RESUMEN
Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.
Asunto(s)
Calpaína/metabolismo , Talina/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Biotina/metabolismo , Plaquetas/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Cromatografía de Gases y Espectrometría de Masas , Humanos , Integrina beta3 , Integrinas/metabolismo , Cinética , Datos de Secuencia Molecular , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Rapid modulation of ligand binding affinity ("activation") is a central property of the integrin cell adhesion receptors. Using a screen for suppressors of integrin activation, we identified the small GTP-binding protein, H-Ras, and its effector kinase, Raf-1, as negative regulators of integrin activation. H-Ras inhibited the activation of integrins with three distinct alpha and beta subunit cytoplasmic domains. Suppression was not associated with integrin phosphorylation and was independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlated with activation of the ERK MAP kinase pathway. Thus, regulation of integrin affinity state is a novel, transcription-independent function of a Ras-linked MAP kinase pathway that may mediate a negative feedback loop in integrin function.