*Mechanisms of integration of cells and extracellular matrices by integrins

Abstract

While it is self-evident that all extracellular molecules are an integral part of a multicellular organism, it is paradoxical that they are often considered to be dissociated from cells. The reality is that a continuum of dynamic, bi-directional interactions links the intracellular environment through cell-surface receptors to multimolecular extracellular assemblies. These interactions not only control the behaviour of individual cells, but also determine tissue architecture. Adhesion receptor function is partly determined by an ability to tether the contractile cytoskeleton to the plasma membrane, but there is also evidence that integrin receptors modulate signalling events that are essential for cellular differentiation. A major challenge is now to integrate work at the atomic, molecular and cellular levels, and obtain holistic insights into the mechanisms controlling cell adhesion. In the present study, we review current knowledge of the molecular mechanisms employed by cells to integrate with the extracellular matrix. Two main topics are covered: the adaptation of integrin structure for bi-directional signalling and the integration of integrin signalling with other receptors.

Full text

822 Biochemical Society Transactions (2004) Volume 32, part 5
Mechanisms of integration of cells and
extracellular matrices by integrins
M.J. Humphries1, M.A. Travis, K. Clark and A.P. Mould
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, 2.205 Stopford Building,
Oxford Road, Manchester M13 9PT, U.K.
Abstract
While it is self-evident that all extracellular molecules are an integral part of a multicellular organism, it
is paradoxical that they are often considered to be dissociated from cells. The reality is that a continuum
of dynamic, bi-directional interactions links the intracellular environment through cell-surface receptors
to multimolecular extracellular assemblies. These interactions not only control the behaviour of individual
cells, but also determine tissue architecture. Adhesion receptor function is partly determined by an ability
to tether the contractile cytoskeleton to the plasma membrane, but there is also evidence that integrin
receptors modulate signalling events that are essential for cellular differentiation. A major challenge is now
to integrate work at the atomic, molecular and cellular levels, and obtain holistic insights into the mech-
anisms controlling cell adhesion. In the present study, we review current knowledge of the molecular
mechanisms employed by cells to integrate with the extracellular matrix. Two main topics are covered: the
adaptation of integrin structure for bi-directional signalling and the integration of integrin signalling with
other receptors.
Integrin structure, priming and activation
In mammals, 18 α-and8β-integrin genes encode poly-
peptides that combine to form 24 α,βheterodimeric receptors
[1]. Both subunits are non-covalently associated, type I
transmembrane proteins with large extracellular and mostly
short cytoplasmic domains. On the basis of their primary
structure, integrins fall into two subfamilies determined by
the presence or absence of a von Willebrand factor A-domain
in the αsubunit. Beginning with the X-ray crystal structure of
the isolated αM integrin A-domain in 1995 [2], recent years
have seen major advances in our understanding of integrin
tertiary structure. A step-change came with the solution of
the first structure of a full-length extracellular dimer (for
αVβ3) in 2001 [3], followed closely by the structure of
a ligand mimetic–receptor complex, formed by soaking an
RGD peptide into pre-existing αVβ3crystals[4].Theoverall
shape of the crystallized conformer of αVβ3 resembles a
large ‘head’ on two ‘legs’ (Figure 1). In contrast with models
derived from electron microscopic and biophysical analyses
[5], both legs are bent in the crystal structure. The head of the
integrin, which is the minimal fragment that contains ligand-
binding activity, comprises a β-propeller from the αsubunit,
an A-domain from the βsubunit (the βA-domain) and an
immunoglobulin ‘hybrid’ domain below the βA-domain.
The α-subunit leg of the integrin comprises three β-sandwich
domains, termed ‘thigh’, ‘calf1’ and ‘calf2’. The β-subunit
leg contains a plexin–semaphorin–integrin domain, which
Key words: activation, adhesion, conformation, cytoskeleton, integrin, signalling.
