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Identification of NSF as a beta-arrestin1-binding protein. Implications for beta2-adrenergic receptor regulation.

Previous studies have demonstrated that beta-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 beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization. Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization. The demonstration of beta-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.

Pubmed ID: 10196135

Authors

  • McDonald PH
  • Cote NL
  • Lin FT
  • Premont RT
  • Pitcher JA
  • Lefkowitz RJ

Journal

The Journal of biological chemistry

Publication Data

April 16, 1999

Associated Grants

  • Agency: NHLBI NIH HHS, Id: HL16037

Mesh Terms

  • Adenosine Triphosphate
  • Amino Acid Sequence
  • Animals
  • Arrestins
  • COS Cells
  • Carrier Proteins
  • Endocytosis
  • Molecular Sequence Data
  • N-Ethylmaleimide-Sensitive Proteins
  • Protein Binding
  • Rats
  • Recombinant Proteins
  • Vesicular Transport Proteins