Back to Search
Start Over
The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein–coupled receptor signaling in platelets
- Publication Year :
- 2017
- Publisher :
- American Society for Biochemistry and Molecular Biology, 2017.
-
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.
- Subjects :
- 0301 basic medicine
Blood Platelets
Male
Models, Molecular
animal structures
Platelet Aggregation
G protein
Recombinant Fusion Proteins
Phospholipase C beta
Bone Marrow Cells
Mice, Transgenic
Biology
Biochemistry
03 medical and health sciences
0302 clinical medicine
Heterotrimeric G protein
Protein Phosphatase 1
Two-Hybrid System Techniques
Animals
Humans
Point Mutation
Protease-activated receptor
Protein Interaction Domains and Motifs
Molecular Biology
Cells, Cultured
Crosses, Genetic
G protein-coupled receptor
Mice, Knockout
GTP-Binding Protein beta Subunits
Protein phosphatase 1
Cell Biology
Heterotrimeric GTP-Binding Proteins
G Protein-Coupled Receptor Signaling
Cell biology
G beta-gamma complex
030104 developmental biology
Amino Acid Substitution
Mutagenesis, Site-Directed
Female
GNB1
Megakaryocytes
030217 neurology & neurosurgery
Signal Transduction
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....52c8b061100cf16e09568510910ff49b