Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by l-DOPA treatment.

Parkinson’s disease (PD) is a neurodegenerative disorder caused by degeneration of dopaminergic neurons that provide dopamine (DA) to the striatum. DA replacement therapy with L-DOPA, although still the best treatment available, often results in motor complications such as dyskinesia (Cotzias et al. 1969; Stocchi et al. 1997). Loss of DA in the basal ganglia alters signaling via DA receptors in a complex manner. In particular, numerous signaling pathways in the DA-depleted basal ganglia show exaggerated responses to dopaminergic stimulation (Gerfen 2000; Gerfen et al. 2002; Brown et al. 2005; Sgambato-Faure et al. 2005; Bychkov et al. 2007). Chronic treatment with L-DOPA suppresses supersensitivity of the MAPK pathway (Bezard et al. 2005; Brown et al. 2005; Bychkov et al. 2006; Kim et al. 2006), while augmenting supersensitivity of others (Sgambato-Faure et al. 2005; Bychkov et al. 2006). The molecular mechanisms of signaling alterations caused by DA depletion remain largely unknown, and even the extent of these changes is not yet fully appreciated. Abnormal signaling via DA receptors in the basal ganglia caused by DA depletion and subsequent dopaminergic therapy undoubtedly play an important role in the pathophysiology of motor disturbances in PD and L-DOPA-induced dyskinesia. Therefore, the studies of signaling pathways in the DA-depleted striatum may prove critical for the understanding of the pathology of PD and dyskinesia and for the success of the antiparkinsonian and/or antidyskinetic therapy. Dopamine receptors belong to the superfamily of G protein-coupled receptors (GPCRs). Upon persistent stimulation, many GPCRs undergo desensitization because of activation-dependent receptor phosphorylation by G protein-coupled receptor kinases (GRKs) followed by arrestin binding. The arrestin binding ‘arrests’ signaling via G proteins and induces receptor internalization (Gurevich and Gurevich 2006; DeWire et al. 2007). GPCR signaling is heavily dependent on the rate and extent of the desensitization process. In cultured cells and in vivo, an increase in arrestin and/or GRK concentration facilitates GPCR desensitization, whereas the reduction of arrestin/GRK levels leads to deficits in GPCR desensitization and internalization and to exaggerated G protein-mediated signaling via affected receptors (Menard et al. 1997; Xu et al. 1997; Iaccarino et al. 1998b; Bohn et al. 1999; Gainetdinov et al. 1999; Willets et al. 1999, 2004; Kim et al. 2001; Bohn et al. 2003; Gainetdinov et al. 2003; Pan et al. 2003). Arrestins, in addition to their role in quenching G protein-mediated signaling, also act as multifunctional adaptors redirecting the GPCR signaling to multiple alternative pathways (Shenoy and Lefkowitz 2003; Gurevich and Gurevich 2006; DeWire et al. 2007). The up-regulation of arrestins reduces G protein-mediated signaling while enhancing arrestin-dependent extracellular signal–regulated kinase (ERK) activation (Ahn et al. 2003, 2004a). Arrestins bind and redistribute signaling molecules among subcellular compartments (Ahn et al. 2004b; Song et al. 2006; Wang et al. 2006; Hanson et al. 2007) affecting not only the intensity but also the direction and timing of the signaling. Therefore, changes in concentrations of arrestins and GRKs and their subcellular localization may contribute to perturbations in dopaminergic signaling caused by loss of DA alone or in combination with L-DOPA therapy. Two out of four arrestins, arrestin2 and arrestin3, and five out of seven GRKs, GRK2, 3, 4, 5, and 6, are expressed in the brain (Arriza et al. 1992; Gurevich et al. 2002, 2004; Bezard et al. 2005; Bychkov et al. 2006) and appear to interact with multiple GPCRs. Studies with genetically engineered mice and in living cells suggest that in vivo receptors may be preferentially phosphorylated by specific GRKs and differentially interact with arrestin isoforms (Koch et al. 1995; Rockman et al. 1996; Iaccarino et al. 1998b,a; Gainetdinov et al. 1999; Oakley et al. 2000; Kohout et al. 2001). In neostriatal neurons, DA D1 receptor interacts mostly with arrestin3 (Macey et al. 2005), whereas D2 receptor prefers arrestin2 (Macey et al. 2004). Recent studies indicate that phosphorylation of GPCRs by different GRKs have distinct functional consequences for both G protein-mediated and G protein-independent signaling (Kim et al. 2005; Ren et al. 2005). Arrestin isoforms differentially bind, activate, or move signaling proteins (McDonald et al. 2000; Scott et al. 2002; Wang et al. 2003; Witherow et al. 2004; Song et al. 2006). Thus, GPCR signaling is regulated in a complex manner by cellular complement of arrestin and GRKs isoforms. The expression of arrestins and GRKs can be regulated in vitro and in vivo by persistent stimulation, blockade, or lack of stimulation of specific GPCRs (Iaccarino et al. 1998a; Ozaita et al. 1998; Hurle 2001; Diaz et al. 2002; Fan et al. 2002; Bezard et al. 2005; Iaccarino et al. 2005; Rubino et al. 2006; Salim et al. 2007). Here we tested the hypothesis that loss of DA in PD and/or excessive stimulation of DA receptors during L-DOPA therapy induce specific changes in the level of expression of arrestin or GRK subtypes. Importantly, as arrestins and GRKs interact with multiple GPCRs, changes in their expression caused by DA depletion would affect signaling via many receptors. We have previously described the up-regulation of arrestins and GRKs in the MPTP-treated monkeys reversed by chronic L-DOPA treatment (Bezard et al. 2005). Here we report that depletion of DA by the 6-hydroxydopamine lesion of dopaminergic neurons causes multiple region- and subcellular compartment-specific changes in the concentrations of arrestins and GRKs in the rat basal ganglia. Chronic treatment with L-DOPA failed to restore normal arrestin and/or GRK levels but in some cases caused additional alterations. In contrast, treatment with the long-lived DA agonist pergolide, which has lower propensity to elicit dyskinesia in humans and sensitization in animals (Foley et al. 2004; Stocchi and Olanow 2004; Stocchi et al. 2005), tended to normalize the expression of these regulatory proteins.