Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates β-1 adrenergic receptor endocytosis through different pathways

Agonist-induced phosphorylation of β-adrenergic receptors (βARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of βARs, particularly the β₁AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type β₁AR (WTβ₁AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA^-β₁AR), the putative GRK phosphorylation sites (GRK^-β₁AR), and both sets of phosphorylation sites (PKA^-/ GRK^-β₁AR). Following agonist stimulation, both PKA^-β₁AR and GRK ^-β₁AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WTβ₁AR and PKA^-β₁AR, sequestration of the GRK^-β₁AR and PKA^-/GRK ^-β₁AR was independent of β-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WTβ₁AR and PKA^-β₁AR, whereas caveolae inhibitors prevented internalization only of the GRK ^-β₁AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the β₁AR and 2) the pathway selected for β₁AR 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.