Abstract
Homologous desensitization of beta-adrenergic receptors, as well as
adaptation of rhodopsin, are thought to be triggered by specific
phosphorylation of the receptor proteins. However, phosphorylation
alone seems insufficient to inhibit receptor function, and it has
been proposed that the inhibition is mediated, following receptor
phosphorylation, by the additional proteins beta-arrestin in the
case of beta-adrenergic receptors and arrestin in the case of rhodopsin.
In order to test this hypothesis with isolated proteins, beta-arrestin
and arrestin were produced by transient overexpression of their cDNAs
in COS7 cells and purified to apparent homogeneity. Their functional
effects were assessed in reconstituted receptor/G protein systems
using either beta 2-adrenergic receptors with Gs or rhodopsin with
Gt. Prior to the assays, beta 2-receptors and rhodopsin were phosphorylated
by their specific kinases beta-adrenergic receptor kinase (beta ARK)
and rhodopsin kinase, respectively. beta-Arrestin was a potent inhibitor
of the function of beta ARK-phosphorylated beta 2-receptors. Half-maximal
inhibition occurred at a beta-arrestin:beta 2-receptor stoichiometry
of about 1:1. More than 100-fold higher concentrations of arrestin
were required to inhibit beta 2-receptor function. Conversely, arrestin
caused half-maximal inhibition of the function of rhodopsin kinase-phosphorylated
rhodopsin when present in concentrations about equal to those of
rhodopsin, whereas beta-arrestin at 100-fold higher concentrations
had little inhibitory effect. The potency of beta-arrestin in inhibiting
beta 2-receptor function was increased over 10-fold following phosphorylation
of the receptors by beta ARK, but was not affected by receptor phosphorylation
using protein kinase A. This suggests that beta-arrestin plays a
role in beta ARK-mediated homologous, but not in protein kinase A-mediated
heterologous desensitization of beta-adrenergic receptors. It is
concluded that even though arrestin and beta-arrestin are similar
proteins, they display marked specificity for their respective receptors
and that phosphorylation of the receptors by the receptor-specific
kinases serves to permit the inhibitory effects of the ärresting"
proteins by allowing them to bind to the receptors and thereby inhibit
their signaling properties. Furthermore, it is shown that this mechanism
of receptor inhibition can be reproduced with isolated purified proteins.
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