Two novel mutations in the αIIb calcium-binding domains identify hydrophobic regions essential for αIIbβ3 biogenesis

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Abstract
The recently published crystal structure of the external domains of αVβ3 confirms the prediction that the aminoterminal portion of αV, which shares 40% homology with αIIb, folds into a β-propeller structure and that the 4 calcium-binding domains are positioned on the bottom of the propeller. To gain insight into the role of the calcium-binding domains in αIIb biogenesis, we characterized mutations in the second and third calcium-binding domains of αIIb in 2 patients with Glanzmann thrombasthenia. One patient inherited a Val298Phe mutation in the second domain, and the other patient inherited an Ile374Thr mutation in the third domain. Mammalian cell expression studies were performed with normal and mutant αIIb and β3 cDNA constructs. By flow cytometry, expression of αIIb Val298Phe/β3 in transfected cells was 28% of control, and expression of αIIbIle374Thr/β3 was 11% of control. Pulse-chase analyses showed that both mutant pro-αIIb subunits are retained in the endoplasmic reticulum and degraded. Mutagenesis studies of the Val298 and Ile374 residues showed that these highly conserved, branch-chained hydrophobic residues are essential at these positions and that biogenesis and expression of αIIbβ3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the αIIb β-propeller indicate that these mutations interfere with calcium binding. These data suggest that the αIIb calcium-binding domains play a key structural role in the β-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogenesis.