PMC

Fluorescence microscopy of CHO cells expressing WT αIIbβ3 and the αIIb mutants G972L, G976L, G976A, and L983A. Cells were fixed with paraformaldehyde, incubated sequentially with the mAb SSA6 and FITC-labeled anti-mouse IgG, and examined by fluorescence microscopy. Representative images are shown.

Structural model of an αIIb/β3 TM domain heterodimer. Shown are space-filling models of the αIIb (A) and β3 (B) TM helices with the interfacial αIIb glycines 972 and 976 highlighted in red and interfacial β3 residues Val-700, Ile-704, and Leu-705 highlighted in yellow.

The αIIb mutation G972N induces αIIbβ3 clustering. (A) Fluorescence microscopy of CHO cells expressing WT αIIbβ3 and the αIIb mutant G972N. Cells were fixed with paraformaldehyde, incubated sequentially with the mAb SSA6 and FITC-labeled anti-mouse IgG, and examined by fluorescence microscopy. Thirty to forty stained cells were randomly selected and photographed. Representative images are shown. (B) FAK phosphorylation in adherent and resuspended CHO cells expressing WT αIIbβ3 and the αIIb mutants G972N and L974N. (Upper) FAK phosphorylation detected by using an antiphosphotyrosine antibody. (Lower) FAK protein detected by using an anti-FAK antibody.

An L980A mutation in the αIIb TM domain induces constitutive αIIbβ3 activity. (A) Dot plots of fibrinogen and anti-β3 mouse mAb SSA6 binding to cells expressing WT human αIIbβ3 and the enhancing αIIb TM domain mutant L980A. FITC fluorescence, representing fibrinogen binding is shown on the y axis, and PE fluorescence, representing β3 expression, is shown on the x axis. Fibrinogen binding was measured in the absence or presence of 5 mM DTT. (B) Fluorescence microscopy of CHO cells expressing WT αIIbβ3 and the αIIb L980A mutant. Cells were fixed, incubated with the mAb SSA6 and FITC-labeled anti-mouse IgG, and examined by fluorescence microscopy. Representative images are shown.

Effect of mutations that disrupt αIIb TM domain dimerization on αIIbβ3 function. (A) Measurement of TM helix association using the TOXCAT assay. CAT expression induced by chimeric proteins containing the indicated αIIb and β3 TM helices was determined by CAT-ELISA and was compared to that induced by chimeric proteins containing the GpA-WT helix and the poorly dimerizing GpA mutant G83I. Data shown are the mean and 1 SE of four to six experiments. (B) Dot plots of fibrinogen and anti-β3 mouse mAb SSA6 binding to cells expressing the disruptive αIIb TM domain mutants G972N, G976L, and G976A and the permissive mutation L983A. FITC fluorescence, representing fibrinogen binding, is shown on the y axis, and PE fluorescence, representing β3 expression, is shown on the x axis.

Effect of Asn substitutions in the αIIb TM domain on αIIbβ3 function. (A) Constitutive fibrinogen binding to CHO cells expressing WT αIIbβ3 and various αIIb TM domain Asn mutants. Data are expressed as the ratio of cells constitutively binding fibrinogen to cells not binding fibrinogen determined from dot plots of two color FACS analysis and were normalized to data obtained from the cells expressing WT αIIbβ3. The data are the mean and SE of two to seven experiments. (B) Dot plots of fibrinogen and anti-β3 mouse mAb SSA6 binding to cells expressing WT human αIIbβ3 and the αIIb mutant G972N. FITC fluorescence, representing fibrinogen binding, is shown on the y axis, and PE fluorescence, representing β3 expression, is shown on the x axis. Fibrinogen binding was measured in the absence or presence of 5 mM DTT and in the presence of 5 mM EDTA.