These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Detection of integrin alpha IIbbeta 3 clustering in living cells.
    Author: Buensuceso C, de Virgilio M, Shattil SJ.
    Journal: J Biol Chem; 2003 Apr 25; 278(17):15217-24. PubMed ID: 12595537.
    Abstract:
    In platelets, bidirectional signaling across integrin alpha(IIb)beta(3) regulates fibrinogen binding, cytoskeletal reorganization, cell aggregation, and spreading. Because these responses may be influenced by the clustering of alpha(IIb)beta(3) heterodimers into larger oligomers, we established two independent methods to detect integrin clustering and evaluate factors that regulate this process. In the first, weakly complementing beta-galactosidase mutants were fused to the C terminus of individual alpha(IIb) subunits, and the chimeras were stably expressed with beta(3) in Chinese hamster ovary cells. Clustering of alpha(IIb)beta(3) should bring the mutants into proximity and reconstitute beta-galactosidase activity. In the second method, alpha(IIb) was fused to either a green fluorescent protein (GFP) or Renilla luciferase and transiently expressed with beta(3). Here, integrin clustering should stimulate bioluminescence resonance energy transfer between a cell-permeable luciferase substrate and GFP. These methods successfully detected integrin clustering induced by anti-alpha(IIb)beta(3) antibodies. Significantly, they also detected clustering upon soluble fibrinogen binding to alpha(IIb)beta(3). In contrast, no clustering was observed following direct activation of alpha(IIb)beta(3) by MnCl(2) or an anti-alpha(IIb)beta(3)-activating antibody Fab in the absence of fibrinogen. Intracellular events also influenced alpha(IIb)beta(3) clustering. For example, a cell-permeable, bivalent FK506-binding protein (FKBP) ligand stimulated clustering when added to cells expressing an alpha(IIb)(FKBP)(2) chimera complexed with beta(3). Furthermore, alpha(IIb)beta(3) clustering occurred in the presence of latrunculin A or cytochalasin D, inhibitors of actin polymerization. These effects were enhanced by fibrinogen, suggesting that actin-regulated clustering modulates alpha(IIb)beta(3) interaction with ligands. These studies in living cells establish that alpha(IIb)beta(3) clustering is modulated by fibrinogen and actin dynamics. More broadly, they should facilitate investigations of the mechanisms and consequences of integrin clustering.
    [Abstract] [Full Text] [Related] [New Search]