The endocytic adaptor protein Disabled-2 is required for cellular uptake of fibrinogen.
Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbβ3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2 ± 9.8% and 59.0 ± 10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2 ± 0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6 ± 2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization.
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan, ROC.
Wei-Shan
WS
Hung
Chien-Ling
CL
Huang
Jui-Ting
JT
Fan
Ding-Yuan
DY
Huang
Chun-Fung
CF
Yeh
Ju-Chien
JC
Cheng
Ching-Ping
CP
Tseng
D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D013194:Staining and Labeling; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D025521:Tumor Suppressor Proteins
D033962:Adaptor Protein Complex 2; D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002966:Clathrin; C102885:DAB2 protein, human; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D005340:Fibrinogen; D013755:Tetradecanoylphorbol Acetate