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

Agonist-induced phosphorylation of beta-adrenergic receptors (betaARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of betaARs, particularly the beta(1)AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta(1)AR (WTbeta(1)AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA- beta(1)AR), the putative GRK phosphorylation sites (GRK(-)beta(1)AR), and both sets of phosphorylation sites (PKA(-)/ GRK(-)beta(1)AR). Following agonist stimulation, both PKA(-)beta(1)AR and GRK(-)beta(1)AR 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 WTbeta(1)AR and PKA(-)beta(1)AR, sequestration of the GRK(-)beta(1)AR and PKA-/GRK(-)beta(1)AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WTbeta(1)AR and PKA(-)beta(1)AR, whereas caveolae inhibitors prevented internalization only of the GRK(-)beta(1)AR mutant. Taken together, these data demonstrate that: 1) PKA- mediated phosphorylation can trigger agonist-induced internalization of the beta(1)AR and 2) the pathway selected for beta(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.