Divalent Cations Differentially Regulate Integrin alpha sub(IIb) Cytoplasmic Tail Binding to beta sub(3) and to Calcium- and Integrin-binding Protein

• Published in: The Journal of biological chemistry Vol. 274; no. 24; pp. 17257 - 17266
• Main Authors: Vallar, L, Melchior, C, Plancon, S, Drobecq, H, Lippens, G, Regnault, V, Kieffer, N
• Format: Journal Article
• Language: English
• Published: 11-06-1999
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.24.17257

We have used recombinant or synthetic alpha sub(IIb) and beta sub(3) integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha super(.) beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha super(.) beta complex by decreasing the dissociation rate. alpha super(.) beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alpha sub(IIb) but not by the beta sub(3) S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alpha sub(IIb)-specific ligand, bound to the alpha sub(IIb) cytoplasmic peptide in a Ca super(2+)- or Mn super(2+)-independent, one-to-one reaction with a KD value of 12 mu M. In contrast in vitro liquid phase binding of CIB to intact alpha sub(IIb) beta sub(3) occurred preferentially with Mn super(2+)-activated alpha sub(IIb) beta sub(3) conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn super(2+)-activated alpha sub(IIb) beta sub(3), suggesting that Mn super(2+) activation of intact alpha sub(IIb) beta sub(3) induces the exposure of a CIB-binding site, spontaneously exposed by the free alpha sub(IIb) peptide. Since CIB did not stimulate PAC-1 binding to inactive alpha sub(IIb) beta sub(3) nor prevented activated alpha sub(IIb) beta sub(3) occupancy by PAC-1, we conclude that CIB does not regulate alpha sub(IIb) beta sub(3) inside-out signaling, but rather is involved in an alpha sub(IIb) beta sub(3) post-receptor occupancy event.