RESUMO
Kindlins play an important role in supporting integrin activation by cooperating with talin; however, the mechanistic details remain unclear. Here, we show that kindlins interacted directly with paxillin and that this interaction could support integrin αIIbß3 activation. An exposed loop in the N-terminal F0 subdomain of kindlins was involved in mediating the interaction. Disruption of kindlin binding to paxillin by structure-based mutations significantly impaired the function of kindlins in supporting integrin αIIbß3 activation. Both kindlin and talin were required for paxillin to enhance integrin activation. Interestingly, a direct interaction between paxillin and the talin head domain was also detectable. Mechanistically, paxillin, together with kindlin, was able to promote the binding of the talin head domain to integrin, suggesting that paxillin complexes with kindlin and talin to strengthen integrin activation. Specifically, we observed that crosstalk between kindlin-3 and the paxillin family in mouse platelets was involved in supporting integrin αIIbß3 activation and in vivo platelet thrombus formation. Taken together, our findings uncover a novel mechanism by which kindlin supports integrin αIIbß3 activation, which might be beneficial for developing safer anti-thrombotic therapies.
Assuntos
Plaquetas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Paxilina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Talina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Plaquetas/citologia , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Paxilina/genética , Ativação Plaquetária/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Talina/genética , Trombose/genética , Trombose/metabolismo , Trombose/patologiaRESUMO
BACKGROUND: Integrins are essential regulators of angiogenesis. However, the antiangiogenic potential of peptides derived from the integrin cytoplasmic tails (CT) remains mostly undetermined. METHODS: Here we designed a panel of membrane-penetrating peptides (termed as mßCTPs), each comprising a C-terminal NxxY motif from one of the conserved integrin ß CTs, and evaluated their antiangiogenic ability using both in vitro and in vivo approaches. RESULTS: We found that mß3CTP, mß5CTP and mß6CTP, derived respectively from the integrin ß3, ß5 and ß6 CTs, but not others, exhibit antiangiogenic ability. Interestingly, we observed that the integrin ß3, ß5 and ß6 CTs but not others are able to interact with ß3-endonexin. In addition, the antiangiogenic core in mß3CTP is identical to a previously identified ß3-endonexin binding region in the integrin ß3 CT, indicating that the antiangiogenic mßCTPs may function via their binding to ß3-endonexin. Consistently, knockdown of endogenous ß3-endonexin in HUVECs significantly suppresses tube formation, suggesting that ß3-endonexin is proangiogenic. However, neither treatment with the antiangiogenic mßCTPs nor knockdown of endogenous ß3-endonexin affects integrin-mediated HUVEC adhesion and migration, indicating that their antiangiogenic effect may not rely on directly regulating integrin activity. Importantly, both treatment with the antiangiogenic mßCTPs and knockdown of endogenous ß3-endonexin in HUVECs inhibit VEGF expression and cell proliferation, thereby providing mechanistic explanations for the functional consequences. CONCLUSION: Our results suggest that the antiangiogenic mßCTPs can interact with ß3-endonexin in vascular endothelial cells and suppress its function in regulating VEGF expression and cell proliferation, thus disclosing a unique pathway that may be useful for developing novel antiangiogenic strategies.