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

Abstract

The recently published crystal structure of the external domains of ␣V␤3 confirms the prediction that the aminoterminal portion of ␣V, which shares 40% homology with ␣IIb, folds into a ␤-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 ␣IIb biogenesis, we characterized mutations in the second and third calcium-binding domains of ␣IIb 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 ␣IIb and ␤3 cDNA constructs. By flow cytometry, expression of ␣IIb Val298Phe/␤3 in transfected cells was 28% of control, and expression of ␣IIbIle374Thr/␤3 was 11% of control. Pulse-chase analyses showed that both mutant pro-␣IIb subunits are retained in the endoplasmic reticulum and degraded. Mutagenesis studies of the Val298 and Ile374 residues showed that these highly conserved, branchchained hydrophobic residues are essential at these positions and that biogenesis and expression of ␣IIb␤3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the ␣IIb ␤-propeller indicate that these mutations interfere with calcium binding. These data suggest that the ␣IIb calcium-binding domains play a key structural role in the ␤-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogen-

Mapping early conformational changes in αIIb and β3 during biogenesis reveals a potential mechanism for αIIbβ3 adopting its bent conformation

“…34 Briefly, cells were incubated for 30 minutes at 37°C in methionine/cysteine-free medium, followed by pulse-labeling for 15 minutes at 37°C in medium containing 35 S-methionine/cysteine (300 Ci [11.1 MBq]/10 cm plate). The pulse was terminated by incubation in medium containing unlabeled methionine/ cysteine (1 mg/mL each), and the cells were incubated at 37°C until lysis in 1% Triton-X 100 lysis buffer.…”

Integrin β3 regions controlling binding of murine mAb 7E3: Implications for the mechanism of integrin αIIbβ3 activation

“…For some experiments cells were labeled for 30 min with a sulfo-N- Molecular Modeling. The structural effects of the substitutions were analyzed by using a molecular modeling approach using the CHARMM molecular program as described (16). Briefly, the individual amino acids were substituted into the ␤-A domain of ␤3 and then energy minimization was performed with the Adopted Basis Newton-Raphson method (17).…”

Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state

“…4A, all mutants except for W3 and W6 could be expressed and secreted into the medium, indicating the compatibility of these eight insertions to the native structure. The location of the W3 and W6 insertions were very close to the Ca 2+ -binding sites known to be important for the correct folding of the β-propeller domain of α integrin subunits (Mitchell et al, 2003), suggesting that the correct metal ion coordination geometry was severely perturbed by these two mutations. Among the eight mutants that could be expressed and secreted successfully, seven were immunoprecipitated with NZ-1 at the same efficiency as that with the pan-β 3 -integrin monoclonal antibody 7E3 (Fig.…”