Tyrosine Phosphorylation of the β3 Cytoplasmic Domain Mediates Integrin-Cytoskeletal Interactions

Abstract

Tyrosine phosphorylation of the ␤ 3 subunit of the major platelet integrin ␣ IIb ␤ 3 has been shown to occur during thrombin-induced platelet aggregation (1). We now show that a wide variety of platelet stimuli induced ␤ 3 tyrosine phosphorylation, but that this phosphorylation occurred only following platelet aggregation. Several lines of evidence suggest that the ␤ 3 cytoplasmic domain tyrosine residues and/or their phosphorylation function to mediate interactions between ␤ 3 integrins and cytoskeletal proteins. First, phospho-␤ 3 was retained preferentially in a Triton X-100 insoluble cytoskeletal fraction of thrombin-aggregated platelets. Second, in vitro experiments show that the cytoskeletal protein, myosin, associated in a phosphotyrosine-dependent manner with a diphosphorylated peptide corresponding to residues 740 -762 of ␤ 3 . Third, mutation of both tyrosines in the ␤ 3 cytoplasmic domain to phenylalanines markedly reduced ␤ 3 -dependent fibrin clot retraction. Thus, our data indicate that platelet aggregation is both necessary and sufficient for ␤ 3 tyrosine phosphorylation, and this phosphorylation results in the physical linkage of ␣ IIb ␤ 3 to the cytoskeleton. We hypothesize that this linkage may involve direct binding of the phosphorylated integrin to the contractile protein myosin in order to mediate transmission of force to the fibrin clot during the process of clot retraction.

Ligand-dependent Activation of Integrin αvβ3

“…Integrin activation can be defined as the events necessary to permit firm adhesion, including a possible enhancement of integrin affinity, avidity, and integrin-cytoskeletal interactions. A requirement for ␤ 3 -tyrosine phosphorylation during ␣ v ␤ 3 activation has been established; however, the mechanism and the circumstances under which this modification contributes to the overall activation state of ␣ v ␤ 3 have not been have not been completely established (5,12,20,21).…”

Phosphorylation of the Integrin α4 Cytoplasmic Domain Regulates Paxillin Binding

“…For example, Tyr phosphorylation of ␤ 3 and ␤ 1A tails regulates integrin signaling and migration (23,24). The effects of ␤ 3 Tyr phosophorylation may be because of altered interactions with either myosin or the SHC adaptor (25,26). In addition, ␤ 3 phosphorylation at Thr 753 has been proposed to regulate bi-directional integrin signaling (27,28).…”

Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain

“…In immunoblot, Ab pY759 had essentially no reactivity with ␤ 3 from resting platelets but reacted strongly with ␤ 3 from platelets aggregated in response to thrombin, which induces ␤ 3 tyrosine phosphorylation. Prevention of platelet aggregation by the integrin inhibitor, RGDS, inhibited the reaction of Ab pY759 with ␤ 3 , confirming that ␤ 3 phosphorylation is induced by integrin outside-in signaling (20).…”

“…Detection of Tyrosine Phosphorylation and Calpain Cleavage of Integrin ␤ 3 in Platelets-Blood from healthy human donors or from wild type and knock-in mice with both Tyr 747 and Tyr 759 mutated to phenolalanine (20,21) was anticoagulated with 1/7 volume of ACD (2.5% trisodium citrate, 2.0% D-glucose, 1.5% citric acid) (26). Washed platelets in Tyrode's buffer were allowed to stay at 25°C for 1 h (26).…”

“…Inside-out signaling requires the membrane proximal region and the two NXXY motifs in the ␤ 3 cytoplasmic domain (3)(4)(5)(6)(15)(16)(17)(18)(19). Outside-in signaling requires the intact cytoplasmic domain of ␤ 3 (15) and also requires tyrosine phosphorylation in NXXY motifs (20,21). However, the mechanism responsible for the role of tyrosine phosphorylation of ␤ 3 in outside-in signaling is unclear.…”