Integrins are αβ heterodimers. Each subunit crosses the membrane once, with most of each polypeptide in the extracellular space and two short cytoplasmic domains. The figure depicts the mammalian subunits and their αβ associations; 8 β subunits can assort with 18 α subunits to form 24 distinct integrins. These can be considered in several subfamilies based on evolutionary relationships (coloring of α subunits), ligand specificity and, in the case of β2 and β7 integrins, restricted expression on white blood cells. α subunits with gray hatching or stippling have inserted I/A domains. Such α subunits are restricted to chordates, as are α4 and α9 (green) and subunits β2-β8. In contrast, α subunits with specificity for laminins (purple) or RGD (blue) are found throughout the metazoa and are clearly ancient. Asterisks denote alternatively spliced cytoplasmic domains. A few extracellular domains are also alternatively spliced. Each of the 24 integrins shown appears to have a specific, nonredundant function.

In addition to their roles in adhesion to ECM ligands or counterreceptors on adjacent cells, integrins serve as transmembrane mechanical links from those extracellular contacts to the cytoskeleton inside cells. For all integrins except α6β4, the linkage is to the actin-based microfilament system, which integrins also regulate and modulate. The β4 subunit differs from all the others; its cytoplasmic domain being much larger, ~1,000 amino acids long instead of around 50, and making connections to intermediate filaments instead of to actin. The submembrane linker proteins connecting the cytoplasmic domains of integrins to the cytoskeleton are multiple and their interactions are complex. In part related to the integrin-mediated assembly of cytoskeletal linkages, ligation of integrins also triggers a large variety of signal transduction events that serve to modulate many aspects of cell behavior including proliferation, survival/apoptosis, shape, polarity, motility, gene expression, and differentiation. These signal transduction pathways are complex, like those emanating from receptors for soluble factors (e.g., G protein-coupled and kinase receptors). Indeed, many integrin-stimulated pathways are very similar to those triggered by growth factor receptors and are intimately coupled with them. In fact, many cellular responses to soluble growth factors, such as EGF, PDGF, LPA, and thrombin, etc., are dependent on the cell's being adherent to a substrate via integrins. That is the essence of anchorage dependence of cell survival and proliferation and integrins lie at the basis of these phenomena.

The major signal transduction pathways and many of the key players in them leading to the major effects on cell behavior mediated by integrins, often acting in concert with G protein-coupled or kinase receptors for soluble factors. The major submembranous, integrin-associated links between integrins and these signal transduction pathways are contained within the pink-purple pentagon beneath the clustered integrins. Details of the interactions of these linker/adaptor proteins and of the signal transduction pathways are omitted, as are other known players in these processes.


Integrin αVβ3

Integrins use bidirectional signaling to integrate the intracellular and extracellular environments. In outside-in signaling, ligand binding activates intracellular signaling pathways. In inside-out signaling, signals received by other receptors activate intracellular signaling pathways that impinge on integrin cytoplasmic domains, and make the extracellular domain competent for ligand binding on a time-scale of less than 1 second. These signaling pathways expose activation-dependent or ligand-induced binding site (LIBS) epitopes in the integrin extracellular domains, and may also regulate integrin clustering. Despite two crystal structures of the integrin αVβ3 it remained unknown how conformational signals are communicated in integrins. Both crystals were obtained in Ca2+, a cation that stabilizes integrins in the inactive or low affinity conformation. It had been expected based on previous electron micrographs that integrins would have an extended conformation. However, the crystal structures revealed an unexpected bent conformation, in which the headpiece is folded over the tailpiece, with each leg bent at a knee. The extreme bend causes the ligand binding site in the headpiece to come down close to the C-terminal, membrane-proximal end of the two legs. The bent conformation was suggested to represent an inactive, physiologically relevant conformation and that activation would be accompanied by a switchblade-like opening of the headpiece-tailpiece interface. Inside-out signals are thought to activate integrins by breaking a clasp between the α and β subunit cytoplasmic or transmembrane domains. However, how this could be communicated to the extracellular domains was unclear, just as it was unclear how in the reverse direction, ligand binding to the extracellular domain can be communicated to the cytoplasmic domains. The question was particularly perplexing, since many domains in the α and β subunit legs intervene between the ligand binding headpiece and the membrane, and transmission of conformational change through so many domains was unprecedented. In collaboration with Timothy Springer (The Center for Blood Research), we could describe the rearrangements in integrin architecture that communicate signals between the extracellular and intracellular environments, and that regulate affinity for biological ligands.

Comparison of negatively stained αVβ3 projection averages to the αVβ3 crystal structure. Top left: projection average of clasped αVβ3 particles in the presence of Ca2+ used for cross-correlation. Bottom left: best correlating projected view calculated from the αVβ3 crystal structure. Top middle: representative projection average of an extended integrin with a closed headpiece (in the presence of Mn2+) used in cross-correlation. Bottom middle: best correlating projected view calculated from the headpiece of the αVβ3 crystal structure (1JV2); the corresponding headpiece from the ligand-complexed structure (1L5G) gave an identical cross-correlation rotation function and projected view. Top right: representative projection average of an extended integrin with an open headpiece (in the presence of Ca2+ and cyclo-RGDfV) used in cross-correlation. Bottom right: best correlating projected view calculated from the model of the open headpiece crystal structure.

