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Overexpression of the partially activated α IIb β 3 D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule‐dependent proplatelet–like extensions in Chinese hamster ovary cells
Author(s) -
SCHAFFNERRECKINGER E.,
SALSMANN A.,
DEBILI N.,
BELLIS J.,
DE MEY J.,
VAINCHENKER W.,
OUWEHAND W. H.,
KIEFFER N.
Publication year - 2009
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2009.03494.x
Subject(s) - microbiology and biotechnology , rhoa , chinese hamster ovary cell , biology , focal adhesion , integrin , signal transduction , chemistry , cell , biochemistry , receptor
Summary. Background: We have recently reported a novel mutation in the β 3 subunit of the platelet fibrinogen receptor (α IIb β 3 D723H) 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 α IIb β 3 D723H or α IIb β 3 D723A salt bridge mutants, but not fully activated α IIb β 3 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 α IIb β 3 , the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34 + ‐derived megakaryocyte proplatelets. Mutant α IIb β 3 D723H signaling was independent of Src, protein kinase C or phosphoinositide 3‐kinase, but correlated with decreased RhoA activity as compared with wild‐type α IIb β 3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO α IIb β 3 D723H 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 α IIb β 3 D723H 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.