Fig 1: GRK2 is not required for ciliogenesis or IFT A–CPrimary cilia are normal in GRK2 -/- fibroblasts. Cells were serum starved for 24 or 48 h to induce cilia, and (A) immunostained for acetylated tubulin (AcTu, red), ARL13B (green) (upper panel; insets show the individual signals), or AcTu (red), pericentrin (red) and GLI3 (green) (lower panel). Control and GRK2 -/- cilia were positive for AcTu and ARL13B, and showed similar localization of GLI3 to the tips (lower panel, arrows). Acetylated tubulin and pericentrin staining were used to visualize the axoneme and centrioles, respectively. Scale bar, 2 µm. (B) Length distribution of cilia did not differ between control and GRK2 -/- cells. Red bars show medians; dots represent individual cilia. Mann–Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. (C) There was no difference in the efficiency of ciliogenesis between control and GRK2 -/- cells. Mean ± SEM. Welch's t-test; number of biological experiments and the total numbers of analyzed cells are indicated.DLoss of GRK2 did not affect localization of ciliary and IFT components. Immunofluorescence of serum-starved (48 hr) control and GRK2 -/- fibroblasts immunostained with AcTu or detyrosinated tubulin (DetyrTu) to mark the cilium (red), and IFT43, IFT88, KIF3A, TRAF3IP1, WDR34, or ICK (green). No significant differences in staining were found between control and GRK2 -/- cilia, suggesting normal IFT. Scale bars, 5 µm and 1 µm (insets).E–GInhibition of GRK2 activity did not affect primary cilia in chondrocytes. (E) Control human R92-284 and R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either 20 µM CMPD101 or 10 µM paroxetine, for 24 h before they were fixed and immunostained for cilia (ARL13B; gamma tubulin, ?Tu; acetylated tubulin, AcTu) and the retrograde IFT-B proteins IFT81 or IFT88. Scale bar, 1 µm. (F) Cilia length was measured using the ARL13B signal. Red bar, median. Mann–Whitney U-test; number of biological experiments and total cilia numbers are indicated. (G) The percentage of ciliated cells was calculated. Mean ± SEM. Welch's t-test; number of biological experiments and total cell numbers are indicated. Source data are available online for this figure.
Fig 2: IPoC‐induced desensitization of β‐ARs. In the short term, rapid and transient activation of the GRK2 could reduce the reperfusion injury and protect the myocardium against hypercontractions. In the long term, however, stabilization of GRK2 and its phosphorylated fraction leads to a desensitization or decoupling of β‐ARs with a resultant loss of the positive inotropic functional reserve. AR indicates adrenergic receptor; GRK2, G protein‐coupled receptor kinase 2; MDM2, mouse double minute 2 homolog/E3 ubiquitin‐protein ligase Mdm2; PI3, phosphoinositide 3‐kinase; PKC, protein kinase C.
Fig 3: Loss of Grk2 protein or its activity does not prevent ciliary SMO accumulation in the murine NIH3T3 cells ASerum‐starved Grk2 +/+ and Grk2 −/− NIH3T3 cells were treated with 500 nM SAG for 4 h; the cells were immunostained for ARL13B and SMO, and the intensity of ciliary SMO was analyzed and plotted. Note the normal ciliary SMO intensity in Grk2 −/− cells. Central band, median. Box, 1st–3rd quartile. Whiskers, 10–90% percentile. Mann–Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bars, 1 μm.BSerum‐starved Grk2 +/+ and Grk2 −/− NIH3T3 cells were treated with 500 nM SAG for 10–16 h, and immunoblotted for Gli3 and Gli1. The optical density of the bands was normalized to that of actin, and the Gli3‐FL/R ratios and Gli1 protein levels were plotted. Mean ± SEM. Mann–Whitney U‐test; number of biological experiments is indicated. Gli3FL and Gli3R, full‐length and repressor GLI3 variants, respectively.C–F Grk2 +/+ NIH3T3 cells were serum starved together with 25 μM CMPD101 (C, D) or 1 μM paroxetine (E, F) for 12 h, and treated with 500 nM SAG for 4 hours (C, E) or 12 h (D, F). (C, E) Inhibition of Grk2 activity by neither CMPD101 