Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors

Eur J Biochem. 1995 Sep 1;232(2):464-72.


beta-arrestin is a cytosolic protein thought to be responsible for uncoupling agonist-activated beta 2-adrenergic receptors from their guanine-nucleotide-binding proteins (G-protein) subsequent to receptor phosphorylation by the beta-adrenergic receptor kinase (beta ARK). In order to investigate this interaction, we generated a recombinant baculovirus for the expression of beta-arrestin in Sf9 insect cells. Apparently homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rhodopsin in a phosphorylation-dependent plus light-dependent manner. Binding to beta 2-adrenergic receptors was investigated using purified receptors reconstituted into lipid vesicles. The accessibility of the reconstituted receptors was determined using the agonist isoproterenol for the ligand-binding site and an antibody binding to an attached myc tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta ARK-phosphorylated beta 2-adrenergic receptors was found to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta-arrestin also bound to receptors which had been completely dephosphorylated with acid phosphatase, but the affinity was approximately 30-fold lower. In contrast to regulation by phosphorylation, binding of agonists or antagonists to the receptors had negligible effects on beta-arrestin binding. Finally, beta-arrestin and beta ARK were shown to be capable of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic receptors occurs in a beta ARK-dependent manner and is sufficient to impair adenylyl cyclase stimulation by the receptors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Animals
  • Antigens / genetics
  • Antigens / metabolism*
  • Arrestins*
  • Cattle
  • Cell Line
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Eye Proteins / genetics
  • Eye Proteins / metabolism*
  • GTP-Binding Proteins / metabolism*
  • Gene Expression
  • Humans
  • Kinetics
  • Phosphorylation
  • Protein Binding
  • Receptors, Adrenergic, beta-2 / chemistry
  • Receptors, Adrenergic, beta-2 / genetics
  • Receptors, Adrenergic, beta-2 / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Rhodopsin / metabolism
  • Rod Cell Outer Segment / metabolism
  • Spodoptera
  • beta-Adrenergic Receptor Kinases
  • beta-Arrestins


  • Antigens
  • Arrestins
  • Eye Proteins
  • Receptors, Adrenergic, beta-2
  • Recombinant Proteins
  • beta-Arrestins
  • Rhodopsin
  • Cyclic AMP-Dependent Protein Kinases
  • beta-Adrenergic Receptor Kinases
  • GTP-Binding Proteins
  • Adenylyl Cyclases