Other Signaling Pathways

In addition to coupling to G proteins, GPCRs have been shown to mediate signal transduction events through interactions with other cellular proteins (Figure 2.2). For example, c-Src tyrosine kinase interacts with the proline-rich SH3 (Src homol-ogy-3) domain in the third intracellular loop of the p3-adrenergic receptor, which leads to the activation of the extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) cascade.15 It has also been demonstrated that the interaction between p-arrestin-1 and c-Src can facilitate the p2-adrenergic receptor-dependent activation of the ERK-MAPK pathway.16

The p-arrestin-c-Src interaction plays an important role in the glucose transport mediated by endothelin receptors through its stimulation of the GLUT4 glucose transporter.17 In a similar way, the interleukin-8-stimulated granule release is facilitated.18 Another example of a receptor-protein interaction relates to the activation

Ste 7

MEK 1

MKK 4/7

MKK 3/6

Ste 7

MEK 1

MKK 4/7

MKK 3/6

ERK 1/2

JNK 1-3

ERK 1/2

Transcription factors

NFkB

AP-1

FIGURE 2.2 Other signalling pathways. GPCRs can also trigger the activation of other signalling pathways such as the ERK/MAPK and JNK pathways, which leads to the induction of transcription factors.

MLBBSO-

Transcription factors

JNK 1-3

MLBBSO-

Nucleus

Nucleus

NFkB

AP-1

FIGURE 2.2 Other signalling pathways. GPCRs can also trigger the activation of other signalling pathways such as the ERK/MAPK and JNK pathways, which leads to the induction of transcription factors.

of the STAT (signal transducer and activator of transcription) transcription factor.19 In this case, the angiotensin 1A receptor interacts through its C-terminus with JAK2 (Janus kinase).

Several other examples demonstrate interactions of GPCRs and cellular proteins. Typically, the interaction of p2-adrenergic receptor and the NHERF1 and NHERF2 sodium-hydrogen exchanger regulatory proteins is mediated by PDZ2021 Metabotro-pic glutamate receptors such as mGluR7 also can interact with PDZ-domain-con-taining proteins.22 The biological and pharmacological significance of non-G protein signaling is probably to function as a means to enhance the specificity of GPCR signaling. It may also allow distinct physiological functions of two different GPCRs otherwise activated by the same ligand and coupled to the same G protein.

In addition to the interactions of individual receptors and individual cellular proteins, more general interactions can occur between GPCRs and adaptor/scaffolding proteins. The function of p-arrestin as an adaptor/scaffolding protein for the activation of MAPK cascades has been described above. Recently, it was discovered that p-arrestin can interact with the last three kinases in both the ERK/MAPK pathway and the JNK3 (c-Jun amino-terminal kinase 3) cascade.2324 The function of p-arrestin seems to be establishment of the contacts of the kinases in both the ERK/MAPK and JNK3 cascades with each other and the GPCRs, which facilitates the signal transduction from receptor to effector. Interestingly, the p-arrestin-medi-

ated scaffolding of MAPK cascades results in kinase retention in the cytoplasm, which promotes the internalization of receptors as described below.25 In summary, scaffolding proteins can enhance the overall activation of proteins in signal transduction pathways and can increase specificity by interaction with only those proteins that will be activated. In this context, p-arrestin-2 only induces the activation of JNK3, although two other JNK isoforms exist.24

An important fairly recent phenomenon observed for the signal transduction of GPCRs is the dimerization of receptors.26 In case of signaling through receptor tyrosine kinases, for dimeric receptors each monomer can phosphorylate the other monomer during ligand-stimulated autophosphorylation to achieve a fully active state of the kinase. GPCR dimerization is described in more detail in Chapter 15.

2.6 TRAFFICKING OF GPCRs

Although the localization of GPCRs has been associated with the plasma membrane, several studies have demonstrated that they possess the means of being internalized and present various ways of trafficking in different cellular compartments (Figure 2.3). In principle, at least two endocytic GPCR pathways exist; both lead to the formation of specific membrane vesicles but different involvement of p-arrestins.27 The internalization can be mediated by flask-shaped membrane invaginations called caveolae, which contain large amounts of caveolin proteins and cholesterol. It is

FIGURE 2.3 Trafficking of GPCRs. Upon agonist activation, GPCRs are internalized in endosomes with the aid of p-arrestins. Depending on the receptor, they are either recycled to the plasma membrane or subjected to degradation.

Receptor Mediated Caveolin Endocytosis

FIGURE 2.3 Trafficking of GPCRs. Upon agonist activation, GPCRs are internalized in endosomes with the aid of p-arrestins. Depending on the receptor, they are either recycled to the plasma membrane or subjected to degradation.

thought that signaling molecules such as receptor-activated G proteins transiently bind to caveolin and this protein-protein interaction decreases the signaling capacity of the GPCR, which results in desensitization of the receptor.28 In addition, caveolin has been shown to interact with GRKs, which results in a modulated kinase activity.29 Agonist-dependent endocytosis into caveolae has been described for endothelin-A,30 bradykinin B2,31 p2-adrenergic receptors,32 and adenosine A1 receptors.28

GPCRs can also be internalized through specialized plasma membrane structures called clathrin-coated pits.33 These complexes first invaginate and then detach from the plasma membrane, forming clathrin-coated vesicles. Two components, clathrin and adapter protein complex AP2, are responsible for the assembly of clathrin-coated pits. In the case of GPCR endocytosis, p-arrestin forms a bridge between the activated receptor and the clathrin-coated pit.34 Studies of agonist-activated thyrotro-phin-releasing hormone receptor-1 demonstrated that receptor-arrestin complexes are internalized into pre-existing clathrin-coated pits and do not promote the formation of specific coated pits for endocytosis.35 The involvement of dynamin has been demonstrated for both clathrin- and caveolin-mediated sequestration of GPCRs.3637 It has also been revealed that p-arrestin can regulate GPCR internalization and degradation through ubiquitination — a process by which ubiquitin is added to lysine residues of the target protein.38 The process requires three enzymes: ubiquitin-acti-vating enzyme, ubiquitin-carrying enzyme, and ubiquitin-ligase. A direct interaction occurs between p-arrestin and the ubiquitin-ligase mouse double minute 2 (MDM2) that results in ubiquitinated of both p-arrestin and p2-adrenergic receptor. The ubiquitination p-arrestin regulates the internalization of the p2-adrenergic receptor. p-arrestin can also modulate receptor internalization through an interaction with proteins involved in vesicle budding. For instance, p-arrestin interaction occurs with ARF6 (ADP-ribosylation factor 6) and the N-ethylmaleimide-sensitive factor (NSF).39

The overexpression of NSF in cells expressing recombinant p2-adrenergic receptor increases the amount of internalized receptor and the recycling of receptor to the plasma membrane is promoted after agonist removal. It has also been suggested that the p-arrestin-dependent internalization aids in receptor downregulation. In this context, the internalization and downregulation of the p2-adrenergic receptors are blocked in cells that lack p-arrestin-1 and p-arrestin 2.40 Receptor downregulation requires ubiquitination. A p2-adrenergic receptor that lacks all potential ubiquitination sites is not downregulated because it is not targeted to lysosomes.38

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