The heterotrimeric G protein G1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein–coupled receptor signaling in platelets

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 and G subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1c and the heterotrimeric G protein G1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and G1 revealed that G1 interacts with the PP1c , , and 1 isoforms. Purified PP1c bound to recombinant G1-GST protein, and PP1c co-immunoprecipitated with G1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-G1 complex, which correlated with an association of PP1c with phospholipase C 3 (PLC3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLC3. siRNA-mediated depletion of GNB1 (encoding G1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1c/ murine platelets and in human platelets treated with a small-molecule inhibitor of G. Finally, disruption of PP1c-G1 complexes with myristoylated G1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that G1 protein enlists PP1c to modulate GPCR signaling in platelets.