Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length.
The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha 1 at GPIIIa 63-87 and 303-350; gamma 1 at GPIIIa 9-37; alpha 2 at GPIIIa 151-191; and gamma 2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets.(ABSTRACT TRUNCATED AT 400 WORDS)
Calvete
J J
JJ
Instituto de Química Física, C. S. I. C., Madrid, Spain.
Schäfer
W
W
Mann
K
K
Henschen
A
A
González-Rodríguez
J
J
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D004801:Eosine Yellowish-(YS); D005340:Fibrinogen / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009842:Oligopeptides; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D014357:Trypsin; D002118:Calcium; D004801:Eosine Yellowish-(YS)