Characterization of a gain of function mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa).
Integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) is a prototype of integrins involved in cellular adhesive functions. As part of a structure-function analysis of this molecule, we constructed a mutant, designated alpha IIb beta 3 (beta 1-2), by replacing 6 amino acids within a putative ligand binding domain of the beta 3 subunit with sequences derived from beta 1. The alteration did not affect the capacity of beta 3(beta 1-2) to combine with transfected alpha IIb, nor did it cause it to combine with endogenous alpha 5. Integrin alpha IIb beta 3(beta 1-2) was in a "resting" state on Chinese hamster ovary cells as judged by minimal binding of an activation-specific anti-alpha IIb beta 3, PAC1. Nevertheless, cells expressing alpha IIb beta 3(beta 1-2) spontaneously bound fibrinogen with low affinity (Ka = (4.85 +/- 0.84) x 10(6) M-1). Activation with an anti-beta 3 antibody (monoclonal antibody 62) resulted in a 10-fold increase in fibrinogen binding affinity (Ka = (4.55 +/- 0.77) x 10(7) M-1), which was 3-fold greater than fibrinogen binding to activated wild type alpha IIb beta 3 (Ka = (1.66 +/- 0.33) x 10(7) M-1, F = 7.46, p = 0.008). The mutant receptor also bound fibrinogen mimetic peptide ligands with enhanced affinity as measured by the conformation-specific antibody, anti-LIBS1. This indicates that the increased affinity for fibrinogen was caused by enhanced interaction of alpha IIb beta 3(beta 1-2) with known recognition sequences in fibrinogen. Thus, this gain of function mutant augments ligand binding function, supporting a role for this region of the beta subunit in ligand binding to integrins.
Bajt
M L
ML
Committee on Vascular Biology, Scripps Research Institute, La Jolla, California 920037.
Loftus
J C
JC
Gawaz
M P
MP
Ginsberg
M H
MH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002448:Cell Adhesion; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen