Ligand Binding to Integrin αIIbβ3 Is Dependent on a MIDAS-like Domain in the β3 Subunit

doi:10.1074/jbc.271.36.21978

Ligand Binding to Integrin α_IIbβ₃ Is Dependent on a MIDAS-like Domain in the β₃ Subunit*

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, the
^‡ Department of Biochemistry, and the
^§ Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom

** To whom correspondence should be addressed. Tel.: 619-784-7120; Fax: 619-784-7360; E-mail: loftus{at}scripps.edu.

Abstract

Substitution of β₃ residue Asp¹¹⁹, Ser¹²¹, or Ser¹²³ results in a loss of the ligand binding function of integrin α_IIbβ₃. Homologous residues in other integrin β subunits are similarly critical for ligand binding function. This DXSXS motif is also present in the I domain of certain integrin α subunits, where it constitutes a portion of the unique metal ion-dependent adhesion site (MIDAS). In this report, we have utilized the crystal structure of the recombinant α_M I domain to produce a three-dimensional model of the homologous region in the integrin β₃ subunit. We performed mutagenesis of candidate amino acid residues predicted from this model to be involved in cation coordination and ligand binding. We report the identification of Asp²¹⁷ and Glu²²⁰ as residues essential for the ligand binding function of α_IIbβ₃. Alanine substitution of these residues did not affect receptor expression but abolished the binding of activation-dependent (PAC1) and -independent (OPG2) ligand mimetic antibodies. In our proposed model, β₃ Asp²¹⁷ is analogous to a metal-coordinating residue in the α_M MIDAS domain, while Glu²²⁰ does not correspond to a functional MIDAS domain residue. Substitution of the highly conserved β₃ residue Thr¹⁹⁷ corresponding to a critical MIDAS metal-coordinating Thr residue did not affect ligand binding function, suggesting that this region of β₃ adopts a structure that is very similar to but not identical to that of the MIDAS domain. These data support a functional linkage between these two sequences and further define a common feature of ligand binding to integrins.

Footnotes

↵^¶ A Royal Society University Research Fellow.
↵^∥ Supported by an EPSRC studentship.
↵* This work was supported in part by National Institutes of Health Grant HL48728 (to J. C. L.) and was done during the tenure of an Established Investigatorship of the American Heart Association (to J. C. L.). This is publication 9957VB from The Scripps Research Institute. 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.
↵¹ The abbreviations used are:

mAb

monoclonal antibody

MIDAS

metal ion-dependent adhesion site.
↵²R. Liddington, unpublished observations.
- Received April 1, 1996.
- Revision received June 27, 1996.