Overexpression of the partially activated αIIbβ3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet–like extensions in Chinese hamster ovary cells |
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| Authors: | E SCHAFFNER-RECKINGER A SALSMANN N DEBILI† J BELLIS‡ J DE MEY‡ W VAINCHENKER† W H OUWEHAND§¶ N KIEFFER# |
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| Institution: | Laboratoire de Biologie et Physiologie Intégrée (CNRS/GDRE-ITI), University of Luxembourg, Grand-Duchy of Luxembourg, Luxembourg;;INSERM U790, Institut Gustave Roussy, UniversitéParis XI, Villejuif;;ESBS, Institut Gilbert Laustriat (CNRS/UMR-7175), UniversitéLouis Pasteur (Strasbourg I), Illkirch, France;;Department of Haematology, University of Cambridge, Cambridge;;National Health Service Blood and Transplant, Cambridge, UK;;and French-Chinese Research Center for Life Sciences and Genomics, Shanghai JiaoTong University School of Medicine, Shanghai, China |
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| Abstract: | Summary. Background: We have recently reported a novel mutation in the β3 subunit of the platelet fibrinogen receptor (αIIbβ3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen‐dependent, microtubule‐driven proplatelet‐like cell extensions. Results: Here we demonstrate that the partially activated αIIbβ3D723H or αIIbβ3D723A salt bridge mutants, but not fully activated αIIbβ3 mutants, cause this phenotype. Time‐lapse videomicroscopy clearly differentiated these stable microtubule‐driven and nocodazole‐sensitive extensions from common dynamic actin‐driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of αIIbβ3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+‐derived megakaryocyte proplatelets. Mutant αIIbβ3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3‐kinase, but correlated with decreased RhoA activity as compared with wild‐type αIIbβ3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO αIIbβ3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO αIIbβ3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin‐dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule‐driven formation of cytoplasmic extensions. |
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