The heterotrimeric G protein Gβ1 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 Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ₁ subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ₁ revealed that Gβ₁ interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ₁-GST protein, and PP1c co-immunoprecipitated with Gβ₁ in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ₁ complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser¹¹⁰⁵ residue in PLCβ3. siRNA-mediated depletion of <i>GNB1</i> (encoding Gβ₁) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα^-/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ₁ complexes with myristoylated Gβ₁ peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ₁ protein enlists PP1c to modulate GPCR signaling in platelets.