Arrestin-2 Regulates Akt Activation by G Protein-Coupled Receptors in Platelets.

Abstract

Arrestins have been shown to play important roles in G Protein-Coupled Receptor (GPCR) function in many cells, but their roles in platelets remain uncharacterized. While the classical role of arrestins is considered to be the internalization and desensitization of GPCRs, more recent studies suggest that arrestins can serve as scaffolds to recruit phosphatidyl inositol-3 kinases (PI3K)s to Gq-coupled receptors and promote PI3K-dependent signaling. Thrombin stimulates the PI3K-dependent activation of Akt in platelets in a Gq-dependent manner. Therefore, we sought to determine whether arrestins are involved in the PI3K-dependent activation of Akt in platelets. Comparative immunoblots show that of the two non-visual mammalian arrestins, only one, arrestin-2 (β-arrestin-1), is expressed in human and mouse platelets. Immunoprecipitation of arrestin-2 or p85-PI3K from platelet lysates demonstrated that arrestin-2 associates with the p85 subunit of PI3Ka/b in thrombin or ADP-stimulated platelets, but not resting cells. The association can be inhibited by inhibitors of the P2Y12 receptor for ADP, but not by P2Y1 inhibitors. p85-arrestin association is also blocked by inhibitors of src family kinases, as is Akt phosphorylation. To determine whether src family members were part of the p85-arrestin complexes, immunoblots were re-probed with antibodies to src, lyn and fyn. The results show that Lyn is incorporated into thrombin-stimulated arrestin complexes in a P2Y12-dependent manner. To determine whether arrestin-2 is important for Akt phosphorylation in platelets, megakaryocytes differentiated in culture from mouse embryonic stem cells were used as models of platelet signaling, since these cells are amenable to genetic manipulation. Arrestin-2 was inhibited in the cultured megakaryocytes using a siRNA approach, then cells were stimulated with thrombin and Akt phosphorylation was assessed by immunoblotting. Arrestin-2 expression in the cultured megakaryocytes treated with arrestin-2 specific siRNA was suppressed by an average of 53% compared to cells treated with scrambled siRNA, while thrombin-stimulated Akt phosphorylation was suppressed by 98% compared to scrambled siRNA-treated control cells (n=3 experiments, difference is significant, p=.01, unpaired student's t-test). In conclusion, the results show that arrestin-2, lyn and PI3Kform a tri-molecular complex following stimulation of platelets with ADP or thrombin. Formation of arrestin complexes at activated receptor sites is important for the localized recruitment and src-dependent activation of p85-PI3K, thus promoting activation of Akt by G protein-coupled receptors.