Expression, activation, and subcellular localization of the Rap1 GTPase in cord blood-derived human megakaryocytes.
The expression of the small GTPase Rap1 in human megakaryocytes (MKs) differentiated from cord blood (CB)-derived progenitors was investigated. High levels of Rap1 were detected in the majority of mature megakaryocytes independently of days of culture, while a very low percentage of immature megakaryocytes was found to express a small amount of the protein. Rap1 was predominantly detected on internal alpha-granule but not on the plasma membrane. By contrast, CD41 was clearly present on the peripheral plasma membrane, although it also displayed an intracellular localization similar to that of Rap1. Upon thrombin stimulation, both Rap1 and CD41 translocated to the periphery of the cell. At the opposite, RhoA GTPase and glycoprotein Ibalpha were predominantly located at the plasma membrane and did not undergo relocation upon thrombin stimulation. Thrombin induced a dose- and time-dependent activation of Rap1 in mature megakaryocytes. By using a confocal microscopy approach with a specific probe, active Rap1 was detected exclusively at the peripheral plasma membrane. These results demonstrate that expression of Rap1 occurs during maturation rather than differentiation of megakaryocytes from cord blood progenitor cells. Moreover, we demonstrate that thrombin-activated Rap1 is exclusively localized at the peripheral plasma membrane.
Balduini
Alessandra
A
Center of Excellence for Applied Biology, Department of Biochemistry, University of Pavia, Pavia, Italy.
Pecci
Alessandro
A
Lova
Paolo
P
Arezzi
Nicoletta
N
Marseglia
Carmela
C
Bellora
Federica
F
Perotti
Cesare
C
Balduini
Cesare
C
Balduini
Carlo L
CL
Torti
Mauro
M
D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005312:Fetal Blood / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000187:drug effects / Q000378:metabolism; D040201:Platelet Membrane Glycoprotein IIb / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D020827:rap1 GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000187:drug effects / Q000378:metabolism
D019038:Platelet Glycoprotein GPIb-IX Complex; D040201:Platelet Membrane Glycoprotein IIb; D013917:Thrombin; D020827:rap1 GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein