Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin a IIb b 3 , 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. b 3 -Endonexin is a novel 111–amino acid protein that binds selectively to the b 3 tail. Since b 3 -endonexin is present in platelets, we asked whether it can affect a IIb b 3 function. When b 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 a IIb b 3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and a IIb b 3 bound little or no PAC1. However, those transfected with GFP/ b 3 -endonexin and a IIb b 3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/ b 3 -endonexin did not affect levels of surface expression of a IIb b 3 nor did it modulate the affinity of an a IIb b 3 mutant that is defective in binding to b 3 -endonexin. Affinity modulation of a IIb b 3 by GFP/ b 3 -endonexin was inhibited by coexpression of either a monomeric b 3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that b 3 -endonexin can modulate the affinity state of a IIb b 3 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 a IIb b 3 . I ntegrins are ab heterodimers and each subunit contains a relatively large extracellular domain, a membrane-spanning domain, and a 20–70–amino acid cytoplasmic tail. They function in cell adhesion and signaling by interacting with extracellular matrix proteins or cellular counter-receptors on the one hand, and with intracellular proteins on the other (8, 34, 59). The adhesive function of many integrins is subject to rapid regulation by two processes collectively referred to as “inside-out” signaling: ( a ) a structural change intrinsic to the heterodimer, and ( b ) clustering of heterodimers within the plane of the plasma membrane. The former modulates the affinity of the ligand–receptor interaction and thus is often referred to as “affinity modulation.” The latter increases the valency and, therefore, the avidity of the interaction. These two types of regulation are not mutually exclusive, and their relative contributions probably vary with the integrin and the cell type (12, 20, 62, 71). A good example of the pathophysiological significance of rapid integrin regulation involves platelet a IIb b 3 . Circulating platelets ordinarily do not interact with each other or with the blood vessel wall. However, when the vessel is damaged by trauma or disease, platelets become activated and a IIb b 3 is converted within seconds into a functional receptor for several Arg-Gly-Asp–containing ligands, including fibrinogen and von Willebrand factor. Since ligand binding is required for platelet aggregation, inside-out signaling is a prerequisite for primary hemostasis and for formation of occlusive platelet thrombi in vascular diseases (9, 27). Affinity modulation is thought to be responsible for the initial, reversible phase of fibrinogen binding to platelets, while integrin clustering may be involved in stabilizing the interaction (14, 52). Studies with intact and permeabilized platelets indicate that specific intracellular mediators promote rapid increases or decreases in ligand binding to a IIb b 3 . Excitatory platelet agonists, such as thrombin, increase ligand binding by a process that involves heterotrimeric G proteins and protein and lipid kinases (38, 61, 69, 74). On the other hand, substances such as prostacyclin and nitric oxide, 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. 88:140 a ). on F ebuary 3, 2013 jcb.rress.org D ow nladed fom Published June 16, 1997