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. 2017 Aug 11;292(32):13133-13142.
doi: 10.1074/jbc.M117.796656. Epub 2017 Jun 14.

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets

Subhashree Pradhan  1   2 , Tanvir Khatlani  1   2 , Angus C Nairn  3 , K Vinod Vijayan  4   2   5   6
Affiliations

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets

Subhashree Pradhan et al. J Biol Chem. .

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 GNB1 (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.

Keywords: ADP; G protein-coupled receptor (GPCR); megakaryocytes; phosphoprotein phosphatase 1 (PP1); platelet; protein phosphatase 1; thrombin.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Gβ1 is a novel PP1c interacting protein. A, lysate from 293 cells transfected with HA-tagged PP1c α, β, and γ and FLAG-tagged Gβ1 or empty vector were immunoprecipitated (IP) and immunoblotted (IB) with anti-HA and FLAG antibodies. Blots are representative of three experiments. B, characterization of Gβ1-GST protein by immunoblotting with anti-Gβ1 antibody. C, Gβ1-GST or GST beads coupled to glutathione beads were used to pull down purified PP1c. Blots are representative of two experiments.
Figure 2.
Figure 2.
Platelet activation disrupts endogenous PP1c-Gβ1 complex, facilitates PP1c-PLCβ3 interaction, and dephosphorylates PLCβ3 at Ser1105. A, C, and E, platelets were maintained in resting (Res) state or treated with thrombin (Thr) (0.5 unit/ml) or ADP (10 μm) for 2 mins. A, lysate was immunoprecipitated with control IgG or Gβ1 antibodies. Samples separated on SDS-PAGE were immunoblotted with anti-PP1c or Gβ1 antibodies. B, densitometry of the relative intensity of PP1c in arbitrary units (AU) from four experiments. C, lysate was immunoprecipitated with control IgG or PP1c antibodies. Samples were loaded in alternate lanes and separated on SDS-PAGE and immunoblotted with anti-PLCβ3 and PP1c antibodies. **, nonspecific bands. D, densitometry of the relative intensity of PLCβ3 in arbitrary units (AU) from four experiments. E, lysate was loaded in alternate lanes and separated on SDS-PAGE and immunoblotted with anti-Ser 1105 PLCβ3 or PLCβ3 antibodies. F, densitometric quantification of PLCβ3 Ser1105/total PLCβ3 from three to four experiments. Error bars, mean ± S. E.
Figure 3.
Figure 3.
Genetic depletion of Gβ1 and pharmacological inhibition of Gβγ signaling reduce thrombin receptor–induced fibrinogen binding and platelet aggregation. A, Gβ1 expression in control and Gβ1 siRNA-treated murine megakaryocytes. Blots were reprobed for actin (loading control). Blots are representative of two to three experiments. B, megakaryocytes were mixed with 1 mm PAR4-AP, nonblocking αIIb antibody, 7-AAD dye, and Alexa 488 conjugated fibrinogen. Specific fibrinogen binding was evaluated as mean fluorescence intensity in a gated population of large megakaryocytes that are viable and expressed αIIb. n = 4. C, aggregation profile of washed platelets preincubated with DMSO and 10 μm or 20 μm gallein and then stimulated with thrombin (0.05 unit/ml). The time to achieve 50% aggregation was increased by gallein treatment as seen by the rightward shift in the tracings. D, final platelet aggregation from four subjects is shown. Error bars, mean ± S. E.
Figure 4.
Figure 4.
Knock-out of PP1cα in platelets decreases thrombin-induced platelet aggregation and soluble fibrinogen binding. A, characterization of platelet-specific PP1cα knock-out mice. Lysate from platelets and non-megakaryocytic tissue (lymphocytes) from Pf4Cre− and PP1cαfl/fl (WT+/+) and Pf4Cre+ and PP1cαfl/fl (PP1cα−/−) were immunoblotted with anti-PP1cα, PP1cβ, PP1cγ, and actin antibodies. B, aggregation profile of washed WT and PP1cα−/− platelets stimulated with 0.02 unit/ml and 1 unit/ml thrombin. C, final platelet aggregation from six experiments is shown. D, platelets (basal, resting) stimulated with thrombin (0.02 unit/ml) in the presence of Alexa 488 conjugated fibrinogen were analyzed by flow cytometry, and soluble fibrinogen binding is shown as mean fluorescence intensity from six experiments. Error bars, mean ± S. E.
Figure 5.
Figure 5.
Identification of PP1c binding site on Gβ1 and use of myristoylated Gβ1 peptide to disrupt PP1c-Gβ1 complex in platelets. A, 293 cells were transfected with either empty vector (EV), FLAG-tagged wild-type Gβ1 (WT), or FLAG-tagged Gβ1 with single alanine point mutations at residue Lys27, Ile29, Trp31, or triple alanine mutations at these locations along with HA-tagged PP1c α, β, and γ. Lysate was immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted (IB) with anti-FLAG and HA antibodies. Input panel shows PP1c HA expression in cells transfected with Gβ1 full-length and mutant constructs. Similar results were obtained from two independent experiments. B, platelets were treated with vehicle DMSO, 200 μΜ Myr Gβ1 peptide, or Myr control scrambled (Scr) peptide, and lysate was immunoprecipitated with control (IgG) and anti-PP1c antibodies and blotted with anti-Gβ1 and PP1c antibodies. Blots are representative of three independent experiments.
Figure 6.
Figure 6.
Myristoylated Gβ1 peptide reduces thrombin-induced platelet aggregation. A, aggregation profile of washed platelets preincubated with DMSO, Myr control scrambled (Scr) peptide, and Myr Gβ1 peptide and then stimulated with thrombin (0.02 unit/ml). B, final platelet aggregation from six subjects is shown. Error bars, mean ± SEM.
Figure 7.
Figure 7.
Proposed model depicting the role of PP1c-Gβ1 and PP1c-PLCβ3 complexes in GPCR-induced platelet activation. A multiprotein complex of Gβ1 and PP1c exists presumably in the vicinity of GPCR. Engagement of GPCR with thrombin or ADP leads to the disruption of this complex. A new signal-induced complex of PP1c-PLCβ3 with a concomitant dephosphorylation of PLCβ3 Ser1105 occurs. Based on previous findings, dephosphorylation of PLCβ3 may aid in signal transmission. Forced disruption of PP1c-Gβ1 complex prior to thrombin stimulation reduced platelet aggregation. Collectively, these studies suggest that PP1 facilitates thrombin signaling by coupling with Gβ1 and PLCβ3 in the GPCR pathway.
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