Figure 1.
All αIIbβ3 mutants were successfully expressed with high disulfide-bridge formation efficiency.
(A)Integrin αIIbβ3 constructs used in our study. B1: Wild type integrin αIIbβ3; B2: Disulfide-bonded αIIbβ3(αIIb_W968C, β3_I693C); B3: α truncated αIIbβ3(αIIb1-990); B4: α truncated, disulfide-bonded αIIbβ3 (αIIb1-990_W968C, β3_I693C). (B) Cell lines stably expressing WT and mutated integrin αIIbβ3 as evaluated by three different monoclonal antibodies. B1 (WT αIIbβ3), B2 (disulfide-bonded αIIbβ3 mutant), B3 (α-truncated αIIbβ3), and B4 (disulfide-bonded α-truncated αIIbβ3). The solid line and dashed line represent untransfected CHO-K1 cells and stable transfectants, respectively. AP3, 7E3, and 10E5 are mAbs targeting different domains of the αIIbβ3 heterodimer. (C) Disulfide bonds formed between α and β subunits with high efficiency. Cells labeled with S35 were lysed and subjected to immunoprecipitation by anti-αIIb mAb 10E5. Immunoprecipitated protein was then resolved by SDS-PAGE and visualized by radioautography. 2mM DTT was used to reduce the disulfide bridge in TM-clasped mutants.
Figure 2.
TM domain disassociation is required for cytoplasmic domain dissociation-induced high affinity integrin as measured by soluble ligand binding.
Binding of ligand mimetic mAb PAC-1(A) and fibrinogen (Fbg, B) to B1 through B4 cells in the presence of either EDTA (5mM), Ca2+ (5mM), Mn2+ (1mM), or Ca2+(5mM) with DTT (4mM) as indicated. Error bars represent standard deviation (S.D.) from three independent assays.
Figure 3.
TM domain separation is required for cell spreading.
(A) Stably transfected cells were seeded on immobilized Fbg (20μg/mL) with or without 1mM DTT for 30min at 37°C and visualized using differential interference contrast (DIC). Images are representatives from one of three independent assays. White bar: 10μm. (B) Average areas of adherent cells were quantified by pixel. The error bars represent S.D. from 100 randomly chosen cells.
Figure 4.
TM domain separation is required for FA formation, actin fiber organization, FAK activation and recruitment to FA sites in outside-in signaling.
(A) Clasping of TM domains ablated FA formation and disrupted actin filaments organization. Note that α truncation led to an even distribution of FAs around adherent cells. (B) Treatment with 1mM DTT largely restored cell spreading and FA assembly. Green: focal adhesions (labeled with anti-vinculin antibody); Red: actin filaments (labeled with TRITC-conjugated Phalloidin). White bar: 10μm. (C) Activated FAK (FAKpY397) was recruited normally to FAs in WT (B1) and the α-truncated mutant (B3), but recruitment was abolished by clasping the TM domains (B2 and B4). (D) Treatment with 1mM DTT restored recruitment of phosphorylated FAK to FAs. Green: FAKpY397; Red: actin filaments; Blue: FA marker vinculin. White bar: 10μm.
Figure 5.
TM domain separation but not αIIb cytoplasmic domain is required for recruitment and phosphorylation of paxillin.
(A) Recruitment of phosphorylated paxillin (PaxpY31, green) to FA sites (blue) was observed in both wild type (B1) and αIIb-truncated mutant (B3) but not in the two disulfide-bonded mutants (B2 and B4). Note that recruitment of phosphorylated paxillin was normal in the αIIb-truncated mutant indicating a dispensable role of the αIIb cytoplasmic domain in paxillin recruitment. (B) Addition of 1mM DTT restored recruitment of phosphorylated paxillin in both of the disulfide-bonded mutants. White bar: 10μm.
Figure 6.
TM separation promotes activation of FAK whereas PI3K regulates Akt and Erk1/2 but not FAK in outside-in signaling.
Cells were seeded on Fbg (20μg/mL)-coated dishes with or without 1mM DTT or with 1.5μM/mL Wortmannin at 37°C for 1h and then lysed and subjected to western blot. Cells seeded on 1% BSA coated dishes were used as control. (A)FAK activation induced by immobilized ligand Fbg is TM domain separation dependent. TM-clasped mutants expressed reduced phosphorylation on Y397. (B) Treatment with Wortmannin ablated Akt and Erk1/2 activation. Removing Wortmannin before cell spreading restored Erk1/2 activation without inducing Akt activation. This may implicate that PI3K but not Akt activity is required for cell survival and proliferation mediated by Erk1/2 in outside-in signaling. (C) Wortmannin treatment did not alter activation of FAK, implicating parallel pathways in outside-in signaling.
Figure 7.
Inhibition of PI3K and Src did not affect cell spreading, FAK/paxillin recruitment, or phosphorylation.
(A, B) FAK and paxillin were phosphorylated and recruited to FAs normally in WT (B1) and the αIIb-truncated mutant (B3) with control treatment (DMSO). TM clasping abolished the events as previously observed; (C, D) Treatment with 1.5μM/mL Wortmannin did not affect recruitment of phosphorylated FAK and paxillin. (E, F) PP1 (15μM/mL) had little to no effect on cell spreading with regard to FAK/paxillin phosphorylation and recruitment. White bar: 10μm.