Detection of Integrin alpha IIbbeta 3 Clustering in Living Cells Export

@article{citeulike:966694, 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 MnCl2 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. 10.1074/jbc.M213234200}, author = {Buensuceso, Charito and de Virgilio, Maddalena and Shattil, Sanford J.}, citeulike-article-id = {966694}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M213234200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/278/17/15217}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/12595537}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=12595537}, day = {18}, doi = {10.1074/jbc.M213234200}, journal = {J. Biol. Chem.}, keywords = {gfp, integrin, interaction, luciferase, protein}, month = {April}, number = {17}, pages = {15217--15224}, posted-at = {2006-11-29 13:40:29}, priority = {2}, title = {Detection of Integrin alpha IIbbeta 3 Clustering in Living Cells}, url = {http://dx.doi.org/10.1074/jbc.M213234200}, volume = {278}, year = {2003} }

TY - JOUR ID - citeulike:966694 L3 - citeulike-article-id:966694 N2 - 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 MnCl2 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. 10.1074/jbc.M213234200 IS - 17 JF - J. Biol. Chem. EP - 15224 TI - Detection of Integrin alpha IIbbeta 3 Clustering in Living Cells VL - 278 SP - 15217 KW - gfp KW - integrin KW - interaction KW - luciferase KW - protein AU - Buensuceso, Charito AU - de Virgilio, Maddalena AU - Shattil, Sanford PY - 2003/04/18/ UR - http://dx.doi.org/10.1074/jbc.M213234200 ER -