Previous studies have demonstrated that β-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel β-arrestin1-binding protein, NSF ( N-ethylmaleimide-sensitivefusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant β-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. β-Arrestin1-NSF complex formation exhibits a conformational dependence with β-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the β-arrestin1-clathrin interaction, however, the phosphorylation state of β-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated β2-adrenergic receptor (β2-AR) internalization. Furthermore, when coexpressed with a β-arrestin1 mutant (βarr1S412D) that mimics a constitutively phosphorylated form of β-arrestin1 and that acts as a dominant negative with regards to β2-AR internalization, NSF rescues the βarr1S412D-mediated inhibition of β2-AR internalization. The demonstration of β-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization.
This work is supported in part by National Institutes of Health Grant HL16037 (to R. J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.