Palmitoylation Regulates Intracellular Trafficking of β2 Adrenergic Receptor/Arrestin/Phosphodiesterase 4D Complexes in Cardiomyocytes

β2 adrenergic receptor (β2AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β2AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β2AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β2AR association with arrestin/PDE4D. Replacing cysteine 341 on β2AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β2AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β2AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β2AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β2AR.β2肾上腺素能受体（β2 adrenergic receptor, β2AR）是一类经典的G蛋白偶联受体，可激活经典的环腺苷酸-蛋白激酶A（cAMP-protein kinase A, PKA）信号通路。近期研究表明，cAMP-PKA的活性受到时空调控，部分原因在于β2AR与环腺苷酸降解酶家族成员磷酸二酯酶4D（phosphodiesterase 4D, PDE4D）的动态结合。本研究证实，在心肌细胞中，β2AR的棕榈酰化修饰——即其半胱氨酸残基341位点的共价酰化修饰——通过调控β2AR与抑制蛋白/PDE4D的结合，对心肌细胞内的亚细胞水平cAMP-PKA活性塑造发挥关键作用。将β2AR的341位半胱氨酸替换为丙氨酸（C341A）后，其与β抑制蛋白2的结合能力受损。令人意外的是，在激动剂刺激达到饱和浓度时，C341A突变体仍可通过不依赖抑制蛋白的途径发生内化；该内化过程依赖于小窝蛋白，且需要发动蛋白GTP酶的参与。然而，与β抑制蛋白2的结合受损同时也导致PDE4D向C341A突变体的招募受阻。因此，C341A突变型β2AR无法招募PDE4D，只能单独从细胞膜转运至内体。这一改变使得在β2AR受到刺激时，用于激活PKA的胞质cAMP信号增强。功能层面，C341位点突变或β2AR棕榈酰化修饰抑制，均可增强受体诱导的胞质PKA活性，并提高心肌细胞的收缩速率。本研究数据揭示了棕榈酰化修饰的全新功能：通过调控β抑制蛋白2-PDE4D复合物向激动剂激活的β2AR的招募，塑造心肌细胞内的亚细胞水平cAMP-PKA信号通路。