G protein-coupled receptor kinases phosphorylate activated G protein-coupled receptors, which promotes the binding of an arrestin protein to the receptor. Arrestin binding to phosphorylated, active receptor prevents receptor stimulation of heterotrimeric G protein transducer proteins, blocking their cellular signaling and resulting in receptor desensitization. Arrestin binding also directs receptors to specific cellular internalization pathways, removing the receptors from the cell surface and also preventing additional activation. Arrestin binding to phosphorylated, active receptor also enables receptor signaling through arrestin partner proteins. Thus the GRK/arrestin system serves as a complex signaling switch for G protein-coupled receptors.
Background References
1. Del Rincon S.V. et. al. Development and validation of a method for profiling post-translational modification activities using protein microarrays. PLoS ONE 5:E11332-E11332(2010).
ICC staining of GRK3 in MG-63 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (HA500317, 1/200) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ICC staining of GRK3 in SiHa cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (HA500317, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
Flow cytometric analysis of GRK3 was done on SH-SY5Y cells. The cells were fixed, permeabilized and stained with the primary antibody (HA500317, 1ug/ml) (red) compared with Rabbit IgG, monoclonal - Isotype Control (green). After incubation of the primary antibody at +4℃ for 1 hour, the cells were stained with a Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃ (dark incubation).Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Western blot analysis of GRK3 on different lysates with Rabbit anti-GRK3 antibody (HA500317) at 1/5,000 dilution.
Lane 1: Jurkat cell lysate(20 µg/Lane) Lane 2: Mouse lung tissue lysate(40 µg/Lane) Lane 3: Rat lung tissue lysate(40 µg/Lane)
Exposure time: 10 seconds; ECL: K1801
Blocking: 5% NFDM/TBST, 1 hour at room temperature Primary antibody: HA500317, 1/5,000 in primary antibody dilution buffer (K1803), overnight at 4 ℃ Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature
Predicted band size: 80 kDa Observed band size: 75 kDa
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