J. Cell Biol.
© The Rockefeller University Press
0021-9525/97/06/1433/11 $2.00
Volume 137, Number 6, June 16, 1997 1433-1443

Affinity Modulation of Platelet Integrin _IIb3 by ₃-Endonexin, a Selective Binding Partner of the ₃ Integrin Cytoplasmic Tail

Hirokazu Kashiwagi,^* Martin A. Schwartz,^* Martin Eigenthaler,^* K.A. Davis,^§ Mark H. Ginsberg,^* and Sanford J. Shattil^*

^* Department of Vascular Biology,  Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037; and ^§ Becton-Dickinson Immunocytometry Systems, San Jose, California 95131

Platelet agonists increase the affinity state of integrin _IIb3, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. ₃-Endonexin is a novel 111-amino acid protein that binds selectively to the ₃ tail. Since ₃-endonexin is present in platelets, we asked whether it can affect _IIb3 function. When ₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor _IIb3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and _IIb3 bound little or no PAC1. However, those transfected with GFP/₃-endonexin and _IIb3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/₃-endonexin did not affect levels of surface expression of _IIb3 nor did it modulate the affinity of an _IIb3 mutant that is defective in binding to ₃-endonexin. Affinity modulation of _IIb3 by GFP/₃-endonexin was inhibited by coexpression of either a monomeric ₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that ₃-endonexin can modulate the affinity state of _IIb3 in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein-protein interactions at the level of the cytoplasmic tails of _IIb3.

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