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Requirement of alpha and beta subunit transmembrane helix separation for integrin outside-in signaling.

ABSTRACT: Adhesion to extracellular ligands through integrins regulates cell shape, migration, growth, and survival. How integrins transmit signals in the outside-to-in direction remains unknown. Whereas in resting integrins the alpha and beta subunit transmembrane domains are associated, ligand binding promotes dissociation and separation of these domains. Here we address whether such separation is required for outside-in signaling. By introduction of an intersubunit disulfide bond, we generated mutant integrin alphaIIbbeta3 with blocked transmembrane separation that binds ligand, mediates adhesion, adopts an extended conformation after ligand binding, and forms antibody-induced macroclusters on the cell surface similarly to wild type. However, the mutant integrin exhibits a profound defect in adhesion-induced outside-in signaling as measured by cell spreading, actin stress-fiber and focal adhesion formation, and focal adhesion kinase activation. This defect was rescued by reduction of the disulfide bond. Our results demonstrate that the separation of transmembrane domains is required for integrin outside-in signal transduction.

SUBMITTER: Zhu J

PROVIDER: S-EPMC1988953 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Requirement of alpha and beta subunit transmembrane helix separation for integrin outside-in signaling.

Zhu Jieqing J Carman Christopher V CV Kim Minsoo M Shimaoka Motomu M Springer Timothy A TA Luo Bing-Hao BH

Blood 20070705 7

Adhesion to extracellular ligands through integrins regulates cell shape, migration, growth, and survival. How integrins transmit signals in the outside-to-in direction remains unknown. Whereas in resting integrins the alpha and beta subunit transmembrane domains are associated, ligand binding promotes dissociation and separation of these domains. Here we address whether such separation is required for outside-in signaling. By introduction of an intersubunit disulfide bond, we generated mutant i ...[more]

PMID: 17615290

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Project description:Protein interactions with the integrin beta-subunit cytoplasmic domain (beta-tail) are essential for adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. Talin-1 binds to the conserved membrane-proximal NPxY motif of beta-tails (NPIY in beta1 integrin) promoting the inside-out activation of integrins and providing a linkage between integrins and the actin cytoskeleton. Here, we characterize the role of interactions between talin-1 and beta-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant beta1A tail, with a tyrosine to alanine substitution in the NPIY motif. In addition to inhibiting integrin activation, the YA mutation suppresses cell spreading, integrin signaling, focal adhesion and stress-fiber formation, as well as microtubule assembly. Constitutive activation of the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif downstream of integrin activation. Depletion of talin-1 using TLN1 siRNA demonstrated that talin-1 is required for cell spreading, focal adhesion and stress-fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin-1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin-1 is depleted. We identified filamin A (FLNa) as this inhibitory protein; it binds to the beta1A tail in an NPIY-dependent manner and inhibition of FLNa expression in talin-1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac activation. Expression of the dominant inhibitory mutant, FilGAP(DeltaGAP), which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the beta1A tail, but not by integrins containing the beta1D tail, which is known to bind poorly to FLNa. Together, these results suggest that the binding of talin-1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the beta1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the beta1 tail downstream of integrin activation in promoting integrin function.

Dataset Information

Requirement of alpha and beta subunit transmembrane helix separation for integrin outside-in signaling.

Publications

Requirement of alpha and beta subunit transmembrane helix separation for integrin outside-in signaling.

Similar Datasets

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