It goes without saying that the cellular plasma membrane effectively creates a barrier
between the inside (intracellular area) and outside (extracellular area) of the cell it
defines. In order for the cell to sense and respond to its environment (including other
cells and the supporting structures that comprise the extracellular matrix [ECM]) and for
the environment to influence cell function (including cell growth and movement),
bidirectional signaling across the plasma membrane has to be mediated by receptors and
other structures. About two decades ago, it became widely appreciated that many of the cell
surface receptors that mediate cell–cell and cell–ECM interactions were structurally and
functionally related, and the term “integrins” was coined to reflect the capacity of
members of this family to integrate the extracellular and intracellular environment (Hynes
1987). Integrin-mediated interactions are vital to the maintenance of normal cell
functioning because of their ability to mediate inside-out (intracellular to extracellular)
and outside-in (extracellular to intracellular) signaling. Integrin dysfunctions are
associated with numerous human disorders such as thrombosis, atherosclerosis, cancer, and
chronic inflammatory diseases. Despite a total of nearly 30,000 integrin-related articles
in the literature, intensive effort—more than 200 articles per month—continues to focus on
understanding the roles of integrins in both physiological and pathological processes.
The Integrin Family
The integrin family comprises 20 or more members that are found in many animal species,
ranging from sponges to mammals (Hynes 2002). They consist of two distinct, associated
subunits (noncovalent heterodimers), where each subunit (α, β) consists of a single
transmembrane domain, a large extracellular domain of several hundred amino acids (composed
of multiple structural domains), and typically, a small cytoplasmic domain of somewhere
between 20–70 residues (Figure 1). The extracellular domains bind a wide variety of
ligands, whereas the intracellular cytoplasmic domains anchor to cytoskeletal proteins. In
this manner, the exterior and interior of a cell are physically linked, which allows for
bidirectional transmission of mechanical and biochemical signals across the plasma
membrane, and leads to a cooperative regulation of cell functions, including adhesion,
migration, growth, and differentiation. A central topic in the integrin research over the
past decade has been the mechanism of inside-out activation (Liddington and Ginsberg 2002).
In their resting state, integrins normally bind the molecules that activate them with low
affinity. Upon stimulation, a cellular signal induces a conformational change in the
integrin cytoplasmic domain that propagates to the extracellular domain. Integrins are
transformed from a low- to a highaffinity ligand binding state. Such inside-out regulation
of integrin affinity states is distinct from the outside-in signaling observed upon
activation of most other transmembrane receptors (e.g., growth factor–growth factor
receptor interactions), including integrins. The inside-out signaling protects the host
from excessive integrin-mediated cell adhesion, which could, for example, lead to
spontaneous aggregation of blood cells and have profound pathological consequences.
The Heads and Tails of Inside-Out Signaling
Mutational studies provided the initial hints that disruption of the non-covalent clasp
between α and β cytoplasmic tails is clearly the event within the structure of the integrin
that initiates inside-out signaling. Point mutations in the α and β cytoplasmic tails that
are near the membrane or deletion of either region result in constitutive activation of the
receptor (O'Toole et al. 1991, 1994; Hughes et al. 1995). Mutating a single specific
residue in the cytoplasmic tail of either subunit led to integrin activation, but a double
mutation, which would have allowed retention of a salt bridge between the subunits, did not
(Hughes et al. 1996)—suggesting that integrin inside-out activation is dependent upon
regulation of the interaction between the two subunits. In support of this hypothesis,
peptides corresponding to α and β cytoplasmic tails have been shown to interact with each
other (Haas and Plow 1996). Since these original observations, there has been an intensive
effort to understand the mechanism for regulation of integrin activation by the cytoplasmic
region (for a recent review, see Hynes 2002). On the road toward this goal, Ginsberg and
colleagues discovered that the head domain of a cytoskeletal protein—talin—plays a key role
in binding to integrin β cytoplasmic tails and inducing integrin activation (Calderwood et
al. 1999). Many other intracellular proteins bind to the α and β cytoplasmic tails (Liu et
al. 2000), but the importance of talin in integrin activation is particularly convincing
since it has been confirmed by multiple laboratories (Vinogradova et al. 2002; Kim et al.
2003; Tremuth et al. 2004) using various methods including overexpression and gene
knockdown (siRNA) approaches (Tadokoro et al. 2003). In 2001, Springer and coworkers
provided evidence for a model by which separation of the C-terminal portions of the α and β
subunits results in inside-out activation. They showed that replacement of the
cytoplasmic-transmembrane regions by an artificial linkage between the tails inactivates
the receptor, whereas breakage of the clasp activates the receptor (Lu et al. 2001; Takagi
et al. 2001). Shortly thereafter, the model gained direct and strong experimental support
from a structural analysis in which the membrane-proximal helices of the two subunits were
found to clasp in a weak “handshake” that could be disrupted by talin or constitutively
activating mutations (Vinogradova et al. 2002). The model has been further verified by
other biophysical studies (Kim et al. 2003) and extended to other integrins (Vinogradova et
al. 2004). Since the membrane-proximal regions of integrin α and β cytoplasmic tails are
highly conserved, the generalization of this signaling mechanism to all integrins was to be
anticipated. A dynamic image of how such cytoplasmic unclasping occurs at the membrane
surface can now be modeled (Figure 1) (Vinogradova et al. 2004).
