Dataset Information

Three-dimensional model of human platelet integrin alphaIIb beta3 in solution obtained by small angle neutron scattering.

ABSTRACT: Integrin alphaIIbbeta3 is the major membrane protein and adhesion receptor at the surface of blood platelets, which after activation plays a key role in platelet plug formation in hemostasis and thrombosis. Small angle neutron scattering (SANS) and shape reconstruction algorithms allowed formation of a low resolution three-dimensional model of whole alphaIIb beta3 in Ca(2+)/detergent solutions. Model projections after 90 degrees rotation along its long axis show an elongated and "arched" form (135 degrees) not observed before and a "handgun" form. This 20-nm-long structure is well defined, despite alphaIIb beta3 multidomain nature and expected segmental flexibility, with the largest region at the top, followed by two narrower and smaller regions at the bottom. Docking of this SANS envelope into the high resolution structure of alphaIIb beta3, reconstructed from crystallographic and NMR data, shows that the solution structure is less constrained, allows tentative assignment of the disposition of the alphaIIb and beta3 subunits and their domains within the model, and points out the structural analogies and differences of the SANS model with the crystallographic models of the recombinant ectodomains of alphaIIb beta3 and alphaV beta3 and with the cryo-electron microscopy model of whole alphaIIb beta3. The ectodomain is in the bent configuration at the top of the model, where alphaIIb and beta3 occupy the concave and convex sides, respectively, at the arched projection, with their bent knees at its apex. It follows the narrower transmembrane region and the cytoplasmic domains at the bottom end. AlphaIIb beta3 aggregated in Mn(2+)/detergent solutions, which impeded to get its SANS model.

SUBMITTER: Nogales A

PROVIDER: S-EPMC2801229 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Three-dimensional model of human platelet integrin alphaIIb beta3 in solution obtained by small angle neutron scattering.

Nogales Aurora A García Carolina C Pérez Javier J Callow Phil P Ezquerra Tiberio A TA González-Rodríguez José J

The Journal of biological chemistry 20091106 2

Integrin alphaIIbbeta3 is the major membrane protein and adhesion receptor at the surface of blood platelets, which after activation plays a key role in platelet plug formation in hemostasis and thrombosis. Small angle neutron scattering (SANS) and shape reconstruction algorithms allowed formation of a low resolution three-dimensional model of whole alphaIIb beta3 in Ca(2+)/detergent solutions. Model projections after 90 degrees rotation along its long axis show an elongated and "arched" form (1 ...[more]

PMID: 19897481

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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.

Dataset Information

Three-dimensional model of human platelet integrin alphaIIb beta3 in solution obtained by small angle neutron scattering.

Publications

Three-dimensional model of human platelet integrin alphaIIb beta3 in solution obtained by small angle neutron scattering.

Similar Datasets

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