Journal: Journal of Biological Chemistry

Article Title: G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

doi: 10.1074/jbc.m109.003665

Figure Lengend Snippet: FIGURE 1. Distribution of endogenous D3R and GRK4 in membrane microdomains of hPTCs. Lipid and non-lipid membrane fractions of hPTCs were prepared by sucrose gradient centrifugation to determine the basal membrane distribution of both D3R and GRK4. Twelve fractions were obtained (fractions 1–6 correspond to lipid rafts and fractions 7–12 to non- lipid rafts) and immunoblotted for D3R, GRK4, and caveolin-1, a commonly used marker of lipid rafts. Cells pretreated with MCD, a cholesterol-depleting and lipid membrane-disrupting agent, were used as control. n 3 independ- ent experiments.

Article Snippet: The cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 for 10 min, double-immunostained for D3R and GRK4 using monoclonal anti-D3R antibody (Zymed Laboratories Inc.) and rabbit polyclonal anti-GRK4 antibody (Santa Cruz Biotechnol- 21426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 32 • AUGUST 7, 2009 ogy) for 1 h, and re-probed with goat anti-mouse (H L)-Alexa 555 and goat anti-rabbit (H L)-Alexa 633 (Molecular Probes) secondary antibodies.

Techniques: Membrane, Gradient Centrifugation, Marker, Control

Journal: Journal of Biological Chemistry

Article Title: G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

doi: 10.1074/jbc.m109.003665

Figure Lengend Snippet: FIGURE 2. Co-localization of D3R and GRK4 in hPTCs and kidney sections from Wistar Kyoto rats. A, hPTCs grown on poly-D-lysine-coated coverslips were serum-starved for 1 h and treated with the D3R agonist PD128907 (1 M) at the indicated duration of treatment. The cell membrane was labeled with a membrane- impermeant biotin, after which the cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100 in PBS. The cells were double-immunostained for D3R (pseudocolored red) and GRK4 (pseudocol- ored green). The membrane (pseudocolored blue) was probed with Cy3-conjugated avidin. The distribution and co-localization of D3R and GRK4 (shown as discrete yellow areas in merge and inset images) and of D3R or GRK4 and the cell membrane (magenta and cyan in inset images, respectively) were evaluated by laser scan- ning confocal microscopy. B, formalin-fixed, paraffin-embedded kidney sections of WKY rats were prepared to determine in vivo co-localization of D3R (pseudocolored red) and GRK4 (pseudocolored green) by confocal microscopy. Differential interference contrast (DIC) images were also obtained to show the cellular and histo- logical boundaries. G indicates glomerulus; yellow arrow indicates proximal tubule (S1 segment); red arrow indicates proximal tubule (S3 segment), and white arrow indicates distal tubule. Scale bar, 10 m, 600 mag- nification, n 3–5 independent experiments.

Article Snippet: The cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 for 10 min, double-immunostained for D3R and GRK4 using monoclonal anti-D3R antibody (Zymed Laboratories Inc.) and rabbit polyclonal anti-GRK4 antibody (Santa Cruz Biotechnol- 21426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 32 • AUGUST 7, 2009 ogy) for 1 h, and re-probed with goat anti-mouse (H L)-Alexa 555 and goat anti-rabbit (H L)-Alexa 633 (Molecular Probes) secondary antibodies.

Techniques: Membrane, Labeling, Avidin-Biotin Assay, Confocal Microscopy, Formalin-fixed Paraffin-Embedded, In Vivo

Journal: Journal of Biological Chemistry

Article Title: G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

doi: 10.1074/jbc.m109.003665

Figure Lengend Snippet: FIGURE 3. Interaction of D3R and GRK4. A, capacity for D3R and GRK4 to physically interact was determined in hPTCs treated with the D3R agonist PD128907 (1 M) at the indicated duration of treatment. Total cell lysates were prepared, and a monoclonal anti-D3R Ab, or normal mouse IgG as neg- ative control, or polyclonal anti-GRK4 Ab as positive control was used as immunoprecipitant. The immune complexes were precipitated using agar- ose-A/G beads and immunoblotted for GRK4. A 55-kDa band correspond- ing to GRK4 was visualized. B, hPTCs were double-transfected with D3R and GRK4-tagged with the C and N termini of the fluorescent protein EYFP, respectively. The cells were grown for 48 h post-transfection, serum-starved for2h,stimulatedwiththeD3RagonistPD128907(1M)attheindicatedtime points, and then prepared for confocal microscopy. The cell membrane (CM) was biotinylated with a membrane-impermeant biotin to allow visualization (pseudocolored red) and co-localization with the BiFC signal (pseudocolored green). Co-localization of the BiFC signal with the CM is indicated by yellow punctate areas in the merged images. An overlay of the BiFC signal and the nucleus (pseudocolored blue) is shown to indicate intracellular distribution. Untransfected cells treated with the D3R agonist for 5 min were used as neg- ative control (control). Scale bar, 10 m, 600 magnification, n 3–4 inde- pendent experiments.

Article Snippet: The cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 for 10 min, double-immunostained for D3R and GRK4 using monoclonal anti-D3R antibody (Zymed Laboratories Inc.) and rabbit polyclonal anti-GRK4 antibody (Santa Cruz Biotechnol- 21426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 32 • AUGUST 7, 2009 ogy) for 1 h, and re-probed with goat anti-mouse (H L)-Alexa 555 and goat anti-rabbit (H L)-Alexa 633 (Molecular Probes) secondary antibodies.

