Abstract
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.
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