Download Studies of focal adhesion assembly

Iain D. Campbell
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
Abstract
Recent studies of some proteins involved in the formation of focal adhesions are described. These include
fibronectin, integrins, talin, Dok1 and filamin. Emphasis is placed on features that facilitate regulated
assembly of complexes; these include a modular construction and flexible regions that provide interaction
sites whose affinity can be adjusted by conformational masking and phosphorylation.
Introduction
Cell migration depends on the formation of transient
self-assembling complexes called focal adhesions. Integrin
adhesion receptors are central components of these complexes
since they span the cell membrane to link the ECM
(extracellular matrix) with numerous intracellular molecules,
including the cytoskeleton [1,2]. A schematic view of some
of these components is shown in Figure 1. A more complete
description of the interaction network involved can be found
in Zaidel-Bar et al. [3].
Focal adhesion complexes are formed by proteins with a
wide range of functions, including adhesion receptors for
the ECM, such as integrins, signalling molecules, such as
Src and FAK (focal adhesion kinase), and adaptors that
facilitate intracellular complex formation, such as paxillin.
Focal adhesions are dynamic, but are formed by multiple
specific protein–protein interactions. Some general structural
features of the component proteins can be discerned: (i)
they are usually modular, constructed from a relatively
limited repertoire of folded domains of similar structure [4];
(ii) the domains are often connected by intrinsically unfolded
linker regions that provide binding sites for other proteins;
and (iii) protein–protein interactions can be modulated by
phosphorylation and by changes in site exposure caused, for
example, by cell-mediated tension. A multidomain structure
with flexible joining regions appears to be very well suited for
providing a variety of binding sites in different conditions.
I will briefly illustrate some of these general features with
examples from our own recent work.
Fibronectin
Fibronectin (Fn) is a large dimeric glycoprotein found in
relatively high concentrations in plasma as well as the
ECM [5]. Fn performs a wide range of functions, including
provision of binding sites for collagen and integrins. It is
Key words: Dok1, fibronectin, filamin, focal adhesion, integrin, talin.
Abbreviations used: Dok1, downstream of kinase 1; ECM, extracellular matrix; FAK, focal
adhesion kinase; FERM, 4.1/ezrin/radixin/moesin; Fn, fibronectin; Fn30, Fn N-terminal 30 kDa
fragment; PTB, phosphotyrosine-binding.
1
email [email protected]
Biochem. Soc. Trans. (2008) 36, 263–266; doi:10.1042/BST0360263
composed of three different domain types: Fn1, Fn2 and
Fn3. High-resolution structures of many of these component
domains and domain combinations have been available for
some time [6]; the main remaining uncertainties are associated
with how Fn forms complexes with its various partners. Fn
has conformational flexibility that exposes different binding
sites in different conditions; for example, it is much more
active in binding integrins when it is in the ECM than when
in its soluble plasma form.
The process by which Fn forms insoluble fibrillar networks
in the ECM is still not well understood. Hidden or ‘cryptic’
sites found in Fn3 domains have been hypothesized to
function as nucleation points, thereby initiating fibrillogenesis [7]. The requirement for integrin function has led
to the suggestion that exposure of these sites arises from
tension-mediated mechanical rearrangement of Fn3 domains.
We showed recently that an interaction complex is formed
between the first two Fn3 domains, 1 Fn3 and 2 Fn3 [8],
although the two domains are connected covalently by a
long flexible linker. Tension-induced separation of the two
domains could thus induce a considerable lengthening of
this domain pair without any required domain unfolding.
For recent papers on the controversial topic of the role
of domain unfolding, see, for example, [9,10]. The weak,
but specific, interdomain interaction maintains 1 Fn2 and
2
Fn3 in a closed conformation, a form that associates
only weakly with the Fn N-terminal 30 kDa fragment
(Fn30). It was observed, however, that disruption of this
interdomain interaction by amino acid substitutions, to
make an ‘open’ form of 1 Fn3–2 Fn3, dramatically enhances
association with Fn30. Truncation analysis of 1 Fn3–2 Fn3
reveals that the interdomain linker is also necessary for this
interaction between 1 Fn3–2 Fn3 and Fn30 [8]. This example
illustrates how cryptic sites could be exposed by mechanical
tension, followed by fibril formation arising from multiple
interactions between different regions of Fn.
