Activation of the Integrin Involves a Discrete Cation-binding Site That Regulates Conformation

doi:10.1074/jbc.271.3.1364

Activation of the Integrin Involves a Discrete Cation-binding Site That Regulates Conformation (*)

From the (1)Department of Cell Biology and the
(2)Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

^§ To whom correspondence should be addressed:
Dept. of Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037.
Tel.: 619-554-9871; Fax: 619-554-6251.

Abstract

“Activation” of integrins is involved in the dramatic transition of leukocytes and platelets from suspension to adhesion. The integrin αβ is not known to take part in this sort of transition, even though it shares its β subunit with αβ, the activable integrin on platelets. In the context of a constitutively adhered cell, changes in activation state may be more subtle in their effects, but nonetheless important in regulating cell behavior. We hypothesized that αβ can undergo conformational changes analogous to those associated with αβ activation. Accordingly, we examined αβ on the surface of M21 cells (a human melanoma cell line) and found that, like αβ, it can undergo conformational changes upon binding of a ligand analog and can be activated for ligand binding and migration by a monoclonal antibody directed against β. Modulation of the binding of this activating antibody, AP5, ligand binding, and antibody-mediated activation all are associated with a discrete cation-binding site shared in both αβ and αβ. Based on a measured K, this site has an apparent K for calcium of approximately 20 μM. At physiological levels of calcium, about 40% of the total αβ on a cell's surface is in a conformation detected by AP5. The data suggest a model for both αβ and αβ function in which the molecule can exist in either of (at least) two conformational states, one stabilized either by AP5 or ligand binding, refractory to calcium binding, and enhanced for ligand recognition, the other stabilized by calcium binding and refractory to AP5 and ligand binding. Functional analysis suggests that AP5 activates αβ by preventing occupation of this calcium site, and that the activated form of αβ differs functionally from the basal form. The active form is more conducive to migration and the basal to tight adhesion.

Footnotes

↵* This work was supported by National Institutes of Health Training Grant AI 07244 and Markey Fellowship 92-10 (to A. J. P.) and National Institutes of Health Grants HL 46979 and DE 10063 (to T. K. and V. Q., respectively). 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.
↵¹A. J. Pelletier, unpublished data.
↵² The abbreviations used are:

DPBS

Dulbecco's phosphate-buffered saline

PBS

phosphate-buffered saline

FITC

fluorescein isothiocyanate

MFI

mean fluorescence intensity

Opn

osteopontin

Vn

vitronectin.
- Received May 11, 1995.
- Revision received September 7, 1995.