Regulation of RalA GTPase Activity by Taste Receptor Agonist Quinine

Objectives: 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 the most bitter compound known and acts as an agonist for a number of G protein-coupled bitter taste receptors 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 and its activity is regulated by 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.
Methods: We have utilized the immortalized megakaryocyte cell line, CHRF-288-11, in these studies. Pull-down assays using the Ral-binding domain of Ral-interacting protein 1 were used to assess RalA activation. To investigate if CHRF cells express bitter taste receptor, T2R4, RT-PCR and western blot analysis was carried out.
Results: 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 is required for RalA activation. Thus, we investigated if quinine altered the interaction between calmodulin and RalA. Incubation of CHRF cell lysate prior to incubation with CaM-Sepharose beads demonstrated that quinine did not affect the interaction between RalA and calmodulin. Studies are underway to elucidate the mechanism responsible for the regulation of RalA activity in megakaryocytes.
Conclusions: The results demonstrate that Quinine activates RalA. Further 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.