ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.
Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) 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 P2Y(1) and P2Y(12) 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 P2Y(1) or P2Y(12) 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 endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.
Institute of Life Science, College of Medicine, Swansea University, Swansea, United Kingdom.
Venkateswarlu
V
Kanamarlapudi
Sian E
SE
Owens
Keya
K
Saha
Robert J
RJ
Pope
Stuart J
SJ
Mundell
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000502:physiology; D058470:Receptors, Purinergic P2Y / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12 / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D058470:Receptors, Purinergic P2Y; D058925:Receptors, Purinergic P2Y12; D000242:Cyclic AMP; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human