GRK3 suppresses L-DOPA-induced dyskinesia in the rat model of Parkinson’s disease via its RGS homology domain

Degeneration of dopaminergic neurons causes Parkinson’s disease. Dopamine replacement therapy with L-DOPA is the best available treatment. However, patients develop L-DOPA-induced dyskinesia (LID). In the hemiparkinsonian rat, chronic L-DOPA increases rotations and abnormal involuntary movements modeling LID, via supersensitive dopamine receptors. Dopamine receptors are controlled by G protein-coupled receptor kinases (GRKs). Here we demonstrate that LID is attenuated by overexpression of GRK3 in the striatum, whereas knockdown of GRK3 by microRNA exacerbated it. Kinase-dead GRK3 and its separated RGS homology domain (RH) suppressed sensitization to L-DOPA, whereas GRK3 with disabled RH did not. RH alleviated LID without compromising anti-akinetic effect of L-DOPA. RH binds striatal Gq. GRK3, kinase-dead GRK3, and RH inhibited accumulation of ∆FosB, a marker of LID. RH-dead mutant was ineffective, whereas GRK3 knockdown exacerbated ∆FosB accumulation. Our findings reveal a novel mechanism of GRK3 control of the dopamine receptor signaling and the role of Gq in LID.

. The lentivirally-transferred isolated RH domain is expressed in striatal neurons and has subcellular distribution similar to full-length GRK3. (A) Domain structure of rat GRK3 and construction of the lentivirus containing isolated RH of GRK3 1 . Residues 1 to 180 containing N-terminal helix a0 and N-terminal helices 1-9 of RH were subcloned into the lentiviral vector as shown. RH -RGS homology domain; RHc -C-terminal helices 10 and 11 of the RH; kinase -kinase domain; PH -pleckstrin homology domain; (B) The expression of RH in medium spiny striatal neurons was detected by immunohistochemistry to co-cistronically expressed GFP (green) with anti-GFP antibody combined with the staining for FOXP1 (red) as described in Methods. (C) Subcellular localization of isolated RH with myc N-terminal tag was compared with that of myc-tagged wild type GRK3. Rats were injected into the striatum with lentiviruses encoding either RH of GRK3, striata were isolated, and subcellular fractionation was performed as previously described 2, 3 . Caspase-3 was used as the marker for cytosol fraction (S3); Na + ,K + ,ATPase -for light membrane fraction (P3); PSD95 -for synaptic membrane fraction (LP1); synaptophysin is detected in P3, LP1, and crude synaptic vesicle fraction (LS1); H -homogenate. Left lanes in GRK3-myc and RH-myc blots show standards in serial dilutions obtained by infecting HEK293 cells with respective lentiviruses.
Cell culture, transfection, and stimulation. HEK-293-FT cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2. Lipofectamin2000 (Invitrogen, Carlsbad, CA) (1:2.5 DNA:lipid) in Opti-MEM was used to transfect cells. DNA amounts in each transfection were kept constant by the addition of empty vector, where necessary. All experiments were conducted 48 h post-transfection.
Antibodies. Anti-HA antibody for immunoprecipitation was from Roche Diagnostics (Indianapolis, IN); anti-phospho-threonine antibodies were from Cell Signaling Technology (Beverly, MA) and anti-phospho-serine from Invitrogen (Carlsbad, CA).
Immunoprecipitation. HEK292FT cells co-transfected with indicated receptor and GRK clones were stimulated with dopamine (10 µM) for 10 min at 37 o C (control cells were exposed to vehicle for the same time), scraped off plates, collected by centrifugation in phosphate-buffered saline and resuspended in the immunoprecipitation buffer (IPB) containing 50 mM Tris-HCl, 2 mM EDTA, 250 mM NaCl, 10% (v/v) glycerol, 0.5% NP-40, 20 mM NaF, 1 mM sodium orthovanadate, and 10 mM N-ethylmaleimide. Benzamidine (2 mM final concentration) and phenylmethylsulfonyl fluoride (1 mM) were added immediately before use. Cells were lysed at 4 o C for 1 h and centrifuged to remove the debris. The supernatant was pre-cleared by incubating with 25-30 µl of Protein G Agarose for 1 h at 4 o C. Receptors were then immunoprecipitated by incubating the supernatant overnight at 4 o C with anti-HA antibody (1-2 µg per 60 mm dish) and 20-25 µl of Protein G agarose. Beads were washed three times with IPB, and bound proteins were eluted by boiling in Laemmli SDS buffer for 5 min.
Western blotting. The proteins were analyzed by reducing SDS-PAGE and Western blotting onto Immobilon-P (Millipore, Bedford, MA, USA) membrane. The membrane was blocked with 5% non-fat dry milk in TBS at room temperature for 1 h, then incubated in TBS supplemented with 0.1% Triton X-100 and 1% BSA and appropriate primary antibody overnight at 4 o C. Blots were incubated with secondary antibodies coupled with horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h at room temperature, and bands were visualized by SuperSignal enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA).

Fractionation.
Rats were injected with lentiviruses encoding either wild type GRK3-myc or RH-myc. The striatal tissue was isolated as fractioned as described previously 2,4 . Briefly, approximately 12 mg of fresh striatum tissue was homogenized in 10 volume of ice-cold HEPES-buffered sucrose (0.32 M sucrose, 4mM HEPES pH7.4, 1mM EGTA) containing protease inhibitor cocktail (Sigma-Aldrich, St.Louis. MO) in glass-teflon homogenizer. Homogenate (H) was centrifuged at 1000xg for 10 min at 4 o C. Supernatant (S1) was centrifuged at 10,000xg for 15 min to obtain crude synaptosomal fraction (P2) and supernatant (S2). The synaptosomal pellet was lysed by hypo-osmotic shock in 9 volume of ice cold HEPES-buffer with protease inhibitor cocktail for 30 min. The lysate was centrifuged at 25,000g for 20 min at 4 o C to obtain synaptosomal membrane fraction (LP1) and crude synaptic vesicle fraction (LS1). Supernatant (S2) was centrifuged at 165,000g for 2 hours to obtain cytosolic fraction (S3) and light membrane fraction (P3). Protein