The Cytoplasmic Domain of 
A TERNARY COMPLEX OF THE INTEGRIN α AND β SUBUNITS AND A DIVALENT CATION (*)
- From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Abstract
Peptides corresponding to the cytoplasmic tails of the α
(α
(985-1008)) and β
(β
(713-762)) subunits of the integrin receptor α
β
(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.
Footnotes
-
↵§ Supported by a fellowship from the American Heart Association, Northeast Ohio Affiliate.
-
↵* This work was supported in part by National Institutes of Health Grants HL38292 and HL54924. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- HPLC
-
high performance liquid chromatography
- Boc
-
tert-butyloxycarbonyl
- ESIM
-
electrospray ionization mass
- HBTU
-
1-hydroxybenzotriazole tetramethyluronium hexafluorophosphate
- GuHCl
-
guanidine hydrochloride
- PIPES
-
1,4-piperazinediethanesulfonic acid
- MOPS
-
4-morpholinepropanesulfonic acid.
-
↵2T. A. Haas and E. F. Plow, manuscript in preparation.
-
- Received August 28, 1995.
- Revision received November 20, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
