A molecular basis for affinity modulation of Fab ligand binding to integrin α(IIb)β3

Thomas J. Kunicki, Douglas S. Annis, Yang Jia Deng, Joseph C. Loftus, Sanford J. Shattil

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Abstract

The Arg-Gly-Asp (RGD) sequence within the third complementarity- determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin α(IIb)β3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (V(H) domain + C(γ)1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 κ chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified α(IIb)β3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1.1 Fab molecules bound per platelet was 17% in the presence of 1 mM Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 μm Mn2+. The ratio of Fab molecules bound after versus before activation (mean ± S.D.; n = 3) was: for AP7.3, 9.8 ± 0.6; for PAC1.1, 8.8 ± 0.3; and for AP7, 1.4 ± 0.2. In addition, AP7 bound to the stably expressed integrin mutant α(IIb)β3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin α(IIb)β3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of α(IIb)β3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.

Original languageEnglish (US)
Pages (from-to)20315-20321
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number34
DOIs
StatePublished - Sep 5 1996

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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