Straightening Out the Outside
On the extracellular side, ground-breaking insights were provided when the crystal
structure of the extracellular domain of integrin α
v β
3 (the nomenclature identifies the particular α and β subunits) was
determined (Xiong et al. 2001). In addition to the exquisite structural details, the
overall conformation was surprisingly bent (Figure 1), which contrasted with structures
revealed by the earlier electron micrographic studies that showed an extended, stalk-like
structure (Weisel et al. 1992). Springer and coworkers used a series of
biochemical/biophysical experiments to suggest that the bent structure represents an
inactive form of integrin (Takagi et al. 2002), whereas activation induces a switchblade
shift that converts the bent form to the extended form (Figure 1). A molecular picture has
emerged for integrin insideout activation where a cellular signal induces the
conformational change of talin exposing its head domain allowing it to bind to the integrin
β cytoplasmic tail. This interaction unclasps the complex between the cytoplasmic tails,
which then allows a conformational shift in the extracellular domain from a bent to a more
extended form for high-affinity ligand binding (Figure 1) (Takagi et al. 2002).
The activated integrins may then undergo clustering whereby the transmembrane domain of
each type of subunit (the α or β) interacts with itself—called homotypic oligomerization of
the transmembrane domains (Figure 1) (Li et al. 2003). Ligand occupancy and receptor
clustering initiates outside-in signaling that, in turn, regulates a variety of cellular
responses (see below). The three steps in Figure 1 occur as part of a dynamic equilibrium,
and perturbation of any step can shift the equilibrium, leading to transient, partial, or
permanent integrin activation/inactivation depending on the extent of perturbation. For
example, deletion of aIIb cytoplasmic tail completely removes the clasp and permanently
activates the receptor (O'Toole et al. 1991), whereas a particular disease mutation may
only impair the clasp and partially activate the receptor (Peyruchaud et al. 1997). While
the model in Figure 1 is based on direct structural evidence for the cytoplasmic face
(Vinogradova et al. 2002; Kim et al. 2003) and the extracellular domain (Takagi et al.
2002), the changes in the transmembrane region remained speculative. In this issue of
PLoS Biology , Luo et al. (2004) provide what is, to our knowledge, the
first experimental evidence for the transmembrane domain separation, an event suggested by
the model shown in Figure 1. By selectively altering the residues that can interact with
one another, the authors defined a specific transmembrane domain interface in resting α
IIb β
3 and showed that this interface is lost upon activation of this
integrin. Backed by extensive structural and biochemical data on the integrin
cytoplasmic/extracellular domains, this transmembrane domain study takes the next vital
step toward a more complete understanding of the unclasping mechanism for integrin
activation. Although the energy required for lateral separation of the transmembrane
domains in membrane appears to be high, the third step in Figure 1 (clustering via
transmembrane domain oligomerization) may compensate for it.
Filling in the Pieces
Despite the molecular level of our understanding of integrin activation, a number of key
questions remain unresolved. Although we know that the membrane-proximal clasp on the
integrin cytoplasmic face controls the integrin activation, the distal side of either the α
or β cytoplasmic tails may also play a role in integrin activation, since other mutations
indicate that the C-terminal membrane distal region is important in regulating integrin
activation via a mechanism that is yet unknown. Thus, the picture for the cytoplasmic
face-controlled inside-out activation may be substantially more complicated than specified
in Figure 1. There may exist other factors, such as negative regulators, in cells that bind
to the cytoplasmic tails or their complex, and control the conformational change required
for integrin activation. Also, there may be pathways other than the talin-mediated one that
lead to integrin activation. Structures of the integrin cytoplasmic face bound to talin and
the many other proteins known to bind to the cytoplasmic tails of integrins will
undoubtedly provide further insights. In the transmembrane region, although there is ample
evidence for heterodimeric transmembrane domain association (Adair and Yeager 2002;
Schneider and Engelman 2003; Gottschalk and Kessler 2004; Luo et al. 2004) and dissociation
upon integrin activation (Luo et al. 2004), a definitive structural view is missing. Some
studies have proposed that homo-oligomerization is essential for inducing integrin
activation (Li et al. 2003). However, the data provided by Luo et al. do not appear to
support this model. On the extracellular side, while the C-terminal unclasping and
separation of the cytoplasmic and transmembrane regions appears to relieve the structural
constraint and may allow the unbending of the extracellular domain to attain the
high-affinity ligand binding state (Takagi et al. 2002), a thorough molecular understanding
of this process awaits high resolution structures of the intact receptor in inactive and
active forms.
What About Outside-In?
Upon the inside-out activation, integrins bind to specific extracellular matrix
proteins. However, for the integrins to grip tightly to the extracellular matrix to mediate
cell adhesion and migration, the integrin cytoplasmic domains must be anchored to the
cytoskeleton (Giancotti and Ruoslahti 1999). This is achieved by “outside-in” signaling,
i.e., when an integrin binds to the extracellular ligand, it clusters with other bound
integrins, resulting in the formation of highly organized intracellular complexes known as
focal adhesions that are connected to the cytoskeleton. The focal adhesions incorporate a
variety of molecules, including the cytoplasmic domains of the clustered integrins,
cytoskeletal proteins, and an extensive array of signaling molecules. The high local
concentrations of these molecules facilitate cascades of downstream intracellular responses
via protein–protein interactions, which are linked to the cytoskeleton as well as to
complex intracellular signaling networks. Although many intracellular components involved
in outsidein signaling have been identified, and much has been learned about various
signaling pathways involved in outside-in signaling (Giancotti and Ruoslahti 1999), a
molecular view of how the various events occur in time and space is still very uncertain.
In particular, little structural insight has been obtained for early outside-in
intracellular events following ECM–integrin binding, e.g., upon ECM engagement. How is the
integrin cytoplasmic domain connected to the cytoskeleton? How is this connection regulated
during cell adhesion and migration? The next wave of structural information may provide
insights into these important and fertile areas of investigation.