Techniques: Control, Positive Control, Transfection, Confocal Microscopy, Membrane

Journal: Journal of Biological Chemistry

Article Title: G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

doi: 10.1074/jbc.m109.003665

Figure Lengend Snippet: FIGURE 4. GRK4-mediated phosphorylation of D3R. T-REx CHO cells stably transfected with the tetracycline-inducible GRK4 splice variants, or empty vector as control, were transfected with His-tagged D3R. The expression of the transgenes was induced by the addition of doxycycline, a tetracycline analog, 24 h prior to D3R activation. The transfected cells were metabolically labeled with [32P]H3PO4 before stimulation with the D3R agonist PD128907 (1 M). The heterologously expressed D3R was pulled down from uniform amounts of protein (500 g, confirmed by immunoblotting for glyceralde- hyde-3-phosphate dehydrogenase (GAPDH)) using a His pulldown kit and resolved in 10% SDS-PAGE. Thereafter, the gel was subjected to autoradiog- raphy for 24–48 h. Blots showing the amounts of phosphorylated D3R (phos- pho-D3R) and total His-tagged D3R are shown in the upper panel. Band densi- ties were quantified via Scion densitometry software. Data are expressed as mean S.E. ✦, p 0.05, versus untreated cells transfected with the same GRK4 isoform, t test. *, p 0.05, versus untreated empty vector transfected control, one-way ANOVA followed by Holm-Sidak post hoc test. n 3 inde- pendent experiments.

Article Snippet: The cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 for 10 min, double-immunostained for D3R and GRK4 using monoclonal anti-D3R antibody (Zymed Laboratories Inc.) and rabbit polyclonal anti-GRK4 antibody (Santa Cruz Biotechnol- 21426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 32 • AUGUST 7, 2009 ogy) for 1 h, and re-probed with goat anti-mouse (H L)-Alexa 555 and goat anti-rabbit (H L)-Alexa 633 (Molecular Probes) secondary antibodies.

Techniques: Phospho-proteomics, Stable Transfection, Transfection, Plasmid Preparation, Control, Expressing, Activation Assay, Metabolic Labelling, Labeling, Western Blot, SDS Page, Software

Journal: Journal of Biological Chemistry

Article Title: G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

doi: 10.1074/jbc.m109.003665

Figure Lengend Snippet: FIGURE 5. D3R-mediated mitogenesis. A, hPTCs were grown in 24-well plates and incubated with BrdUrd, a thymidine analog, before treatment with the D3R agonist PD128907 (PD, 1 M) and/or the D3R antagonist GR103691 (GR,1M)orvehicleascontrol(C).BandC,functionorexpressionofGRK4was inhibited by transfecting the hPTCs with heparin (Hep) or GRK4-specific silencing siRNA (siRNA), respectively, prior to BrdUrd incubation and treat- ment with the D3R agonist PD128907 or vehicle (V) as control. Cells trans- fected with vehicle (control) or nonsilencing siRNA (mock) served as controls. Thereafter, the cells were fixed and the extent of cell proliferation was deter- mined by the amount of BrdUrd that was incorporated using an anti-BrdUrd Ab conjugated with europium. Europium fluorescence was determined by time-resolved fluorometry using a Victor3 multilabel reader and normalized for that of the nuclear stain DAPI. Data are expressed as mean S.E. ✦, p 0.05, versus vehicle-treated pair, t test. *, p 0.05, versus vehicle-treated (untransfected) control, one-way ANOVA, and Holm-Sidak post hoc test, n 3 independent experiments performed in duplicates.

Article Snippet: The cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 for 10 min, double-immunostained for D3R and GRK4 using monoclonal anti-D3R antibody (Zymed Laboratories Inc.) and rabbit polyclonal anti-GRK4 antibody (Santa Cruz Biotechnol- 21426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 32 • AUGUST 7, 2009 ogy) for 1 h, and re-probed with goat anti-mouse (H L)-Alexa 555 and goat anti-rabbit (H L)-Alexa 633 (Molecular Probes) secondary antibodies.

Techniques: Incubation, Control, Fluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

doi: 10.1074/jbc.m109.003665

Figure Lengend Snippet: FIGURE 6. GRK4-dependent MAP kinase phosphorylation. hPTCs trans- fected with GRK4-specific siRNA or nonsilencing siRNA (mock) were treated with the D3R agonist PD128907 (1 M, 1 h), and the total cell lysates were immunoblotted using a mixture of Abs that detects the phospho-p44/42 (Erk1/2) MAP kinases, phospho-p90RSK (a downstream target of p44/42), phospho-Akt (which is involved in cell survival and apoptosis), and phos- pho-S6 ribosomal protein (which is phosphorylated by p70 S6 kinase in response to growth factors and mitogens). elF4E detects the total target pro- tein and is used for normalization. Nontransfected (control) cells served as an additional negative control. Nonspecific bands are seen above phospho-S6 and phospho-p44/42 and below phospho-Akt. Band densities were quanti- fied by Scion densitometry software. Data are expressed as mean S.E. *, p 0.05, versus others, one-way ANOVA, and Holm-Sidak post hoc test, n 3 independent experiments.

Article Snippet: The cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 for 10 min, double-immunostained for D3R and GRK4 using monoclonal anti-D3R antibody (Zymed Laboratories Inc.) and rabbit polyclonal anti-GRK4 antibody (Santa Cruz Biotechnol- 21426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 32 • AUGUST 7, 2009 ogy) for 1 h, and re-probed with goat anti-mouse (H L)-Alexa 555 and goat anti-rabbit (H L)-Alexa 633 (Molecular Probes) secondary antibodies.

Techniques: Phospho-proteomics, Control, Negative Control, Software