Abbreviations used: ECM, extracellular matrix; FA, focal adhesion; FB, fibrillar adhesion; FC, focal
complex; FN, fibronectin; LIBS, ligand-induced-binding sites; mAb, monoclonal antibody.
1To whom correspondence should be addressed (email martin.humphries@man.ac.uk).
is disordered in the structure, four EGF-like repeats and a
cystatin-like fold. The bend in the integrin is between thigh
and calf1 (in αV), and at the conjunction of the hybrid domain,
two EGF repeats and the PSI domain (in β3). Since 2001, there
have been no further integrin crystal structures reported, and,
therefore, the conformational diversity of the family remains
unclear.
In the RGD–αVβ3complexstructure,thepeptidelies
at the interface between the αVβ-propeller and the β3A-
domain, and the carboxyl group of the aspartate co-ordinates
a bivalent cation at a so-called MIDAS (metal-ion-dependent
adhesion site) [4]. A similar receptor–cation–ligand interac-
tion had been observed previously in a crystal structure of
acomplexbetweentheα2 A-domain and a triple-helical
collagen peptide [6]. While complementary conformations
of ligand acidic motifs and receptor cation-binding pockets
might determine the specificity of receptor-ligand binding,
there is also evidence that integrin ligands contain accessory
or ‘synergy’ sites for receptor binding. FN (fibronectin) is
the best-characterized ligand in this regard, and although it
has been suggested that receptor engagement of the synergy
site is transient [7], a combination of gain-of-function ana-
lyses with integrin αsubunit chimaeras, anti-functional
mAb (monoclonal antibody) epitope mapping, and structure
determination by small-angle X-ray scattering have led to
a detailed topological model for the FN–integrin complex
[8,9]. Solution of the structure of a macromolecular ligand–
integrin complex remains a priority for understanding
how integrins form stable interactions with ECM (extracel-
lular matrix) proteins.
The dynamic nature of integrin function, by which cells
use adhesion to sample their pericellular environment and
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Signalling Outwards and Inwards 823
Figure 1 Cartoon of the straightened form of an integrin which
lacks an αA-domain
The αsubunit is in red and the βsubunit in blue. The polypeptide
modules comprising both subunits and the major ligand-binding sites
are denoted. The sites of unbending are indicated by green arrows.
The conformational changes that take place during integrin priming and
activation are indicated by magenta arrows. These include movements
of α-helices within the βA-domain, a swing-out of the hybrid domain
away from the αsubunit, closing up of the interfaces between βA
and the hybrid domain, and the βA-domain and the β-propeller, and
leg/cytoplasmic domain separation.
respond by changing their position and differentiated state,
demands a highly responsive receptor structure (Figure 1).
Gross conformational changes in integrins have been detected
by a variety of biophysical and biochemical techniques
(reviewed in [10]), and much work has been performed on
activation-dependent binding of mAbs to integrins. A subset
of these mAbs recognize so-called LIBS (ligand-induced-
binding sites). It has been hypothesized that LIBS mAbs
stimulate integrin-ligand binding by displacing conform-
ational equilibria in favour of ligand-competent and ligand-
occupied forms of the integrin. Shape changes reported by
LIBS mAbs can also be triggered from the cytoplasmic
side of the plasma membrane [11], but the degree of simi-
larity between the changes induced from either side of the
plasma membrane is currently unclear. To separate these two
processes conceptually, we have proposed the term ‘priming’
to indicate the acquisition of ligand-binding ability by in-
tegrins and ‘activation’ to denote ligand-induced changes [12].
The availability of the crystal structure of αVβ3hasstimu-
lated structure–function analyses of integrin priming. The
relevance of the bend in the integrin dimer has received
much attention. Although it has been proposed that integrins
remain bent [13], several lines of evidence indicate that bent
integrins are inactive and extended integrins are primed. For
example, epitopes that become exposed on integrin priming,
and residues that restrain priming, are buried in the bent knee
of β2 integrins [14], and locked-bent integrins containing
engineered disulphide bonds have very low affinity for ligand
[15]. Furthermore, predominantly bent conformers of β3
integrins are observed by electron microscopy in the presence
of Ca2+, whereas extended conformers are found in Mn2+
[15].