nor paroxetine prevents the SAG‐induced ciliary SMO accumulation. Central band, median. Box, 1st–3rd quartile. Whiskers, 10%–90% percentile. Mann–Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bars, 1 μm. (D, F) CMPD101 and paroxetine inhibit SAG‐mediated Gli3 processing and Gli1 upregulation. Mean ± SEM. Mann–Whitney U test; number of biological experiments is indicated.G, HMicromasses derived from Grk2 +/+ and Grk2 −/− NIH3T3 cells were serum starved, treated, and analyzed as in (C–F). (G) Note the inhibition of ciliary SMO intensity in Grk2 −/− micromasses. Central band, median. Box, 1st–3rd quartile. Whiskers, 10%–90% percentile. Mann–Whitney U‐test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bars, 1 μm. (H) Western blots for Gli3 processing and Gli1 upregulation. Mean ± SEM. Mann–Whitney U‐test; number of biological experiments is indicated. Vinc., vinculin.I, JNormal SMO cilia accumulation in murine IMCD3 cells with downregulated Grk2. (I) Doxycycline (DOX)‐inducible Grk2 downregulation, tested by Western blot, normalized to actin and plotted below. Mean ± SEM. Mann–Whitney U‐test; number of biological experiments is indicated. shScr, scramble shRNA. (J) Cells pre‐treated with DOX for 3 days were serum starved, SAG treated, stained, and analyzed as in (A), and the intensity of ciliary SMO was analyzed and plotted. Note the normal SMO accumulation in cilia in cells with Grk2 downregulation. Central band, median. Box, 1st–3rd quartile. Whiskers, 10%–90% percentile. Mann–Whitney – test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 μm.KUnder‐phosphorylation of SMO in Grk2 −/− NIH3T3 cells. Lysates of SMO‐transfected cells were resolved by the phospho(p)‐shift PAGE and immunoblotted for SMO. The portion of pSMO analyzed by densitometry is shown. GRK2 add‐back demonstrates a rescue of the pSMO in Grk2 −/− cells. Mean ± SEM. Welch's t‐test; number of biological replicates is indicated. Source data are available online for this figure.
Fig 4: Regulatory mechanisms for GRK2. A, Representative immunoblots and densitometric data of the (B) phosphorylated and (C) total MDM2 protein. D, Western blot image and (E) analysis of PI3 kinase expression. Immunoblot bands were normalized to GAPDH and expressed as a percentage of Sham. Data are means±SD of n=6 hearts. *P=0.05 vs Sham, # P=0.05 vs IR. GAPDH indicates Glyceraldehyde 3-phosphate dehydrogenase; GRK2, G protein–coupled receptor kinase 2; IPC, ischemic preconditioning; IPoC, ischemic postconditioning; IR, ischemia/reperfusion; MDM2, mouse double minute 2 homolog/E3 ubiquitin-protein ligase Mdm2; p-GRK2, phosphorylated GRK2; PI3K, phosphoinositide 3-kinase; p-MDM2, phosphorylated MDM2.
Fig 5: Inhibition of GRK2 activity inhibits canonical Wnt signaling AT-REx™-293 cells were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual-luciferase assay. Note the less Wnt3A-induced TOPflash trans-activation in cells treated with 20 µM paroxetine. R-spondin-1 (RSPO1) was used to block endogenous inhibition of canonical Wnt. Mean ± SEM. Mann–Whitney U-test; number of biological experiments is indicated.BT-REx™ 293 cells with GRK2 inhibited by 20 µM paroxetine show lower levels of LRP6 and lower pDVL2/DVL2 ratio, as determined by Western blot and quantified by densitometry. No significant Wnt3A-induced LRP6-T1572 phosphorylation was observed in T-REx™ 293 cells. Mean ± SEM. Welch's t-test; number of biological experiments is indicated.C–ERat chondrosarcoma (RCS) cells (C), and control R00-082 (D) and R92-284 (E) chondrocytes were treated with 20 µM paroxetine overnight and then treated with Wnt3A for 1 h. Note the lower levels of LRP6 and less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with paroxetine. #, nonspecific band. Mean ± SEM. Welch's t-test (C) and Mann–Whitney U test (D, E); number of biological experiments is indicated. Source data are available online for this figure.
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