Identification of NSF as a beta-arrestin1-binding protein - Implications for beta(2)-adrenergic receptor regulation

Previous studies have demonstrated that beta-arrestin1 serves to target G p rotein-coupled receptors for internalization via clathrin-coated pits and t hat its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel bet a-arrestin1-binding protein, NSF N-ethylmaleimide-sensitive fusion protein) , an ATPase essential for many intracellular transport reactions. We demons trate that purified recombinant beta-arrestin1 and NSF interact in vitro an d that these proteins can be coimmunoprecipitated from cells. beta-Arrestin 1-NSF complex formation exhibits a conformational dependence with beta-arre stin1 preferentially interacting with the ATP bound form of NSF, In contras t to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding, Functionally, overexpr ession of NSF in HEK 293 cells significantly enhances agonist-mediated beta (2)-adrenergic receptor (beta(2)-AR) internalization. Furthermore, when coe xpressed with a beta-arrestin1 mutant (beta arr1S412D) that mimics a consti tutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta(2)-AR internalization, NSF rescues the beta a rr1S412D-mediated inhibition of beta(2)-AR internalization. The demonstrati on of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in faci litating clathrin coat-mediated G protein-coupled receptor internalization.