title: Overexpression of the partially activated alpha(IIb)beta3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet-like extensions in Chinese hamster ovary cells.
abstract: BACKGROUND
  We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) 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 alpha(IIb)beta3D723H or alpha(IIb)beta3D723A salt bridge mutants, but not fully activated alpha(IIb)beta3 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 alpha(IIb)beta3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant alpha(IIb)beta3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type alpha(IIb)beta3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO alpha(IIb)beta3D723H 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 alpha(IIb)beta3D723H 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.
authors.affiliation: Laboratoire de Biologie et Physiologie Intégrée (CNRS/GDRE-ITI), University of Luxembourg, Grand-Duchy of Luxembourg, Luxembourg. elisabeth.schaffner@uni.lu
authors.forename: E
authors.initials: E
authors.lastname: Schaffner-Reckinger
authors.forename: A
authors.initials: A
authors.lastname: Salsmann
authors.forename: N
authors.initials: N
authors.lastname: Debili
authors.forename: J
authors.initials: J
authors.lastname: Bellis
authors.forename: J
authors.initials: J
authors.lastname: De Mey
authors.forename: W
authors.initials: W
authors.lastname: Vainchenker
authors.forename: W H
authors.initials: WH
authors.lastname: Ouwehand
authors.forename: N
authors.initials: N
authors.lastname: Kieffer
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary; D015536:Down-Regulation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D022001:Focal Adhesions; D008870:Microtubules / Q000378:metabolism* / Q000502:physiology*; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D020742:rhoA GTP-Binding Protein