Functional implications of arrestin recruitment

Arrestin binding to G protein-coupled receptors leads to desensitization (Chapter 1.4.2) of the GPCR, to receptor internalization (Chapter1.4.2) and to non-canonical signal transduction (Chapter1.4.2).

Desensitization

Repetitive stimulation of GPCRs leads to a decrease in receptor response. This means that the same amount of catecholamines will result in a lower amount of produced cAMP as compared to a receptor that is stimulated for the first time. This depicts a negative feedback mechanism to protect the cell from overstimulation (Ferguson, 2001). In case of rhodopsin it is also the basis for the adaptation to different levels of light intensity (Arshavsky,2002; Ridge et al.,2003).

The molecular basis for this desensitization of a GPCR is receptor phosphory-lation by GRKs (Bouvier et al.,1988) (Figure1.9[2]) and subsequent binding of arrestins (Lohse et al.,1990) (Figure1.9 [3]). These proteins physically uncou-ple the receptor from its G protein and cause the cessation of cAMP production.

Since this process is strongly agonist-dependent, it is also termedhomologous desensitization(Lohse et al.,1990) to distinguish it from the agonist-independent

heterologous desensitization, which is found after phosphorylation by kinases other than GRKs such as PKA or PKC. In contrast to homologous desensitization the latter process is more generalized and also switches off unactivated receptors.

Figure 1.9:Desensitization and internalization of a G protein-coupled receptor using the example of theβ₁-adrenoceptor.

After activation (1) the receptor recruits a trimeric G protein and the canonical signaling starts (Figure 1.10). (2) The GPCR is then phosphorylated by different kinases for example G protein-coupled receptor kinases and protein kinase A. (3) This leads to the attraction ofdocking proteins such asβ-arrestins. Binding of arrestins results in desensitizationof the receptor which means that the GPCR stops canonical signal transduction. (4and5) Arrestin recruitment also leads to endocytosis or internalization of the receptor. The receptor is then either degraded (6) or recycled (7) and brought back to the plasma membrane.

Internalization

The second process responsible for a decrease in cAMP production after repetitive agonist stimulation is internalization. Arrestin recruitment leads to endocytosis of the GPCR via caveolae and clathrin-coated pits (Figure1.9[4]).β-arrestins interact directly with clathrin or with the clathrin adapter protein AP2 (Goodman et al.,1996;

Goodman et al.,1997; Oakley et al.,1999; Laporte et al.,2002). The receptor can

then be either degraded through the endosome lysosome system (Figure1.9[6]) or recycled and brought back to the membrane (Figure1.9[7]).

Internalization of theβ₁-adrenoceptor however, is still a highly discussed subject.

Its internalization has been shown to depend on the different kinases, which phoshorylate the receptor. This phosphorylation then leads to clathrin-mediated endocytosis as described above and to internalization via invaginations of the plasma membrane, called caveolae (Rapacciuolo et al., 2003). Other authors have found the ADRB1 not to internalize at all (Eichel et al., 2016), to exhibit an endophilin-dependent internalization (Boucrot et al., 2015) or to internalize depending on the receptor’s PDZ type I domain (Figure1.3[red]). This is a protein-protein interaction domain at the far distal C-terminus which impacts internalization for many GPCRs, as shown for the β₂-adrenoceptor (Cao et al., 1999). PDZ is an acronym for post synaptic density protein 95, Drosophila disc large tumor suppressor and zonula occludens-1 protein. These were the first three proteins which were found to contain this conserved domain (Kennedy,1995). This domain is made up of the following sequence: X-(S/T)-X-Ø, with X at positions -1 and -3 as any amino acid, serine or threonine at position -2 and a bulky hydrophobic amino acid (Ø) at position 0 (Sheng and Sala,2001).

Many different downstream proteins interact with the PDZ type I domain. For the β₁-adrenoceptor there is evidence, that SAP97 (synapse-associated protein 97) is of great importance for receptor internalization and receptor recycling (Gardner et al.,2007). It was reported by the same group, that phosphorylation of serine 312 by protein kinase A acts in concert with the PDZ type I domain regarding the internalization of the ADRB1 (Gardner et al.,2004).

Signal transduction via arrestins

Other effects of β-arrestin binding include GPCR ubiquitination (Shenoy and Lefkowitz, 2003) and the triggering of an alternative signaling pathway where β-arrestin itself acts as a signal transducer (Luttrell and Gesty-Palmer,2010).

Apart from canonical signal transduction via G proteins (Chapter1.2.1) recent data suggests that GPCRs can also signal alternatively viaβ-arrestins (Figure1.10;

Shenoy et al.,2006; Shukla et al.,2014). The idea is thatβ-arrestins also undergo a conformational change after binding to GPCRs. This involves the rotation of the two domains relative to each other (Gurevich and Gurevich,2006), which is thought to uncover binding sites for various other proteins. Thusβ-arrestins act as adaptor proteins and can form scaffolds, to which other proteins can bind to (Rockman et al., 2002). So far this signal transduction pathway is not fully understood, but signaling

via MAP kinase 1/3 (MAPK1/3), PKB, c-src (proto-oncogene tyrosine-protein kinse src) and JNK (c-Jun N-terminal kinase) is proposed (Irannejad et al.,2013; Smith and Rajagopal, 2016). One possible pathway leads to an activation of the MAP kinase pathway involving MAPK1/3 through an EGFR (epidermal growth factor receptor) transactivaton (Noma et al.,2007). This process seems to be G protein-independent. However, it is very likely that other ways of activating MAPK1/3 in a G_sprotein-independent fashion exist, such as G_βγ-dependent MAPK1/3 activation (Hawes et al.,1995).

Figure 1.10:Schematic overview of GPCR signaling

GPCRc can signal via the non-canonical (left hand side) and the canonical (right hand side) pathway. cAMP = cyclic adenosine monophosphate, GRK = G protein-coupled receptor kinase, GTP = guanosine triphosphate, NE = norepinephrine, P = inorganic phosphate and phosphorylation, respectively.

This alternative signal transduction is also known asbiased agonismorfunctional selectivity (Kenakin,2007). This is the case when an agonist only activates one signaling pathway, either the G protein-coupled pathway or the alternative signaling pathway. If the GPCR couples to more than one G protein e.g. theβ₂-adrenoceptor, which is able to couple to G_sand G_i(Daaka et al.,1997), biased agonism can also refer to an agonist, that only activates one of these pathways.

A very well characterized biased agonist is carvedilol (Wisler et al.,2007), which is reported to be an inverse agonist concerning G protein signaling, but a weak

agonist when signaling viaβ-arrestins. The receptor‘s phosphorylation is a key factor for this alternative signaling (Lefkowitz and Shenoy, 2005; Premont and Gainetdinov,2007).

1.4.3 Fluorescence resonance energy transfer-based real time