Platelet agonists increase the affinity state of integrin αIIbβ3, 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. β3-Endonexin is a novel 111–amino acid protein that binds selectively to the β3 tail. Since β3-endonexin is present in platelets, we asked whether it can affect αIIbβ3 function. When β3-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 αIIbβ3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and αIIbβ3 bound little or no PAC1. However, those transfected with GFP/β3-endonexin and αIIbβ3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β3-endonexin did not affect levels of surface expression of αIIbβ3 nor did it modulate the affinity of an αIIbβ3 mutant that is defective in binding to β3-endonexin. Affinity modulation of αIIbβ3 by GFP/β3-endonexin was inhibited by coexpression of either a monomeric β3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β3-endonexin can modulate the affinity state of αIIbβ3 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 αIIbβ3.
Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail
1. Abbreviations used in this paper: GFP, green fluorescent protein; PE, phycoerythrin.
Please address all correspondence to Sanford J. Shattil, Department of Vascular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, VB-5, La Jolla, CA 92037. Tel.: (619) 784-7148. Fax: (619) 784-7422. e-mail: [email protected]
This work was presented in part at the Annual Meeting of the American Society of Hematology on December 8, 1996, in Orlando, FL and published in abstract form (1996. Blood. 88:140a).
Hirokazu Kashiwagi, Martin A. Schwartz, Martin Eigenthaler, K.A. Davis, Mark H. Ginsberg, Sanford J. Shattil; Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail. J Cell Biol 16 June 1997; 137 (6): 1433–1443. doi: https://doi.org/10.1083/jcb.137.6.1433
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