It is now well established that leg separation underpins
both priming and activation of integrins. Thus some stimu-
latory mAb epitopes located in the leg regions are exposed
by activating cytoplasmic domain mutations [16], forced
association of the membrane-proximal regions of the α-and
β-subunit cytoplasmic domains with an engineered coiled-
coil constrains the integrin in an inactive state [16], unclasping
these constrained integrins leads to a spatial separation of the
legs [17], and fluorescence resonance energy transfer studies
show that integrin cytoplasmic domains are close to each
other in the resting state, but undergo spatial separation
during both priming and activation [18].
Using LIBS mAbs as activation state reporters, we have
shown that movement of both the α1andα7 helices of the
β1 A-domain contributes to priming [19,20]. Shape changes
in these elements had previously been observed in αA-domain
crystal structures. βA-domain conformational changes are
coupled with a swing-out of the underlying hybrid domain,
as activating mutations in the α7 helix cause increased
exposure of LIBS mAb epitopes obscured at a hybrid domain-
β-propeller contact [20]. Hybrid domain swing-out has also
been observed using electron microscopy [15] and small-angle
X-ray scattering [9]. The extent to which different ligands
stabilize different integrin conformers and in turn transduce
agonistic effects across the plasma membrane, and the
structural mechanisms that couple extension, leg separation
and βA-domain conformational change, are not clear, but
will underpin our future understanding of how integrins
work as dynamic receptors.
Integrin-receptor cross-talk and signalling
assemblies in migrating cells
Ligand engagement by integrins initiates signalling responses
that include transduction of mechanical force to the cyto-
skeleton and spatial compartmentalization of signalling
complexes. There is now evidence for alterations in the fluxes
of almost all known signalling pathways subsequent to integ-
rin engagement, suggesting that adhesion receptor function
is integrated with other receptor systems. However, the
mechanisms responsible for converting integrin ligation into
an efficacious signal are not known. A hierarchy of recruit-
ment of signalling and cytoskeletal molecules to integrins
has been demonstrated using immunocytochemical ap-
proaches [21], but as yet evidence for ligand/agonist-specific
differences in the composition and/or organization of this
hierarchy is lacking.
Early studies employing receptor chimaeras demonstrated
that some integrin-specific functions were conferred by the
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824 Biochemical Society Transactions (2004) Volume 32, part 5
Figure 2 Indirect immunofluorescence analysis of human fibroblasts showing FAs and FBs
Cells were fixed and stained for β1integrin in a primed conformation (9EG7, green; left panels) and for α5integrin (using
the conformation-dependent mAb SNAKA51, red; middle panels). A merged image is shown on the right. While 9EG7 stains
both focal (white triangles) and FBs (yellow triangles), SNAKA51 localizes specifically to FBs, indicating a conformational
change in α5 during the transition from focal to FB.
cytoplasmic domain. On the basis of an NMR analysis of
a complex between recombinant forms of the αIIb and β3
cytoplasmic domains, it appears that both subunits are largely
unstructured, but are held together by a juxtamembrane
interaction of two α-helical regions [22]. A variety of ap-
proaches including affinity chromatography, equilibrium gel
filtration, IP blotting, synthetic peptide binding and two-
hybrid analysis have been used to identify molecules that
bind integrins (reviewed in [23]). Interacting proteins include
cytoskeletal components such as talin, α-actinin and filamin,
adapters such as paxillin, Rack-1, ICAP-1 and β3-endonexin
and kinases such as Src.
A central role in integrin signalling has been established
for talin. Genetic or translational inhibition of talin disrupts
the cytoskeleton and cell adhesion with a similar phenotype
to integrin deletions, whereas overexpression of an integrin-
binding fragment of talin induces priming triggered by mul-
tiple pathways [24] and causes structural perturbation of the
association between the integrin cytoplasmic domains [22].
