The Cytoplasmic Domain of

Peptides corresponding to the cytoplasmic tails of the α (α(985-1008)) and β (β(713-762)) subunits of the integrin receptor αβ (glycoprotein IIb-IIIa) were synthesized and used to characterize their interaction with cations and with one another. α(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 Tb to α(985-1008) was of high affinity (K = 8.8 ± 5.2 nM), occurred with a 1:1 stoichiometry, and was mediated by its acidic carboxyl terminus (α(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 α(999-1008) with β(713-762) resulted in the formation of a complex, both in the presence and absence of cations. The interactive site for α(999-1008) in β was mapped to β(721-740), and complex formation was associated with a stabilization of secondary structure as assessed by circular dichroism. Both a binary (α(985-1008)•β(721-740)) and a ternary (Tb•α(999-1008)•β(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 α and β and a cation which may constitute a functional intracellular domain.

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