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Three-dimensional reconstruction of intact human integrin ?IIb?3: new implications for activation-dependent ligand binding.

ABSTRACT: Integrin ?IIb?3 plays a central role in hemostasis and thrombosis. We provide the first 3-dimensional reconstruction of intact purified ?IIb?3 in a nanodisc lipid bilayer. Unlike previous models, it shows that the ligand-binding head domain is on top, pointing away from the membrane. Moreover, unlike the crystal structure of the recombinant ectodomain, the lower legs are not parallel, straight, and adjacent. Rather, the ?IIb lower leg is bent between the calf-1 and calf-2 domains and the ?3 Integrin-Epidermal Growth Factor (I-EGF) 2 to 4 domains are freely coiled rather than in a cleft between the ?3 headpiece and the ?IIb lower leg. Our data indicate an important role for the region that links the distal calf-2 and ?-tail domains to their respective transmembrane (TM) domains in transmitting the conformational changes in the TM domains associated with inside-out activation.

SUBMITTER: Choi WS

PROVIDER: S-EPMC3868924 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Three-dimensional reconstruction of intact human integrin αIIbβ3: new implications for activation-dependent ligand binding.

Choi Won-Seok WS Rice William J WJ Stokes David L DL Coller Barry S BS

Blood 20131017 26

Integrin αIIbβ3 plays a central role in hemostasis and thrombosis. We provide the first 3-dimensional reconstruction of intact purified αIIbβ3 in a nanodisc lipid bilayer. Unlike previous models, it shows that the ligand-binding head domain is on top, pointing away from the membrane. Moreover, unlike the crystal structure of the recombinant ectodomain, the lower legs are not parallel, straight, and adjacent. Rather, the αIIb lower leg is bent between the calf-1 and calf-2 domains and the β3 Inte ...[more]

PMID: 24136164

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Project description:Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (?IIb?6B?3) and mutant CHO cells expressing inactive ?IIb?6B?3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and ?-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and ?-parvin, resulting in the restoration of ?IIb?6B?3 activation. In the parental cells expressing active ?IIb?6B?3, ILK, PINCH, and ?-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both ?- and ?-parvin mRNA in the parent cells impaired the ?IIb?6B?3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore ?IIb?6B?3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired ?IIb?6B?3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type ?IIb?3 that is an inactive form, overexpression of a talin head domain (THD) induced ?IIb?3 activation and the THD-induced ?IIb?3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the ?IIb?3-expressing CHO cells further augmented THD-induced ?IIb?3 activation, whereas they did not induce ?IIb?3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.

Dataset Information

Three-dimensional reconstruction of intact human integrin ?IIb?3: new implications for activation-dependent ligand binding.

Publications

Three-dimensional reconstruction of intact human integrin αIIbβ3: new implications for activation-dependent ligand binding.

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