Abstract
Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric G alpha and G beta gamma subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1c alpha and the heterotrimeric G protein G beta(1) subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and G beta(1) revealed that G beta(1) interacts with the PP1c alpha, beta, and gamma 1 isoforms. Purified PP1c bound to recombinant G beta(1)-GST protein, and PP1c co-immunoprecipitated with G beta(1) in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-G beta(1) complex, which correlated with an association of PP1c with phospholipase C beta(3) (PLC beta 3), along with a concomitant dephosphorylation of the inhibitory Ser(1105) residue in PLC beta 3. siRNA-mediated depletion of GNB1 (encoding G beta(1)) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1c alpha(-/-) murine platelets and in human platelets treated with a small-molecule inhibitor of G beta gamma. Finally, disruption of PP1c-G beta(1) complexes with myristoylated G beta(1) peptides containing the PP1c binding site moderately decreased throm-bin-induced human platelet aggregation. These findings suggest that G beta(1) protein enlists PP1c to modulate GPCR signaling in platelets.