l-DOPA reverses the MPTP-induced elevation of the arrestin2 and GRK6 expression and enhanced ERK activation in monkey brain

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Abstract

Dysregulation of dopamine receptors (DARs) is believed to contribute to Parkinson disease (PD) pathology. G protein-coupled receptors (GPCR) undergo desensitization via activation-dependent phosphorylation by G protein-coupled receptor kinases (GRKs) followed by arrestin binding. Using quantitative Western blotting, we detected profound differences in the expression of arrestin2 and GRKs among four experimental groups of nonhuman primates: (1) normal, (2) parkinsonian, (3) parkinsonian treated with levodopa without or (4) with dyskinesia. Arrestin2 and GRK6 expression was significantly elevated in the MPTP-lesioned group in most brain regions; GRK2 was increased in caudal caudate and internal globus pallidus. Neither levodopa-treated group differed significantly from control. The only dyskinesia-specific change was an elevation of GRK3 in the ventral striatum of the dyskinetic group. Changes in arrestin and GRK expression in the MPTP group were accompanied by enhanced ERK activation and elevated total ERK expression, which were also reversed by l-DOPA. The data suggest the involvement of arrestins and GRKs in Parkinson disease pathology and the effects of levodopa treatment.

Introduction

Parkinson disease (PD) is a neurodegenerative disorder caused by degeneration of dopaminergic neurons that provide dopamine (DA) to the striatum (Ehringer and Hornykiewicz, 1960). DA replacement therapy with levodopa (l-DOPA) often results in motor complications such as wearing off or dyskinesia (Cotzias et al., 1969, Stocchi et al., 1997). Denervation-induced supersensitivity of DA receptors (DARs), which are members of the G protein-coupled receptor (GPCR) superfamily, is widely believed to be a contributing factor to lesion-induced motor disturbances as well as to dyskinesia, although the exact mechanisms remain to be elucidated. It has been hypothesized that altered subcellular DAR localization accompanying the complex changes in DAR expression (reviewed in Bezard et al., 2001a) plays a role in dyskinesia (Muriel et al., 1999).

Upon persistent stimulation, many GPCRs undergo desensitization via a two-step process: activation-dependent receptor phosphorylation by G protein-coupled receptor kinases (GRKs) followed by the binding of “uncoupling” proteins termed arrestins. Arrestin binding precludes further signaling via G proteins and induces receptor internalization. Internalized receptor can either be recycled back to the plasma membrane or targeted for degradation, which leads to receptor down-regulation. Two of the four arrestins, rod and cone arrestins, are expressed almost exclusively in the retina. The two nonvisual arrestins, arrestin2 and arrestin3,1 are ubiquitous and appear to participate in the desensitization of various GPCRs. There are seven GRKs, five of which, GRK2, 3, 4, 5, and 6, are expressed in the brain (Arriza et al., 1992, Benovic and Gomez, 1993, Premont et al., 1994). Thus, five GRKs and two arrestins appear to be involved in the desensitization of hundreds of different GPCR subtypes.

Many receptors are phosphorylated equally well in vitro by several GRKs (Ménard et al., 1996, Richardson and Hosey, 1993). Similarly, many receptors in vitro bind both nonvisual arrestins equally well (Gurevich et al., 1995, Krupnick and Benovic, 1998, Richardson and Hosey, 1993). However, studies with mice overexpressing or lacking various GRKs suggest that in vivo receptors may prefer specific GRKs (Gainetdinov et al., 1999, Iaccarino et al., 1998a, Iaccarino et al., 1998b, Koch et al., 1995, Rockman et al., 1996). In living cells, receptors appear to have preferences for specific arrestins (Kohout et al., 2001, Oakley et al., 2000). Thus, the sensitivity of GPCRs may be differentially regulated depending on the cellular complement of arrestins and GRKs. We hypothesized that changes in DA tone caused by nigrostriatal degeneration and/or subsequent pulsatile DA receptor stimulation by l-DOPA lead to alterations in arrestin/GRK gene expression that result in dysregulation of DA receptor signaling.

Here we report multiple changes in the concentrations of arrestins and GRKs within the basal ganglia of four nonhuman primate experimental groups: normal, parkinsonian, parkinsonian chronically treated with l-DOPA without exhibiting dyskinesia, and parkinsonian l-DOPA-treated with overt dyskinesia. Arrestin binding to GRK-phosphorylated GPCRs can initiate second round of signaling via MAP kinase cascades. Therefore, we investigated the changes in the expression and phosphorylation status of extracellular signal-regulated kinase (ERK1/2), which may be connected to modifications in arrestin/GRK expression

Section snippets

Animals

We used 15 female cynomolgus monkeys (Macaca fascicularis, Shared Animal Health, Beijing, PR of China). Animals were housed in individual primate cages under controlled conditions of humidity (50% ± 5%), temperature (24 ± 1°C), and light (12-h light/12-h dark cycle, lights on at 8:00 am); food and water were available ad libitum, and animal care was supervised by veterinarians skilled in the healthcare and maintenance of nonhuman primates. Experiments were carried out in accordance with the

Validation of the experimental model

We previously reported the behavior of the animals and some of the data describing the extent of dopaminergic lesion (Bezard et al., 2003a). Briefly, the parkinsonian syndrome was comparably severe between the different MPTP-treated groups. Four l-DOPA-treated MPTP-intoxicated animals developed severe dyskinesia (dyskinetic l-DOPA-treated MPTP-treated group), which was absent or minimal in the four remaining monkeys (nondyskinetic l-DOPA-treated MPTP-treated group). Because the extent of the DA

MPTP-specific l-DOPA-reversible changes in arrestin and GRK expression

Our data indicate that MPTP lesion in monkeys, which results in extensive loss of dopaminergic neurons and almost complete degeneration of dopaminergic fibers in the basal ganglia, increases the expression of several arrestin and GRK proteins (Table 1). Significant changes in the MPTP-treated monkeys occurred both in the rostral and caudal regions of the basal ganglia, with the cCN and GPi being most often affected. Arrestin2, the ubiquitous arrestin subtype most highly expressed throughout the

Acknowledgments

We are grateful to Dr. R.J. Lefkowitz (Duke University) for the generous gift of purified GRK3. This work was supported by grants from the Michael J. Fox Foundation for Parkinson Research (to EB), the Fédération pour la Recherche sur le Cerveau (to EB and CEG.), Fondation pour la Recherche Médicale (to EB and CEG), NIH grants MH62654 and NS045117 (to EVG), EY11500 and GM63097 (to VVG), and GM44944 and GM47417 (to JLB).

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