Abstract
G protein-coupled receptor kinase 2 (GRK2) plays a fundamental role in the regulation of G protein-coupled receptors (GPCRs), and changes in GRK2 expression levels can have an important impact on cell functions. GRK2 is known to be degraded by the proteasome pathway. We have shown previously that β-arrestins participate in enhanced kinase turnover upon GPCR stimulation by facilitating GRK2 phosphorylation by c-Src or by MAPK or by recruiting the Mdm2 E3 ubiquitin ligase to the receptor complex. In this report, we have investigated how such diverse β-arrestin scaffold functions are integrated to modulate GRK2 degradation. Interestingly, we found that in the absence of GPCR activation, β-arrestins do not perform an adaptor role for GRK2/Mdm2 association, but rather compete with GRK2 for direct Mdm2 binding to regulate basal kinase turnover. Upon agonist stimulation, β-arrestins-mediated phosphorylation of GRK2 at serine 670 by MAPK facilitates Mdm2-mediated GRK2 degradation, whereas c-Src-dependent phosphorylation would support the action of an undetermined β-arrestin-recruited ligase in the absence of GPCR activation. The ability of β-arrestins to play different scaffold functions would allow coordination of both Mdm2-dependent and -independent processes aimed at the specific modulation of GRK2 turnover in different signaling contexts.
Highlights
We have previously described that -arrestins would favor agonist-stimulated G protein-coupled receptor kinase 2 (GRK2)/Mdm2 association by recruiting Mdm2 to the vicinity of GRK2 and that Mdm2 is instrumental for both basal and agonist-induced GRK2 turnover [14]
To define further the contribution of -arrestins to the Mdm2-dependent degradation of GRK2, we first analyzed GRK2 protein decay by pulse-chase assays in the presence of -arrestin2-V54D, a point mutant that fails to interact with Mdm2, and of -arrestin1-V53D, a construct harboring an equivalent mutation within the proposed Mdm2 binding site [21]
Overexpression of -arrestin1-V53D completely blocked the turnover of GRK2 both in basal conditions and upon receptor activation (94 Ϯ 13% and 96 Ϯ 6% of protein remaining after 1 h of chase, respectively, whereas overexpression of -arrestin2-V54D at similar levels abrogated the isoproterenol-induced increase in GRK2 proteolysis but had no effect on the basal turnover of the kinase (Fig. 1B)
Summary
Lysates from HEK-293 cells transiently transfected with GRK2, Mdm2, and different wild-type and mutant constructs of either -arrestin1 (A) or -arrestin2 (B) proteins as indicated were immunoprecipitated (IP) with the specific Mdm2 antibody SMP14. In line with this notion, we found that a mutant -arrestin-1- construct (arrestin1-C-tail), the expression of which has been shown to impair agonist-promoted Src/MAPK activation [29] and which lacks the Mdm2-binding region, abrogates the agonistinduced degradation of GRK2 but does not affect its basal turnover (Fig. 5B), mimicking the effects of the -arrestin2V54D mutant.
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