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
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
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.
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
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.