Neither Free αIIb nor Free β3 Limits αIIbβ3 Biogenesis, and Pro-αIIbβ3 Adopts a Conformation Akin to Ligand-Bound Mature αIIbβ3.
2005
The first steps in the biogenesis of αIIbβ3 involve the individual synthesis of each subunit within the endoplasmic reticulum, followed by complex formation. Previous studies performed in HEL cells and in megakaryocyte-lineage cells cultured from cryopreserved progenitor cells of patients with CML have led to conflicting data with regard to whether αIIb or β3 is produced in limiting amounts that might then control complex formation. To study this question in greater detail, we conducted immunoprecipitation studies on HEK293 cells transfected with αIIb and β3 cDNAs and megakaryocyte-lineage cells derived from human umbilical cord blood grown in the presence of thrombopoietin and IL-11. Cells were labeled for 15 min with 35S-methionine/cysteine, and then lysed after 2 hr with a 1% triton X-100 buffer. The antibodies employed included murine monoclonal antibodies 10E5 (anti-αIIbβ3 complex, which, reacts with αIIb cap domain), 7E3 (anti-αIIbβ3 + αvβ3, which reacts with β3 specificity-determining loop and α1 helix), AP-5 (LIBS anti-β3, which reacts with β3 amino acids 1 - 6), 7H2 (anti-β3, which reacts with PSI domain near C13), 1990 (anti-αIIb, which reacts at unknown site), and LIBS2 (LIBS anti-β3, which reacts with a site within amino acids 602 – 690). The relative amounts of pro-αIIb, mature αIIb, and β3 immunoprecipitated by this panel of antibodies is shown in Table 1. Results were similar in both the HEK293 cells and in megakaryocyte-lineage cells derived from human umbilical cord blood. These data indicate that there are sizeable pools of both free pro-αIIb and free β3 in both cell types, and thus neither subunit controls complex formation by limited availability. Based on the selective precipitation of pro-αIIb + β3 by AP5, it appears that the pro-αIIbβ3 complex adopts a conformation similar to that of ligand-bound mature αIIbβ3. We conclude that αIIbβ3 complex formation is slow relative to the production of the individual subunits, and is probably controlled either by the ability of the subunits to adopt the proper fold and/or to interact with a chaperone(s) that facilitates complex formation. Assuming that AP5 selectively recognizes αIIbβ3 complexes in which the angle of the β3 βA (I-like) domain - hybrid domain interface results in separation of the αIIb and β3 leg domains, these data also suggest that αIIbβ3 head-head interactions precede αIIbβ3 leg-leg interactions during biogenesis, and that αIIb maturation occurs rapidly after αIIbβ3 leg-leg interactions. Such a model is consistent with our previous data demonstrating that pro-αIIb binds to the ER membrane-resident chaperone calnexin via the N-linked glycan at αIIb N15.
| Antibody | Pro- αIIb | Mature αIIb | β3 |
|:--------:| --------- | ----------- | ---- |
| 1990 | ++++ | +++ | +++ |
| 10E5 | ++ | +++ | ++ |
| 7E3 | ++ | +++ | ++ |
| 7H2 | ++ | +++ | ++++ |
| LIBS2 | + | − | ++++ |
| AP5 | ++ | − | ++++ |
Table 1
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