting.

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