Figure 6.
Expression analysis of several mutants suggests that 153aa of β3 with a bulky aromatic and nonpolar structure can maintain normal integrin expression and function. (A) In silico mutagenesis of F153β3 with small and bulky amino acid shows the space filling effect of the sidechain volumes. The F153S and F153A mutations may render αIIbβ3 constitutively active by facilitating an unrestricted inward movement of α2-helix during the conformational transition of the β3 integrin from a resting to an active state, whereas the F153W and F153Y may limit such movement. In silico mutagenesis were made in PyMol using PDB 2vdo and 3fcs. The resting and active conformations of α2-helix are highlighted in green and blue, respectively. (B) Immunofluorescent cytometric analysis to quantitate MFI expression levels of transgenic mutant β3-integrin on the surface of HEK293FT. Integrin αIIb and mutated β3 subunits plus GFP or GFP-TH (talin-head integrin activation construct) were cotransfected into HEK293FT cells. Harvested cells were then labeled with an anti-β3 mAb (AP3). GFP immunofluorescence was used as a control for transfection efficiency and integrin double-positive cells were analyzed for their MFI of either AP3 or GFP by immunocytometry. Results show that there is a significant reduction in the cell surface integrin expression of GT mutation (β3-F153S) compared with the WT control (β3-WT) whereas there is no statistical significance in total β3 surface expression between the WT and the other β3 variants (F153W, F153A, and F153Y). Data are presented as mean MFI + SEM (n ≥ 3) and unpaired 2-tailed Student t test was performed to compare the mutants with WT under the same condition or as indicated (P = .06). (C) Maximum integrin activity after stimulation with GFP-TH is detected by the binding of PAC-1, which is specific for the active conformation of αIIbβ3. Integrin αIIb and indicated β3 subunits plus GFP or GFP-TH are cotransfected into HEK293FT cells. Integrin and GFP double-positive cells were analyzed for the PAC-1 binding via immunocytometry. PAC-1 binding was quantified and normalized to the total integrin expression showing that (1) bulky aromatic, nonpolar (F,W) are activated identically; (2) bulky aromatic polar (Y) activation is negatively disrupted; and (3) small, polar and nonpolar side-chains (S,A) place αIIbβ3 in a constitutively activate confirmation without (red bar) or with (blue bar) talin head domain present. PAC-1 binding is normalized to the total integrin expression. Data are presented as MFI in percentage + SEM (n ≥ 3) and unpaired 2-tailed Student t test was performed to compare the mutants with WT under the same condition or as indicated. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. n.s., not significant.

Expression analysis of several mutants suggests that 153aa of β3 with a bulky aromatic and nonpolar structure can maintain normal integrin expression and function. (A) In silico mutagenesis of F153β3 with small and bulky amino acid shows the space filling effect of the sidechain volumes. The F153S and F153A mutations may render αIIbβ3 constitutively active by facilitating an unrestricted inward movement of α2-helix during the conformational transition of the β3 integrin from a resting to an active state, whereas the F153W and F153Y may limit such movement. In silico mutagenesis were made in PyMol using PDB 2vdo and 3fcs. The resting and active conformations of α2-helix are highlighted in green and blue, respectively. (B) Immunofluorescent cytometric analysis to quantitate MFI expression levels of transgenic mutant β3-integrin on the surface of HEK293FT. Integrin αIIb and mutated β3 subunits plus GFP or GFP-TH (talin-head integrin activation construct) were cotransfected into HEK293FT cells. Harvested cells were then labeled with an anti-β3 mAb (AP3). GFP immunofluorescence was used as a control for transfection efficiency and integrin double-positive cells were analyzed for their MFI of either AP3 or GFP by immunocytometry. Results show that there is a significant reduction in the cell surface integrin expression of GT mutation (β3-F153S) compared with the WT control (β3-WT) whereas there is no statistical significance in total β3 surface expression between the WT and the other β3 variants (F153W, F153A, and F153Y). Data are presented as mean MFI + SEM (n ≥ 3) and unpaired 2-tailed Student t test was performed to compare the mutants with WT under the same condition or as indicated (P = .06). (C) Maximum integrin activity after stimulation with GFP-TH is detected by the binding of PAC-1, which is specific for the active conformation of αIIbβ3. Integrin αIIb and indicated β3 subunits plus GFP or GFP-TH are cotransfected into HEK293FT cells. Integrin and GFP double-positive cells were analyzed for the PAC-1 binding via immunocytometry. PAC-1 binding was quantified and normalized to the total integrin expression showing that (1) bulky aromatic, nonpolar (F,W) are activated identically; (2) bulky aromatic polar (Y) activation is negatively disrupted; and (3) small, polar and nonpolar side-chains (S,A) place αIIbβ3 in a constitutively activate confirmation without (red bar) or with (blue bar) talin head domain present. PAC-1 binding is normalized to the total integrin expression. Data are presented as MFI in percentage + SEM (n ≥ 3) and unpaired 2-tailed Student t test was performed to compare the mutants with WT under the same condition or as indicated. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. n.s., not significant.

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