??2AR (??2 adrenoceptor) is a prototypical G-protein coupled receptor (GPCR) that plays an important role in cardiovascular and pulmonary physiology through activation of the classic Gs-adenylate cyclase-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. In the mammalian heart, increasing cAMP-PKA activity leads to phosphorylation of an array of proteins involved in increasing heart contractility and rate. ??2AR also has a cardiac protective role through utilizing multiple mechanisms to reduce receptor signaling. This includes ??2AR desensitization, ??2AR coupling to G??i, and ??2AR degradation, all of which are tightly regulated by post-translational modifications of the C-terminal region of ??2AR. Over past decades, these modifications have been extensively characterized biochemically in fibroblasts, such as phosphorylation by PKA (serines 261, 262, 345 and 346) and G protein coupled receptor kinases (GRKs) (serines 355, 356 and 364), ubiquitination (lysines 348, 372 and 375) and palmitoylation (cysteine 341). However, the physiological role of these modifications on ??2AR signaling regulation in the heart remains unclear.
This study provides new insight into the role of three post-translational modifications on ??2AR signaling regulation in cardiac myocytes. We find that palmitoylation, the fatty acid modification of ??2AR at cysteine 341 is not required for receptor targeting to the plasma membrane caveolae. Instead, both palmitoylation and GRK phosphorylation are required to mediate the association of ??2AR with ??-arrestin 2/ phosphodiesterase 4D complexes to regulate cAMP signaling. In addition, we provide a new mechanism explaining ??2AR coupling from Gas to Gai, which is agonist dose dependent and controlled by both PKA and GRK phosphorylation of the receptor. Moreover, we demonstrate that mutation of either PKA or GRK phosphorylation sites on ??2AR leads to rapid receptor degradation than that of wild type ??2AR. Interestingly, our data also suggest that degradation of ??2AR is coordinated by both lysosomes and proteasomes: the extracellular domains are degraded by lysosomes and the intracellular domains are degraded by proteasomes. Together, all three post-translational modifications coordinate to regulate ??2AR signaling in cardiac tissue under physiological conditions
