Clathrin Box in G Protein-coupled Receptor Kinase 2

β₁-Adrenergic receptor (β₁AR) shows the resistance to agonist-induced internalization. However, β₁AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the β₁AR and GRK2 fusion protein (β₁AR/GRK2) is dependent on dynamin but independent of β-arrestin and phosphorylation. The β₁AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of β₁AR and various portions of GRK2 reveal that the residues 498-502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498-502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as β-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.