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Structural requirements for activation in alphaIIb beta3 integrin.

ABSTRACT: Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant ?IIb?3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant ?IIb?3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the ?-head/?-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the ?-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the ?-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.

SUBMITTER: Kamata T

PROVIDER: S-EPMC2992275 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Structural requirements for activation in alphaIIb beta3 integrin.

Kamata Tetsuji T Handa Makoto M Ito Sonomi S Sato Yukiko Y Ohtani Toshimitsu T Kawai Yohko Y Ikeda Yasuo Y Aiso Sadakazu S

The Journal of biological chemistry 20100929 49

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are ...[more]

PMID: 20884611

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Project description:Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

Project description:Integrins link the cytoskeleton to the extracellular matrix and regulate key signaling events that coordinate cellular processes such as secretion, migration, and proliferation. A single integrin molecule can exist in a resting state that does not bind extracellular ligands or in an active state that can engage ligands and form large signaling complexes. Activation signals are transduced between the cytosolic region and the extracellular region by a binary on/off switch in the integrin's transmembrane (TM) domain; the integrin's alpha and beta subunits each have a single TM helix that forms an alpha/beta heterodimer in the resting state, and the TM heterodimer separates to transduce an activation signal across the membrane. In this article, two methods used to generate models of the TM heterodimer, both converging on the same structure, are described. The first model was generated by a Monte Carlo algorithm that selected conformations based on their agreement with published experimental mutagenesis results. The second model was generated by threading the integrin's sequence onto TM helix dimers parsed from the Protein Data Bank and by selecting conformations based on their agreement with published experimental cysteine crosslinking results. The two models have similar structures; however, they differ markedly from some previously published models. To distinguish conformations that reflect the native integrin, we compared the Monte Carlo model, the threaded model, and four published models with experimental mutagenesis and cysteine crosslinking results. The models presented here had high correlation coefficients when compared with experimental findings, and they are in excellent agreement, both in terms of accuracy and in terms of precision, with a recent NMR structure. These results demonstrate that multiple approaches converged on the same structure of the resting integrin's TM heterodimer, and this conformation likely reflects the integrin's native structure.

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Structural requirements for activation in alphaIIb beta3 integrin.

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Structural requirements for activation in alphaIIb beta3 integrin.

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