Regulation of Ral GTPase activity in megakaryocyte by the T2R4 agonist, quinine
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Date
2019-08-16
Authors
Alamri, Abeer
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Abstract
Platelets are anucleated cells derived from megakaryocytes and play a crucial role in circulation including, blood clotting and repair during blood vessel injury. Quinine is one of the most bitter compound known and acts as an agonist for several bitter taste receptors (T2Rs) including, T2R4. When used as an antimalarial drug, quinine is known to cause thrombocytopenia. RalA is a small GTPase that has been shown to play a role in platelet function. RalA activity is regulated by calcium and the calcium-binding protein, calmodulin. The objective of this study is to investigate if quinine regulates RalA activity in platelets independently and/or through T2R4.
The immortalized megakaryocyte cell line, CHRF-288-11, was utilized in these studies. Pull-down assays using Ral-binding domain of Ral-interacting protein 1 were employed to assess RalA activation. To investigate if CHRF cells express bitter taste receptor, T2R4, RT-PCR and western blot analysis was carried out.
Treatment of CHRF cells with quinine resulted in the activation of RalA. The results from PCR and western blot analysis demonstrated the presence of T2R4 in CHRF cells. It has been shown that calmodulin and calcium are required for RalA activation. First, we investigated if quinine altered the interaction between calmodulin and RalA. Incubation of CHRF cell lysate with quinine prior to incubation with CaM-Sepharose beads demonstrated that quinine did not affect the interaction between RalA and calmodulin. Next, we investigated if quinine mediated RalA activation is through a calcium dependent pathway. CHRF cells were treated with quinine and calcium chelator (BAPTA-AM) or calmodulin antagonist (W7). The results obtained demonstrated that quinine activated RalA independently of calcium. Treatment of CHRF cells with BCML, a T2R4 antagonist, inhibited quinine mediated activation of RalA suggesting that quinine action is dependent on this bitter taste receptor. The information gained from these studies may lead to the development of an approach to control thrombocytopenia during therapeutic intervention where quinine is prescribed as the drug of choice.
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Keywords
G protein-coupled receptor, Bitter taste receptors, T2R4, Quinine, Calcium, Calmodulin, RalA GTPase, Megakaryocytes
Citation
APA