Divalent Cations Differentially Regulate Integrin αIIb Cytoplasmic Tail Binding to β3 and to Calcium- and Integrin-binding Protein

doi:10.1074/jbc.274.24.17257

Divalent Cations Differentially Regulate Integrin α_IIb Cytoplasmic Tail Binding to β₃ and to Calcium- and Integrin-binding Protein*

From the ^‡Laboratoire Franco-Luxembourgeois de Recherche Biomédicale (CNRS and CRP-Santé), Centre Universitaire, L-1511 Luxembourg, Grand Duchy of Luxembourg, the ^§Institut de Biologie de Lille, Laboratoire de RMN, F-59000 Lille, France, and the ^¶Laboratoire d’Hématologie, UMR CNRS 7563, Faculté de Médecine, F-54500 Vandoeuvre-lès-Nancy, France

Abstract

We have used recombinant or synthetic α_IIb and β₃ integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. α·β heterodimerization occurred in a 1:1 stoichiometry with a weakK _D in the micromolar range. Divalent cations were not required for this association but stabilized the α·β complex by decreasing the dissociation rate. α·β complexation was impaired by the R995A substitution or the KVGFFKR deletion in α_IIb but not by the β₃ S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an α_IIb-specific ligand, bound to the α_IIbcytoplasmic peptide in a Ca²⁺- or Mn²⁺-independent, one-to-one reaction with aK _D value of 12 μm. In contrast,in vitro liquid phase binding of CIB to intact α_IIbβ₃ occurred preferentially with Mn²⁺-activated α_IIbβ₃conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn²⁺-activated α_IIbβ₃, suggesting that Mn²⁺activation of intact α_IIbβ₃ induces the exposure of a CIB-binding site, spontaneously exposed by the free α_IIb peptide. Since CIB did not stimulate PAC-1 binding to inactive α_IIbβ₃ nor prevented activated α_IIbβ₃ occupancy by PAC-1, we conclude that CIB does not regulate α_IIbβ₃ inside-out signaling, but rather is involved in an α_IIbβ₃ post-receptor occupancy event.

Footnotes

↵* This work was supported by grants from Centre de Recherche Public-Santé (CRP-Santé, Luxembourg), CNRS (France), Fondation Luxembourgeoise Contre le Cancer (Luxembourg), and EC Biomed-Project Grant BMH4-CT98-3517.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: Laboratoire Franco-Luxembourgeois de Recherche Biomédicale, (CNRS and CRP-Santé), Centre Universitaire, 162A Ave. de la Faı̈encerie, L-1511 Luxembourg, Grand Duchy of Luxembourg. Tel.: 352-466644-440; Fax: 352-466644-442; E-mail: kieffer{at}cu.lu.
Abbreviations:

CIB

calcium- and integrin-binding protein

CHO

Chinese hamster ovary

ConA

concanavalin A

GST

glutathione S-transferase

HEL

human erythroleukemia

HPLC

high performance liquid chromatography

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

RT-PCR

reverse transcriptase-polymerase chain reaction

RU

resonance unit

SPR

surface plasmon resonance

TBS

Tris-buffered saline

ELISA

enzyme-linked immunosorbent assay

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
- Received January 27, 1999.
- Revision received March 16, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.