Gq pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets

• Published in: The Journal of biological chemistry 2017-09, Vol.292 (35), p.14516-14531
• Main Authors: Badolia, Rachit, Inamdar, Vaishali, Manne, Bhanu Kanth, Dangelmaier, Carol, Eble, Johannes A., Kunapuli, Satya P.
• Format: Article
• Language: English
• Subjects: cell signaling; CLEC-2 receptor; G-protein-coupled receptor (GPCR); Gq signaling; platelet; platelets; rhodocytin; Signal Transduction; snake venom
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.791012

Platelets play a key role in the physiological hemostasis or pathological process of thrombosis. Rhodocytin, an agonist of the C-type lectin-like receptor-2 (CLEC-2), elicits powerful platelet activation signals in conjunction with Src family kinases (SFKs), spleen tyrosine kinase (Syk), and phospholipase γ2 (PLCγ2). Previous reports have shown that rhodocytin-induced platelet aggregation depends on secondary mediators such as thromboxane A2 (TxA2) and ADP, which are agonists for G-protein-coupled receptors (GPCRs) on platelets. How the secondary mediators regulate CLEC-2-mediated platelet activation in terms of signaling is not clearly defined. In this study, we report that CLEC-2-induced Syk and PLCγ2 phosphorylation is potentiated by TxA2 and that TxA2 plays a critical role in the most proximal event of CLEC-2 signaling, i.e. the CLEC-2 receptor tyrosine phosphorylation. We show that the activation of other GPCRs, such as the ADP receptors and protease-activated receptors, can also potentiate CLEC-2 signaling. By using the specific Gq inhibitor, UBO-QIC, or Gq knock-out murine platelets, we demonstrate that Gq signaling, but not other G-proteins, is essential for GPCR-induced potentiation of Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling downstream of Gq and identified an important role for the PLCβ-PKCα pathway, possibly regulating activation of SFKs, which are crucial for initiation of CLEC-2 signaling. Together, these results provide evidence for novel Gq-PLCβ-PKCα-mediated regulation of proximal CLEC-2 signaling by Gq-coupled receptors.