title: The cytoplasmic domain of alphaIIb beta3. A ternary complex of the integrin alpha and beta subunits and a divalent cation.
abstract: Peptides corresponding to the cytoplasmic tails of the alphaIIb (alphaIIb (985-1008)) and beta3 (beta3 (713-762)) subunits of the integrin receptor alphaIIb beta3 (glycoprotein IIb-IIIa) were synthesized and used to characterize their interaction with cations and with one another. alphaIIb (985-1008) was found to contain a functional cation binding site as assessed by both terbium luminescence and electrospray ionization mass spectroscopy. The binding of Tb3+ to alphaIIb (985-1008) was of high affinity (Kd = 8.8 +/- 5.2 nM), occurred with a 1:1 stoichiometry, and was mediated by its acidic carboxy] terminus (alphaIIb (999-1008), PLEEDDEEGE). The affinity of this site for divalent cations was in the micromolar range, suggesting that this site would be constitutively occupied in the intracellular environment. Incubation of alphaIIb (999-1008) with beta3 (713-762) resulted in the formation of a complex, both in the presence and absence of cations. The interactive site for alphaIIb (999-1008) in beta3 was mapped to beta3 (721-740), and complex formation was associated with a stabilization of secondary structure as assessed by circular dichroism. Both a binary (alphaIIb (985-1008).beta3 (721-740)) and a ternary (Tb3+.alphaIIIb (999-1008).beta3 (721-740)) complex were detected by mass spectroscopy, but the distribution and intensity of the mass/charge peaks were distinct. These difference may reflect the involvement of distinct cation coordination sites and the formation of salt bridges in stabilizing the ternary complex. These data demonstrate the formation of a novel entity composed of the cytoplasmic tails of alphaIIb and beta3 and a cation which may constitute a functional intracellular domain.
authors.affiliation: Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Cleveland Clinic Foundation, Ohio 44195, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Haas
authors.forename: E F
authors.initials: EF
authors.lastname: Plow
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008163:Luminescent Measurements; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013725:Terbium / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D010446:Peptide Fragments; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D013725:Terbium