Fig 3. Mutation of β₂AR S^355/356 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 S^355/356), but not PKA2A β₂AR (mutation at S^345/346), 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 Ser^355/356 (GRK sites) was not observed in GRK2A (mutation of β-AR at Ser^355/356), but was present in PKA2A (mutation of β-AR at Ser^345/346). (D) In contrast, Phospho-β₂AR Ser^345/346 was not observed in PKA2A (mutation of β-AR at Ser^345/346), but was present in GRK2A (mutation of β-AR at Ser^355/356).