Protein Kinase A and G Protein-coupled Receptor Kinase Phosphorylation Mediates β-1 Adrenergic Receptor Endocytosis through Different Pathways

Abstract

Agonist-induced phosphorylation of ␤-adrenergic receptors (␤ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization؊ /GRK ؊ ␤ 1 AR was independent of ␤-arrestin recruitment. Importantly, clathrin inhibitors abolished agonistdependent internalization for both the WT␤ 1 AR and PKA ؊ ␤ 1 AR, whereas caveolae inhibitors prevented internalization only of the GRK ؊ ␤ 1 AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the ␤ 1 AR and 2) the pathway selected for ␤ 1 AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits. ␤-Adrenergic receptors (␤ARs)1 belong to the large family of G protein-coupled receptors (GPCRs) characterized by a typical structure of seven transmembrane domains (1, 2). Three types of ␤ARs, designated ␤ 1 , ␤ 2 , and ␤ 3 ARs, have been cloned from mammalian tissues (1, 3). Both ␤ 1 and ␤ 2 ARs contain phosphorylation sites located in the third intracellular loop and the C-terminal tail of the receptor, which serve as targets for cAMPdependent protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs) (2). Furthermore, site-specific mutagenesis studies of the human ␤ 2 AR suggest that low concentrations of agonist preferentially induce phosphorylation at PKA sites, whereas higher concentrations of agonist induce phosphorylation at both PKA and GRK sites (4).Continuous exposure of cells to a stimulus causes ␤ARs to undergo rapid phosphorylation in a process that dampens receptor signaling known as desensitization (4 -8). ␤ARs demonstrate two different mechanisms of desensitization. Agonistspecific or homologous desensitization of ␤ARs consists of a two-step process in which phosphorylation at the C terminus of the ␤AR is mediated by GRKs followed by binding to an arrestin protein, which sterically interrupts signaling to the G protein (5,8). Heterologous or non-agonist-specific desensitization is mediated by the second messenger-stimulated protein kinases A and C, which phosphorylate the receptor and effect a change in receptor conformation such that interaction with the G protein is impaired (5). An important consequence of agonistmediated receptor phosphorylation and desensitization by GRKs is the subsequent internalization of phosphorylated receptors into the cell (9). This process is mediated by ␤-arrestin, which binds to components of the clathrin-mediated endocytic machinery and targets the ligand-bound receptor to clathrincoated pits for endocytosis (10, 11). Interestingly, PKA phosphorylation, although an important mechanism for desensitization (4 -8), appears to play only a small role in ␤ 2 AR

Kv1.5 Association Modifies Kv1.3 Traffic and Membrane Localization

“…Different phosphorylation mechanisms are also mediated by distinct membrane surface locations. Although PKA-mediated phosphorylation is related to the caveolae pathway, G protein-coupled receptor kinases mediate phosphorylation mainly in clathrin-coated pits (47). However, although ligand-dependent phosphorylation and internalization of epidermal growth factor receptor is via clathrin-coated pits, some phosphorylation of epidermal growth factor receptor localizes in caveolar lipid rafts (48,49).…”

Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50)

“…After 48 h, the cells were split onto collagen-coated 25-mm coverslips. Arrestin translocation in response to PTH ligands was assessed at room temperature using real time live cell confocal microscopy as reported (29).…”

cAMP‐dependent protein kinase A and the dynamics of epithelial cell surface domains: Moving membranes to keep in shape

“…PKA may control endocytosis by associating 14 with compositionally and biophysically distinct plasma membrane domains. For instance, the PKA catalytic subunit interacts with caveolin-1, a key component of caveolar membranes domains (88) , and PKA-mediated phosphorylation triggers the agonist-induced internalization of G-protein coupled beta1-adrenergic receptors via a caveolar pathway in non-polarized cells (89) . PKA also controls protein recycling to the plasma membrane following endocytosis.…”