Crystallization of a talin fragment–integrin β3 cytoplasmic
peptide chimaera has revealed a PTB domain-like mode of
binding that may be common to several integrin effectors
[25,26]. Since talin binds vinculin, actin and FAK, it has
the potential to co-ordinate the recruitment of many key
integrin-signalling molecules. Furthermore, talin binds and
activates PIPKIγ, which leads to increased PIP2production
and in turn activates several cytoskeletal proteins.
Integrins undergo cis interactions with a number of
different receptors, and thereby spatially regulate diverse
signalling responses (reviewed in [27]). Direct extracellular
associations with members of the TM4 family, the urokinase
receptor uPAR and CD47 have been established, as has
indirect co-clustering with several growth factor and cytokine
receptors. Cross-talk at the level of signalling occurs with
semaphorins, ephrins and syndecans. It is remarkable that
most ECM molecules possess both integrin- and syndecan-
binding sites, and a clear synergistic relationship exists
between these two families. Thus α5β1-dependent FA (focal
adhesion) formation on FN requires engagement of, and
signalling through, a syndecan co-receptor [28,29].
In spread fibroblasts adherent to FN in vitro, adhesion
signalling complexes are distributed focally rather than dif-
fusely, and are manifested as asymmetric patches, flecks and
stripes. These contact points are found all over the ventral
surface, and are usually associated with the contractile
polymers of the cytoskeleton. Detailed morphological and
functional analyses have defined three major forms of
adhesion contact: FC (focal complexes), FA and FB (fibril-
lar adhesions; Figure 2; reviewed in [30]). These contacts
reflect different stages of interaction of cells with the ECM,
and each is formed and disrupted in a cyclical manner as
cells translocate. Initially, FC form at the posterior edge of
ruffling membrane, where they anchor the short filopodial
struts and lamellipodial meshes of actomyosin that mediate
membrane protrusion. When protrusion ceases or the lamel-
lipodium retracts, FC transform into larger FA, which pro-
vide a more robust anchorage through transcellular acto-
myosin-containing stress fibres. In turn, FA evolve into FB,
which are the major sites of FN matrix deposition [31,32].
Adhesion contact-like structures have been observed in vivo
in smooth-muscle cell plaques and myotendinous junctions
and, recently, in embryonic three-dimensional ECM [33],
thereby validating the use of cell cultures for analysis.
The members of the Rho family of small GTPases are
recognized to play a central role in modulating the actin cyto-
skeleton (reviewed in [34]). Cdc42 and Rac1 promote FC
formation and membrane protrusions at the leading edge
through filopodia and lamellipodia, whereas RhoA mediates
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Signalling Outwards and Inwards 825
FA formation and causes retraction of the trailing edge. Co-
ordination between these pathways involves activation of
p190RhoGAP, which may permit membrane protrusion by
suppressing RhoA activity. Subsequently, the FC to FA
transition is dependent on RhoA and ROCK-mediated
myosin II contractility through phosphorylation of myosin
light chain. Integrin occupancy controls the activity of Rho
family GTPases and regulates their translocation to plasma
membrane microdomains [35,36], but the molecular links
between integrins and Rho GTPases are not understood.
At least 50 different proteins have been localized to FA
[37], but analyses of the differences between FC, FA and FB
are still in their infancy. FC and FA share many components,
but FC tend to lack zyxin and tensin [38], whereas FB contain
α5β1andtensin,butlackmostoftheotherFAcomponents,
including αVβ3 [32]. Thus while compositional changes do
take place as adhesion contacts mature, the signalling events
that regulate these transitions are not well understood. In
particular, how integrin activation with ligands in the ECM
modulates the assembly, maturation and turnover of different
adhesion contacts will be an important area of future study.
Preliminary evidence from our laboratory (Figure 2) suggests
that integrin conformation can vary with location in the cell,
implying that bi-directional communication is important for
co-ordinating cell–matrix integration.
This work was supported by grants from the Wellcome Trust.
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