Characterization of cation-binding sequences in the platelet integrin GPIIb-IIIa (alpha IIb beta 3) by terbium luminescence.
The binding of cations to purified GPIIb-IIIa (alpha IIb beta 3) and synthetic peptides corresponding to the potential cation-binding sites within this integrin has been assessed by terbium luminescence spectroscopy. Tb3+ supported fibrinogen binding to purified GPIIb-IIIa, at lower concentrations than Ca2+, consistent with its higher affinity for cation-binding motifs. Titration analyses indicated the presence of five Tb(3+)-binding sites of relatively high affinity in the receptor. These sites also could be filled by divalent cations. Six sequences within GPIIb-IIIa have the appropriate spacing of five of the usual six coordination sites for cations in functional Ca(2+)-binding EF-hand motifs. Peptides containing Tyr and/or Trp at selected positions as fluorescence energy donors were synthesized, and their Tb(3+)-binding capacity was assessed. The four potential Ca(2+)-binding sequences in the GPIIb subunit, GPIIb 242-255, 296-309, 364-377, and 425-438, were functional, despite lacking the usual Glu residue at the terminal coordination position. These peptides bound Tb3+ with the same affinity as typical Ca(2+)-binding loop peptides and also bound Ca2+ and other divalent cations without preference. Of the two candidate GPIIIa sequences, 118-131 and 208-221, the former bound Tb3+ and divalent cations with an affinity similar to that of the GPIIb peptides, whereas the latter peptide was not functional. This functional difference, as well as data obtained with substituted peptides, emphasizes the importance of the first coordination position for interaction of synthetic peptide loops with cations. Together, these data identify the five cation-binding sites within intact GPIIb-IIIa.
Cierniewski
C S
CS
Department of Biophysics, Medical University in Lodz, Poland.
Haas
T A
TA
Smith
J W
JW
Plow
E F
EF
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008163:Luminescent Measurements; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D016024:Receptors, Cytoadhesin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013725:Terbium / Q000737:chemistry*
D016023:Integrins; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D016024:Receptors, Cytoadhesin; D013725:Terbium; D008345:Manganese; D005340:Fibrinogen; D008274:Magnesium; D002118:Calcium