β-Adrenergic Receptor Kinase (GRK2) Colocalizes with β-Adrenergic Receptors during Agonist-induced Receptor Internalization

Rapid regulation of G protein-coupled receptors appears to involve agonist-promoted receptor phosphorylation by G protein-coupled receptor kinases (GRKs). This is followed by binding of uncoupling proteins termed arrestins and transient receptor internalization. In this report we show that the β-adrenergic receptor kinase (βARK-1 or GRK2) follows a similar pattern of internalization upon agonist activation of β2-adrenergic receptors (β2AR) and that βARK expression levels modulate receptor sequestration. Stable cotransfected cells expressing an epitope-tagged β2AR and βARK-1 show an increased rate and extent of β2AR internalization compared with cells expressing receptor alone. Moreover, subcellular gradient fractionation studies suggest that βARK colocalizes with the internalized receptors. In fact, double immunofluorescence analysis using confocal microscopy shows extensive colocalization of β2AR and βARK in intracellular vesicles upon receptor stimulation. Our results confirm a functional relationship between receptor phosphorylation and sequestration and indicate that βARK does not only translocates from the cytoplasm to the plasma membrane in response to receptor occupancy, but shares endocytic mechanisms with the β2AR. These data suggest a direct role for βARK in the sequestration process and/or the involvement of receptor internalization in the intracellular trafficking of the kinase. Rapid regulation of G protein-coupled receptors appears to involve agonist-promoted receptor phosphorylation by G protein-coupled receptor kinases (GRKs). This is followed by binding of uncoupling proteins termed arrestins and transient receptor internalization. In this report we show that the β-adrenergic receptor kinase (βARK-1 or GRK2) follows a similar pattern of internalization upon agonist activation of β2-adrenergic receptors (β2AR) and that βARK expression levels modulate receptor sequestration. Stable cotransfected cells expressing an epitope-tagged β2AR and βARK-1 show an increased rate and extent of β2AR internalization compared with cells expressing receptor alone. Moreover, subcellular gradient fractionation studies suggest that βARK colocalizes with the internalized receptors. In fact, double immunofluorescence analysis using confocal microscopy shows extensive colocalization of β2AR and βARK in intracellular vesicles upon receptor stimulation. Our results confirm a functional relationship between receptor phosphorylation and sequestration and indicate that βARK does not only translocates from the cytoplasm to the plasma membrane in response to receptor occupancy, but shares endocytic mechanisms with the β2AR. These data suggest a direct role for βARK in the sequestration process and/or the involvement of receptor internalization in the intracellular trafficking of the kinase.