A 2D-DIGE approach to identify proteins involved in inside-out control of integrins.
Leukocyte integrins are functionally regulated by "inside-out" signaling, meaning that stimulus-induced signaling pathways act on the intracellular integrin tail and induce activation of the receptor at the outside. Both a change in conformation (affinity) and in clustering (avidity/valency) of the receptors has been described to occur. This inside-out signaling is essential for adequate migration of leukocytes to inflammatory sites; however, the exact underlying mechanism is not known. We used two variants of a mouse acute lymphocytic leukemia cell line (L1210), a suspension (L1210-S) and an adherent (L1210-A) variant that were characterized by nonactivated and activated integrins (beta(1), beta(2) and beta(3)), respectively. L1210-S and L1210-A cells were compared on protein expression profiles by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE). We found 86 protein spots that were more than 1.25-fold different between L1210-A and L1210-S. Only 4 protein spots were more than 2.5-fold different. We identified 29 proteins by mass spectrometry among which were gelsolin, L-plastin, and Rho GTPase dissociation inhibitor 2. These proteins were upregulated in the L1210-A cells versus L1210-S, which was verified by Western blot analysis. Overexpression of gelsolin in U937 resulted in increased high affinity integrin expression and cell adhesion. Comparison of functionally different cell lines from similar origin by 2D-DIGE might be a successful approach to identify regulatory proteins involved in integrin inside-out control.
Langereis
Jeroen D
JD
Department of Respiratory Medicine, University Medical Center, Utrecht, The Netherlands.
Prinsen
Berthil H C M T
BH
de Sain-van der Velden
Monique G M
MG
Coppens
Cornelis J C
CJ
Koenderman
Leo
L
Ulfman
Laurien H
LH
D000818:Animals; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D018260:Gelsolin / Q000032:analysis* / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000032:analysis* / Q000378:metabolism; D007939:Leukemia L1210; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction; D020298:U937 Cells; D062950:rho Guanine Nucleotide Dissociation Inhibitor gamma
C569643:ARHGDIG protein, human; C569650:Arhgdig protein, mouse; D018260:Gelsolin; D020730:Guanine Nucleotide Dissociation Inhibitors; D016023:Integrins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; C026742:plastin; D062950:rho Guanine Nucleotide Dissociation Inhibitor gamma