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Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Published on: March 19, 2014
A novel functional role for the highly conserved alpha-subunit KVGFFKR motif distinct from integrin alphaIIbbeta3
1Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin, Ireland.
Background:
The highly conserved integrin alpha-subunit membrane-proximal motif KVGFFKR plays a decisive role in modulating the activation of integrin alphaIIbbeta3. Previously, we have shown that a platelet permeable palmityl (pal)-peptide with this seven amino acid sequence can directly activate alphaIIbbeta3 leading to platelet aggregation.
Objectives:
To investigate further the role of the KVGFFKR motif in integrin alphaIIbbeta3 function.
Methods:
We used two sequence-specific complementary model systems, palmityl pal-peptides in platelets, and mutant alphaIIbbeta3-expressing Chinese Hamster Ovary (CHO) cell lines.
Results:
In platelets we show that the two phenylalanine amino acids in pal-KVGFFKR (pal-FF) peptide are critical for stimulating platelet aggregation. Pal-FF peptide treatment of platelets also gives rise to a tyrosine phosphorylation signal despite the presence of inhibitors of fibrinogen binding. In CHO cells, a double alanine substitution, alphaIIb(F992A, F993A)beta3, induces constitutive integrin activation but prevents actin stress fiber formation upon adhesion to fibrinogen, suggesting that alphaIIbbeta3-mediated cytoskeletal reorganization is also dependent on F992 and F993. This further highlights a critical role for the two phenylalanine residues in both of these alphaIIbbeta3-mediated processes.
Conclusion:
In addition to regulating integrin alphaIIbbeta3 activation state, the KVGFFKR motif also influences cytoskeletal reorganization. This activity is critically determined by F992 and F993 within the seven amino acid sequence.
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