Fig 1.
Cardiomyocyte contraction response to epinephrine or norepinephrine under different stimulation conditions.
Cardiac myocytes isolated from β1AR knockout mice that express endogenous β2AR were stimulated with 10 μM epinephrine (Epi) or norepinephrine (NE) for 10 min and then re-challenged with (B, D) or without (A, C) Epi or NE for 60 min. The Epi- (A, B) or NE- (F, H) induced contraction rates were recorded. Data are shown as means ± SDs from three independent experiments.
Fig 2.
Epinephrine-induced internalization and recycling of β2ARs under different stimulation conditions in cardiomyocytes.
Neonatal cardiomyocytes isolated from β1β2AR double-knockout mice were transfected with FLAG-tagged wild-type β2AR at a multiplicity of infection of 100 for 24 h. After serum starvation for 2 h, the cardiac myocytes were stimulated with 10 μM epinephrine (Epi). Epi was then removed (A) or retained (B) for 60 min followed by Epi re-stimulation for another 10 min. Cardiomyocytes were fixed and stained with anti-FLAG and Alexa 488-conjugated goat anti-mouse antibodies. (A) Punctate intracellular staining of FLAG-tagged β2ARs was observed after 10 min of Epi stimulation. The Epi-activated FLAG-β2AR efficiently recycled back to the cell surface after removal of the drug. Epi-activated FLAG-β2AR was rapidly internalized after secondary stimulation. (B) When Epi was present continually, the receptor was not recycled but remained internalized. Photographs representative of 3 different preparations of cardiomyocytes are shown. The cell surface receptors were quantified by a fluorescence-linked immunosorbent assay (FLISA) with Epi treatment for 10 min and then Epi was removed (C) or retained (D) for 60 min followed by Epi re-stimulation for another 10 min.
Fig 3.
Mutation of β2AR S355/356 sites impaired internalization of β2ARs.
Wild-type and two mutant forms of β2AR (GRK2A and PKA2A) were expressed in neonatal cardiomyocytes isolated from β1β2AR double-knockout mice by adenovirus transfection for 2 days. (A) The cells were stimulated with Epi (10 μM) for 10, 30, or 60 min. Compared to wild-type, GRK2A β2AR (mutation at S355/356), but not PKA2A β2AR (mutation at S345/346), impaired receptor internalization. Photographs representative of 3 different preparations of cardiomyocytes are shown. (B) The cell surface receptors of wild-type or mutant β2AR were quantified by FLISA upon Epi stimulation for the indicated times. The quantitative data represent the means ± SDs of at least 3 different experiments. (C) Cardiomyocytes from β1β2AR double-knockout mice were transfected with GRK2A and PKA2A mutant β2ARs for two days and then lysed with radioimmunoprecipitation assay buffer. The lysates were subjected to SDS-PAGE followed by immunoblot analysis using a polyclonal anti-phosphoserine (355, 356)-specific or anti-phosphoserine (345, 346)-specific β2AR antibody. Phospho-β2AR Ser355/356 (GRK sites) was not observed in GRK2A (mutation of β-AR at Ser355/356), but was present in PKA2A (mutation of β-AR at Ser345/346). (D) In contrast, Phospho-β2AR Ser345/346 was not observed in PKA2A (mutation of β-AR at Ser345/346), but was present in GRK2A (mutation of β-AR at Ser355/356).
Fig 4.
Epinephrine-induced β2AR phosphorylation at S355/356 (GRK) but not at S345/346 (PKA) is related to receptor internalization in cardiac myocytes.
(A) Neonatal cardiomyocytes from β1β2AR double-knockout mice were transfected with the β2AR adenovirus for 2 days and then stimulated with epinephrine (Epi; 10 μM) for 2, 5, 15, 30, or 60 min. Cell lysates were subjected to SDS-PAGE followed by immunoblot analysis using polyclonal anti-phosphoserine-(355, 356) or -(345, 346)-specific β2AR antibodies. The blots were stripped and probed with an anti-C-terminal β2AR antibody to visualize total β2ARs. (B) and (C) Relative quantitative analysis of the levels of phosphor-β2AR was performed by normalizing the phosphor-β2AR signal against the total β2AR. (D) Punctate intracellular staining of FLAG-β2ARs was observed at all time points of Epi stimulation, indicating that Epi induced receptor internalization.
Fig 5.
Blocking of phosphorylation of the β2AR S355/356 sites inhibits the contraction response and impairs internalization of β2AR.
Cardiomyocytes from β1β2AR double-knockout mice were transfected with the β2AR adenovirus for 2 days and then stimulated with epinephrine (Epi) (10 μM) for 10 min with or without okadaic acid (OA) pretreatment. Epi was then removed for 60 min. (A) Phosphorylation of the β2ARs at Ser355,356 and (B) internalization of the β2ARs were examined. (C) Cardiac myocytes isolated from β1AR knockout mice were used for the contraction rate assay. Cells were stimulated with Epi with or without OA pre-treatment. The contraction response curves represent the means ± SDs of n beating dishes from at least 3 different neonatal cardiomyocyte preparations. *p < 0.05 vs. Epi-treated group (two-way ANOVA). **p < 0.05 vs. group without OA treatment (Student’s t-test).
Fig 6.
Epinephrine induces interactions between β2ARs and β-arrestin 2.
Cardiac myocytes from β1β2AR double-knockout mice were transfected to express FLAP-β2AR and β-arrestin-GFP for 2 days, and then stimulated with epinephrine (Epi) for 5, 10, 30, or 60 min. (A) Cell lysates were immunoprecipitated with an anti-Flag-m2 antibody. Immunoblot analyses were performed using anti-GFP or β-arrestin antibodies. (B) Cells were fixed for immunofluorescence staining. Intracellular localization of Flag-mβ2AR and β-arrestin-GFP was observed at the indicated times of Epi stimulation. The results presented are representative of at least 3 repeated experiments. Arrows show co-localization of internalized β2ARs and β-arrestin.