Quaternary structural rearrangements in integrin activation. The three conformers defined by electron microscopy (A, D, and E) and the hypothetical intermediates when the headpiece-tailpiece and α tailpiece-β tailpiece interfaces are destabilized in outside-in (B) or inside-out (C) signaling are schematized. The α headpiece domains (β-propeller and thigh) are red, α tailpiece domains (calf-1 and calf-2) are pink, β headpiece domains (I-like, hybrid, PSI, and I-EGF1) are blue, and β tailpiece domains (I-EGF2, and the I-EGF3, I-EGF4, and β-tail which are shown merged together) are cyan. Black squiggly lines symbolize the transmembrane and cytoplasmic domains. The conformers are shown in the same orientation as the cross-correlated headpiece in the above figure, except for rotation in the plane of the figure. The rotation in the plane of the figure is such as to maximize comparison between the conformers, and is not uniform with regard to expected orientation relative to the cell membrane, especially in (D).

» Takagi et al. (2002) Cell 110: 599-611.

Integrin α5β1

Many members of the integrin family, including α5β1, α8β1, αIIbβ3, αVβ3, αVβ5, αVβ6 and αVβ8, recognize an Arg-Gly-Asp (RGD) motif within their ligands. These ligands include fibronectin (Fn), fibrinogen, vitronectin, von Willebrand factor and many other large glycoproteins. Peptides containing this motif can efficiently block these integrin-ligand interactions. However, it is the residues outside the RGD motif that provide specificity as well as high affinity for each integrin-ligand pair. α5β1 integrin and Fn form a prototypic integrin-ligand pair. This receptor-ligand pair is functionally very important because it mediates fibronectin fibril formation and governs extracellular matrix assembly, which is vital to cell function in vivo. The interaction between α5β1 and Fn is fundamental for vertebrate development. In addition to the RGD sequence present in Fn type III module 10, a set of residues present in Fn type III module 9 (synergy site) contribute to high affinity recognition by α5β1. The crystal structure of the extracellular domain of αVβ3 integrin has established a basis to think about integrin function on the atomic level. The subsequent structure of αVβ3 in complex with a ligand mimetic peptide provided a first glimpse as to how integrins recognize the RGD tripeptide motif, where Arg and Asp side chains bridge the integrin α and β subunits at the center of the ligand binding pocket. We have shown with integrin αVβ3 that binding to a small ligand mimetic peptide converted the extracellular domain from a compact `bent' conformation into a tall extended conformation. These studies utilized small peptides containing RGD; however, structural information using a protein ligand was needed in order to understand the contribution of residues outside the RGD-integrin interface to the specificity and stability of the physiological integrin-ligand complex, and to integrin conformation. In collaboration with Timothy Springer (The Center for Blood Research) we showed how an integrin globular head domain composed of both subunits binds its physiological protein ligand using a recombinant α5β1 headpiece fragment and Fn fragments. Together with binding kinetics measurements, new insights emerged on the contribution of the synergy site in Fn to the binding to α5β1 integrin.

Two different integrin headpiece conformations. A representative projection average from unliganded (top left) and liganded α5β1 headpiece (bottom left) was used to identify the best-correlating projections calculated from a 25 Å density map created from αVβ3 headpiece models (middle panels). The models are shown in CPK representation (right panels). The position of the β1 PSI domain, which is present in the α5β1 headpiece construct but is lacking in the αVβ3 crystal structure, can be clearly identified as an extra density present on the tip of the β tail (dotted ovals).



α5β1 headpiece in complex with Fn fragment. Projection averages are shown for Fn7-10 fragment alone (top left), α5β1 headpiece in the closed unliganded conformation (top middle), and α5β1 headpiece in complex with Fn7-10 (bottom). The projection averages of the α5β1/Fn7-10 complexes vary depending on the orientation of the bound ligand.
Top right: projection average of the α5β1 headpiece in complex with a SG/19 Fab. The binding site of SG/19 centers on the junction between the I-like and hybrid domains. The binding site of SG/19 clearly includes significant portions of both the I-like and hybrid domains. Thus a class of anti-β1 antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain, without directly interfering with ligand docking.

Surface-rendered density maps of the α5β1 headpiece in the unliganded closed conformation (left) and the ligand-bound open conformation (right). The unmodified headpiece segments of the αVβ3 crystal structure or the open αVβ3 model were manually fit into the 3D density map of the unliganded and ligand-bound α5β1 headpiece, respectively. Cα worm tracings for αV, β3 and Fn10 segments are colored in red, blue and white, respectively.


» Takagi et al. (2003) EMBO J. 22: 4607-4615.

» Luo et al. (2004) J. Biol. Chem.279: 27466-27471.