As well as having important structural properties, Fn
signals to intracellular spaces via adhesion receptors. The
best-characterized signalling sequence in Fn is the RGD
(Arg-Gly-Asp) sequence in 10 Fn3 [11]. Formation of an
RGD–integrin complex results in a range of downstream
effects, including phosphorylation of FAK. We have shown
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Studies of focal adhesion assembly
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Biochemical Society Transactions (2008) Volume 36, part 2
Figure 1 Schematic view of a focal adhesion complex
The proteins are drawn so as to indicate their modular nature and some
of the flexible linker regions joining them. The ECM at the top of
the diagram is made up of molecules such as Fn (a multidomain
protein constructed from three main module types: Fn1, Fn2 and
Fn3; the segment shown only contains Fn3 domains). Integrins,
membrane-spanning αβ heterodimers [2], bind to ECM components
using their extracellular regions. The short flexible intracellular tail
regions of integrins are linked to the actin cytoskeleton by talin, which
relationships of various recombinant IGD-containing
Fn fragments showed that even conservative mutations
in the IGD motif have severely reduced potency, although
the structure of the fragments remains essentially the same
[15]. The wide range of potency observed in different contexts
(for example, longer Fn fragments are found to have less
IGD activity than shorter ones) can be partly attributed
to exposure of cryptic sites in some Fn conformations
[15].
has a cloverleaf-like FERM head domain and a rod-like actin-binding
domain [17]. Filamin A, a large two-armed dimer, each arm of which
has an actin-binding domain and 24 Ig-like domains, cross-links actin
filaments as well as binding to integrin tails [22]. Important other
associated molecules include kinases [e.g. Src, PIPKIγ (phosphatidylinositol phosphate kinase type Iγ ) and FAK] and phosphatases. Paxillin
and vinculin facilitate the formation of the dynamic complexes by
cross-linking several different proteins.
previously some examples of structure-induced modulation
of integrin–Fn-binding affinity: 8 Fn3 promotes integrin α5β1
binding via stabilization of the 9 Fn3 domain [12], and the
interdomain tilt angle determines the integrin-dependent
function of 9 Fn3 and 10 Fn3 [13].
Regions other than 9 Fn3–10 Fn3 have also been implicated
in signalling. A truncated form of Fn was found to promote
cell migration via IGD (Ile-Gly-Asp) motifs present in Fn1
modules [14]. The receptor for this induced effect is still not
known, but recent investigations of the structure–function
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Authors Journal compilation Integrin cytoplasmic tails and their role in
affinity modulation
Integrin cytoplasmic domains (tails) are relatively short; ∼50
amino acids compared with ∼1000 in integrin extracellular
domains. In the absence of protein ligands, these tails are
largely unstructured [16]; this means that, although relatively
few amino acids are involved, they expose a relatively large
accessible binding surface. Regulation of integrin affinity
(activation) is essential in normal and pathological cell
migration. Talin, a large intracellular molecule that binds
integrin tails and actin [17], has been shown to be a key
intracellular regulator of integrin activation [18]. Binding
of the PTB (phosphotyrosine-binding) subdomain from
the talin FERM (4.1/ezrin/radixin/moesin) head domain is
sufficient to activate β3 integrins [18]. The structure of a
complex between the talin PTB domain and the integrin β3
tail was determined recently [19]; the membrane-proximal
region of the β3 tail forms a well-defined helix that makes
specific contacts with the talin PTB domain. Structurebased mutagenesis also showed that these specific membrane
proximal contacts with the β tail are required for integrin
activation and that they are unique to the talin PTB domain
[19].
Integrin tails also bind other PTB domains, but these
do not usually activate integrins. Indeed, binding of some
PTB domains appears to reduce integrin activation [19].
