The Journal of Neuroscience, September 6, 2006, ():
The Role of G-Protein-Coupled Receptor Kinase 5 in Pathogenesis of Sporadic Parkinson's Disease
J. Neurosci. Arawaka et al.
26: 9227
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental table 1
Association of sporadic Parkinson’s disease (sPD) with SNPs of G protein coupled-receptor kinase genes and casein kinase genes. SNPs with minor allele frequency of 0.1 or more in the Japanese population were selected from the vicinity of ADRBK1(GRK2), ADRBK2(GRK3), GRK6, CSNK1A1(CK1α1), CSNK2A1(CK2α1), CSNK2A2(CK2α2) and CSNK2B(CK2β) genes, and were genotyped with 287 sPD patients and 496 non-PD controls. SNPs not in Hardy-Weinberg equilibrium (p<0.05) in the control samples were excluded. The difference of allele frequencies between two groups were tested using Fisher's exact test. No SNPs showed significant association with sPD at a significance level of 0.01. Haplotype-based association tests with these seven genes were also performed using HTR program, but no significant association was detected at a significance level of 0.01 (data not shown).
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Supplemental figure 1
Characterization of the antibodies against CKs and GRKs employed for the immunohistochemical experiments. A, Immunoblotting of HEK293 cells lysates (30 μg protein) with primary antibodies for CK1 family. Anti-CK1α, CK1ε, and CK1γ2 antibodies clearly stained each of overexpressed proteins and weakly stained its corresponding endogenous one. Anti-CK1δ and CK1γ1 antibodies stained the bands migrating to their putative molecular weights, respectively. B, Immunoblotting of HEK293 cell lysates overexpressing CK2α1 or CK2β cDNA with anti-CK2 antibodies. C, left panels: Immunoblotting of the lysate of HEK293 cells overexpressing GRK1, 2, 5 or 6 cDNA with the antibody against each of the four GRKs. Anti-GRK1, 2, 5 and 6 antibodies specifically detected overexpressed proteins and had no cross-reactivity with the other GRKs. Anti-GRK1 and 2 antibodies barely detected endogenous corresponding proteins in the HEK293 cells. Right panels: Anti-GRK5 antibody recognized a band corresponding to GRK5, which was rich in the membrane fraction of the homogenate of human mid-brain.
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Supplemental figure 2
The schematic drawings of the DNA constructs for the luciferase assays (Figs. 6, 7).
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Supplemental figure 3
GRK5 promotes α-linolenic acid-induced αS oligomerization in a dose-dependent manner of OA. HEK293-αS cells expressing GRK5-FLAG or CAT were incubated with different concentrations of OA for 16 hrs, and the conditioned with α-linolenic acid for 6 hrs along with OA. OA concentrations were 0, 5, 10, 20 nM. High-speed soluble cytosol fractions (30 μg protein) from cell lysates were heat-treated before gel loading, and immunoblotted with Syn-1 (left upper panels) or psyn#64 (right panels). The levels of dimers and oligomers of phosphorylated αS as well as total αS containing non-phosphorylated and phosphorylated species increased with OA concentrations. The αS oligomerization was enhanced by GRK5 co-expression. For loading control, the same amount of non-heated samples was immunoblotted with anti-actin and anti- SOD1 antibodies (left lower panels).
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Supplemental figure 4
GRK5 protein levels in the frontal cortices of the human brains.
Left: sPD vs. controls, Middle: T/T vs. C/T vs. C/C (m24), Right: A/A vs. G/A vs. G/G (m22.1)
Methods. Frontal cortex tissue of the brain was suspended in 1:5 (wt/vol) homogenization buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM EDTA, 1x protease inhibitor cocktail) and disrupted by a Teflon Potter homogenizer. The homogenate was centrifuged at 800xg for 15 min, and the supernatant was centrifuged at 100,000xg for 30 min. The resultant supernatant was denatured by boiling for 5 min in Laemmli’s sample buffer containing 2-mercaptoethanol, and subjected to 12.5% polyacrylamide gels followed by immunoblotting using anti-GRK5 or anti-actin antibodies. The intensity of the band corresponding to GRK5 was measured by densitometry, and normalized to the ratio of β-actin expression levels for quantitative analysis. Statistical analysis was performed by Student’s t test.
