Ca(2+)-binding properties of the platelet glycoprotein IIb ligand-interacting domain.
Glycoprotein (GP) IIb is the alpha subunit of platelet integrin GPIIb-IIIa. Analysis of the primary structure of this subunit has indicated the presence of four stretches of amino acid residues that are highly conserved among various integrin alpha subunits and that have been suggested to be putative calcium-binding sites. To verify the Ca(2+)-binding capacity of these conserved domains and their implication in integrin adhesive functions, a fragment corresponding to the amino acid sequence of GPIIb from positions 171 to 464 was expressed. The nucleotide sequence coding for this GPIIb domain was generated by polymerase chain reaction, cloned into the pTG1924 expression vector, and expressed in Escherichia coli strain TGE901. The recombinant protein was purified by gel exclusion chromatography and used in equilibrium dialysis experiments. The results demonstrate that the four binding sites can be occupied by Ca2+. Two classes of binding sites can be detected, including two sites with a Kd of 30 microns and two sites of lower affinity with a Kd of 120 microns. Interaction of Ca2+ with these two classes of sites was inhibited by a large excess of Mg2+ or Mn2+, suggesting that these cations are competitive for the same sites on GPIIb. Thus, the four Ca(2+)-binding sites of GPIIb are not similar and exhibit different affinities for divalent ions. To verify the functional implication of these Ca(2+)-binding sites, the effect of Ca2+ on the binding of fibrinogen to the recombinant protein was analyzed using a solid-phase assay. The results indicate that optimal fibrinogen binding occurs when the four calcium-binding sites are occupied and establish the functional importance of this Ca(2+)-binding domain in the ligand-binding activity of GPIIb.
Gulino
D
D
Laboratoire d'Hématologie, Institut National de la Santé et de la Recherche Médicale U217, CENG/DBMS, Grenoble, France.
Boudignon
C
C
Zhang
L Y
LY
Concord
E
E
Rabiet
M J
MJ
Marguerie
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D005340:Fibrinogen; D002118:Calcium