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.
Highlights
Peptides corresponding to the cytoplasmic tails of the ␣IIb (␣IIb(985–1008)) and 3 (3(713–762)) subunits of the integrin receptor ␣IIb3 were synthesized and used to characterize their interaction with cations and with one another. ␣IIb(985–1008) was found to contain a functional cation binding site as assessed by both terbium luminescence and electrospray ionization mass spectroscopy
The interactive site for ␣IIb(999 –1008) in 3 was mapped to 3(721–740), and complex formation was associated with a stabilization of secondary structure as assessed by circular dichroism
The central features of integrin extracellular domains and their ligand binding sites extend to ␣IIb3: the subunits interact with one another and bind divalent cations, and the ligand binding function is conformationally regulated
Summary
Through a process termed inside-out signaling [5, 14, 15]. intracellular signals initiate conformational changes, and the extracellular domains can be transformed from a low to a high affinity ligand binding state. The cytoplasmic tails of integrins must be centrally involved in initiating and propagating the conformational changes that mediate such inside-out signaling. At the same time, binding of ligands to the extracellular domain elicits intracellular responses (outside-in signaling), and activation of such signaling pathways must be dependent upon integrin cytoplasmic tails and their conformation [16]. The cytoplasmic tails of ␣IIb3 and other integrins can be viewed as a second ligand binding domain by virtue of their interactions with cytoskeletal proteins and intracellular signaling molecules and by their predicted conformational sensitivity. Interaction of the cytoplasmic tails with one another and with cations are demonstrated; and, in turn, these interactions are shown to effect conformation These findings may have substantial bearing on integrin structure and function.
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