arf6-dependent regulation of p2y receptor traffic and function in human plateletsarf6-dependent p2y受体调节流量和人类血小板功能.pdfVIP

ARF6-Dependent Regulation of P2Y Receptor Traffic and Function in Human Platelets 1 1 1 2 2 Venkateswarlu Kanamarlapudi , Sian E. Owens , Keya Saha , Robert J. Pope , Stuart J. Mundell * 1 Institute of Life Science, College of Medicine, Swansea University, Swansea, United Kingdom, 2 School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol, United Kingdom Abstract Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein- coupled receptors, the P2Y1 and P2Y12 purinoceptors. Recently, we demonstrated that P2Y1 and P2Y12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y1 and P2Y12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y1 or P2Y12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin- dependent fission of coated vesicles during