BINDING OF PURIFIED RECOMBINANT BETA-ARRESTIN TO GUANINE-NUCLEOTIDE-BINDING-PROTEIN-COUPLED RECEPTORS

beta-arrestin is a cytosolic protein thought to be responsible for unc oupling agonist-activated beta(2)-adrenergic receptors from their guan ine-nucleotide-binding proteins (G-protein) subsequent to receptor pho sphorylation by the beta-adrenergic receptor kinase (beta ARK). In ord er to investigate this intel action, we generated a recombinant baculo virus for the expression of beta-arrestin in Sf9 insect cells. Apparen tly homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rh odopsin in a phosphorylation-dependent plus light-dependent manner. Bi nding 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 my c tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta AR K-phosphorylated beta(2)-adrenergic receptors was found to occur with a K-D of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta-a rrestin also bound to receptors which had been completely dephosphoryl ated with acid phosphatase, but the affinity was approximate to 30-fol d lower. In contrast to regulation by phosphorylation, binding of agon ists or antagonists to the receptors had negligible effects on beta-ar restin binding. Finally, beta-arrestin and beta ARK were shown to be c apable of producing synergistic inhibition of beta(2)-adrenergic-recep tor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to be ta(2)-adrenergic receptors occurs in a beta ARK-dependent manner and i s sufficient to impair adenylyl cyclase stimulation by the receptors.