We have shown recently how the PTB domain of Dok1, a
downstream of kinase signalling protein [20], might provide
further regulation of integrin activation by competition
with talin. The structure and binding affinities of Dok1–β3
complexes were analysed using crystallography and NMR
and compared with those of talin [21]. The unphosphorylated
integrin tail was shown to have a 3-fold preference for
the talin PTB domain over that of Dok1. Phosphorylation
of Tyr747 , however, enhanced binding to Dok1 ∼400-fold
(from 14.3 mM to 37 μM), while the binding affinity to the
talin PTB domain decreased ∼2-fold (from 3.5 mM to
6.5 mM). Phosphorylation of Tyr747 thus switches the binding
preference of the integrin tail from talin to the Dok1. Since
Dok1 interacts exclusively with the canonical NPXY motif in
the β3 integrin tail, and not the membrane proximal region,
its binding does not activate integrins. Phosphorylation of
Tyr747 thus provides a switch that promotes the inactive state
of integrins by increasing the binding of a non-activating PTB
domain.
Biochemical Society Annual Symposium No. 75: Structure and Function in Cell Adhesion
Filamin
References
Filamins are large actin-cross-linking proteins that connect
membrane-spanning receptors to the cytoskeleton [22].
The ability of integrins to transmit biochemical signals
and mechanical force across cell membranes depends on
interactions with the actin cytoskeleton. In a high-resolution
structure, we showed that the integrin β tail forms an
extended β-strand that interacts with β-strands C and D
of the 21st immunoglobulin-like domain from filamin A
(21 IgFln) [23]. This filamin-binding site on β tails partially
overlaps the talin-binding site; filamin and talin also compete
for binding to integrin tails. Such competition is again a
possible mechanism for modulating the degree of talindependent integrin activation. Phosphothreonine-mimicking
mutations of tails inhibited binding of filamin, but not talin,
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In a follow-up structural study of a filamin fragment containing three Ig-like domains (19−21 IgFln), we found that they
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with the CD face of 21 IgFln, the same binding site as that
occupied by integrin tails. Disruption of this 20 IgFln–21 IgFlnmasking interaction enhanced filamin binding to integrin
β tails. Structural and functional analysis of other IgFln
domains suggests that this kind of auto-inhibition by adjacent
IgFln domains may also occur with 18 IgFln–19 IgFln. This
masking effect could explain the observed increased integrin
binding in filamin splice variants [25] and the affinity could
be modulated by tension-induced removal of the masking
strand.
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Conclusions
I have illustrated briefly how various proteins in focal
adhesions form complexes, using examples from our own
recent work. Most of the observed interaction affinities are
relatively weak (1–100 μM), but they are specifically tuned
to perform their various functions; the various protein–
protein interactions are often regulated, for example, by
phosphorylation and induced conformational changes. Some
emphasis has been placed on possible tension-induced
exposure of cryptic sites, but proteolysis is another possible
mechanism [26]. Intrinsically disordered regions in proteins
seem to play an especially important role in regulated
networks of protein–protein interactions [27].
I thank the Wellcome Trust, BBSRC (Biotechnology and Biological
Sciences Research Council) and the NIH (National Institutes of
Health) Cell Migration consortium for financial support. I also
acknowledge the enormous contributions made by my co-workers
and collaborators whose names appear in the given references.
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25 Travis, M.A., van der Flier, A., Kammerer, R.A., Mould, A.P., Sonnenberg,
A. and Humphries, M.J. (2004) Interaction of filamin A with the integrin
β7 cytoplasmic domain: role of alternative splicing and phosphorylation.
FEBS Lett. 569, 185–190
26 Schenk, S. and Quaranta, V. (2003) Tales from the crypt[ic] sites of the
extracellular matrix. Trends Cell Biol. 13, 366–375
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Authors Journal compilation 27 Dunker, A.K., Cortese, M.S., Romero, P., Iakoucheva, L.M. and Uversky,
V.N. (2005) Flexible nets: the roles of intrinsic disorder in protein
interaction networks. FEBS J. 272, 5129–5148
Received 27 December 2007
doi:10.1042/BST0360263