Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors.
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.
Söhlemann
P
P
Laboratory of Molecular Biology, University of Munich, Max-Planck-Institute of Biochemistry, Martinsried, Germany.
Hekman
M
M
Puzicha
M
M
Buchen
C
C
Lohse
M J
MJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000941:Antigens / Q000235:genetics / Q000378:metabolism*; D019390:Arrestins; D002417:Cattle; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D018411:Spodoptera; D051552:beta-Adrenergic Receptor Kinases; D000071557:beta-Arrestins
D000941:Antigens; D019390:Arrestins; D005136:Eye Proteins; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D000071557:beta-Arrestins; D012243:Rhodopsin; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases