Early Endosomal GTPase Rab5 (Ras-Related Protein in Brain 5) Regulates GPIbβ (Glycoprotein Ib Subunit β) Trafficking and Platelet Production In Vitro

TY - JOUR

T1 - Early Endosomal GTPase Rab5 (Ras-Related Protein in Brain 5) Regulates GPIbβ (Glycoprotein Ib Subunit β) Trafficking and Platelet Production In Vitro

AU - Bertović, Ivana

AU - Kurelić, Roberta

AU - Mahmutefendić Lučin, Hana

AU - Jurak Begonja, Antonija

PY - 2022/1

Y1 - 2022/1

N2 - OBJECTIVE: Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 (Ras-related protein in brain 5) regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3-monophosphate production important for binding of effector proteins during early-to-late endosomal/lysosomal maturation. Approach and Results: To investigate the role of Rab5 in megakaryocytes, we expressed GFP (green fluorescent protein)-coupled Rab5 wild type and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived megakaryocytes. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing megakaryocytes, when compared with GFP or Rab5 wild type. Moreover, trafficking of GPIbβ (glycoprotein Ib subunit beta), a subunit of major megakaryocytes receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbβ was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 wild type megakaryocytes, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbβ plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in late endosomes/lysosomes. Localization of GPIbβ in enlarged endosomes was phosphatidylinositol 3-monophosphate dependent.CONCLUSIONS: Taken together, our results demonstrate that Rab5-dependent endocytosis plays an important role in megakaryocytes receptor trafficking, membrane formation, and thrombopoiesis.

AB - OBJECTIVE: Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 (Ras-related protein in brain 5) regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3-monophosphate production important for binding of effector proteins during early-to-late endosomal/lysosomal maturation. Approach and Results: To investigate the role of Rab5 in megakaryocytes, we expressed GFP (green fluorescent protein)-coupled Rab5 wild type and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived megakaryocytes. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing megakaryocytes, when compared with GFP or Rab5 wild type. Moreover, trafficking of GPIbβ (glycoprotein Ib subunit beta), a subunit of major megakaryocytes receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbβ was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 wild type megakaryocytes, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbβ plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in late endosomes/lysosomes. Localization of GPIbβ in enlarged endosomes was phosphatidylinositol 3-monophosphate dependent.CONCLUSIONS: Taken together, our results demonstrate that Rab5-dependent endocytosis plays an important role in megakaryocytes receptor trafficking, membrane formation, and thrombopoiesis.

KW - Animals

KW - Blood Platelets/enzymology

KW - Cells, Cultured

KW - Endocytosis

KW - Endosomes/enzymology

KW - Female

KW - Male

KW - Megakaryocytes/enzymology

KW - Mice, Inbred C57BL

KW - Platelet Glycoprotein GPIb-IX Complex/genetics

KW - Point Mutation

KW - Protein Transport

KW - Thrombopoiesis

KW - Transferrin/metabolism

KW - rab5 GTP-Binding Proteins/genetics

U2 - 10.1161/ATVBAHA.121.316552

DO - 10.1161/ATVBAHA.121.316552

M3 - SCORING: Journal article

C2 - 34732055

VL - 42

SP - e10-e26

JO - ARTERIOSCL THROM VAS

JF - ARTERIOSCL THROM VAS

SN - 1079-5642

IS - 1

ER -