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.
McDonald
P H
PH
Howard Hughes Medical Institute and the Departments of Medicine (Cardiology) and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Cote
N L
NL
Lin
F T
FT
Premont
R T
RT
Pitcher
J A
JA
Lefkowitz
R J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis; D008969:Molecular Sequence Data; D052066:N-Ethylmaleimide-Sensitive Proteins; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D033921:Vesicular Transport Proteins; D000071557:beta-Arrestins
D019390:Arrestins; D002352:Carrier Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D000071557:beta-Arrestins; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat