Gβ1 a Component Of The Heterotrimeric G Protein Is a New Protein Phosphatase 1c Interacting Protein That Regulates Platelet Activation

Platelet activation at the site of injury is tied to signal transduction events that are mediated by protein kinases and phosphatases. Reversible tyrosine, serine/threonine (Ser/Thr) phosphorylation-dependent assembly and/or disassembly of effector (cytoskeletal, signaling and adaptor) protein complexes propagate signaling downstream of G protein coupled receptors (GPCRs). Compared to kinases, the contribution of Ser/Thr phosphatases and its effectors in GPCR signaling studies is not well explored. Our previous studies had revealed that the catalytic subunit of protein phosphatase 1γ (PP1cγ) support GPCR signaling and thrombus formation. Since cell signaling networks are dependent on protein-protein interactions, we sought to identify the potential effectors of PP1cγ. We employed yeast two-hybrid interaction studies with the full length PP1cγ fused to GAL4 activating domain as bait and screened human bone marrow library. A novel interaction of PP1cγ with a protein called Gβ1 (GNB1) was identified. Gβ1 is a component of the heterotrimeric G proteins like the Gα and couple to GPCR. However, unlike Gα subunits, Gβ1 is unexplored in platelets. Co-immunoprecipitation (co-IP) studies validated PP1cγ-Gβ1 interaction in 293 cells expressing PP1cγ-HA and Gβ1-FLAG. Importantly, Gβ1 interacted with all the PP1c isoforms, suggesting that Gβ1 could target all PP1c isoforms to the GPCR complex. Purified PP1c bound to recombinant Gβ1-GST protein but not to GST protein, indicating that the in vitro interaction of PP1c with Gβ1 was direct and independent of Gα and Gγ subunits. A small molecule inhibitor of G protein βγ, gallein decreased thrombin-induced human platelet aggregation and adhesion to immobilized fibrinogen. There is a paucity of Gβ1^-/- platelets because Gβ1^-/- mice die within 2 days of birth due to microencephaly. siRNA mediated depletion of Gβ1 in murine megakaryocytes reduced PAR4-activating peptide induced soluble fibrinogen binding to α_IIbβ₃. These studies suggest a functional role for Gβ1 in GPCR signaling. PP1c co-immunoprecipitated with Gβ1 in resting platelets and agonist (thrombin and ADP) treatment under non-stirring conditions induced dissociation of PP1c from Gβ1. These studies indicate that Gβ1-PP1c complex in platelets is responsive to agonist. Furthermore, PP1c and Gβ1 associated with P2Y12 receptor in resting but not agonist activated platelets in a co-IP assay, suggesting a role for this complex in G protein signaling. Finally, agonist induced dissociation of PP1c from Gβ1 correlated with the association of PP1c with the downstream GPCR effector phospholipase C β3 (PLCβ3) with a concomitant dephosphorylation of PLCβ3 at Ser¹¹⁰⁵. Since previous studies have revealed that PLCβ3 activity is inhibited by Ser¹¹⁰⁵ phosphorylation, our observation suggest that agonist-induced association of PP1c with PLCβ3 facilitates dephosphorylation and activation of PLCβ3. These studies highlight a coupling of GPCR signaling with the phosphatase driven signal transduction during platelet activation.