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1.
Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815-1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843-1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction.
Structured summary
MINT-7722300, MINT-7760951: Talin-1 (uniprotkb:P26039) and Vinculin (uniprotkb:P12003) bind (MI:0407) by molecular sieving (MI:0071) 相似文献2.
Patel B Gingras AR Bobkov AA Fujimoto LM Zhang M Liddington RC Mazzeo D Emsley J Roberts GC Barsukov IL Critchley DR 《The Journal of biological chemistry》2006,281(11):7458-7467
The talin rod contains approximately 11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, talin failed to compete for binding of the vinculin Vd1 domain to an immobilized talin polypeptide containing a constitutively active VBS. However, talin did bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a K(d) of approximately 9 mum. Interestingly, Vd1 binding exposed a trypsin cleavage site in the talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, talin 482-889 binds Vd1 with high affinity (K(d) approximately 0.14 mum), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact talin. In support of this, hemagglutinin-tagged talin 482-889 localized efficiently to focal adhesions, whereas 482-655 did not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold bound Vd1 much less well than wild type. We conclude that the stability of the helical bundles that make up the talin rod is an important factor determining the activity of the individual VBSs. 相似文献
3.
Alana R. Cowell Guillaume Jacquemet Abhimanyu K. Singh Lorena Varela Anna S. Nylund York-Christoph Ammon David G. Brown Anna Akhmanova Johanna Ivaska Benjamin T. Goult 《The Journal of cell biology》2021,220(9)
Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain–containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif–containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly. 相似文献
4.
Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle 总被引:1,自引:0,他引:1
Papagrigoriou E Gingras AR Barsukov IL Bate N Fillingham IJ Patel B Frank R Ziegler WH Roberts GC Critchley DR Emsley J 《The EMBO journal》2004,23(15):2942-2951
The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin-binding site (VBS), folds into a five-helix bundle whereas talin 656–789 is a four-helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five-helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions. 相似文献
5.
Benjamin T. Goult Neil Bate Nicholas J. Anthis Kate L. Wegener Alexandre R. Gingras Bipin Patel Igor L. Barsukov Iain D. Campbell Gordon C. K. Roberts David R. Critchley 《The Journal of biological chemistry》2009,284(22):15097-15106
Talin is a large flexible rod-shaped protein that activates the integrin
family of cell adhesion molecules and couples them to cytoskeletal actin. It
exists in both globular and extended conformations, and an intramolecular
interaction between the N-terminal F3 FERM subdomain and the C-terminal part
of the talin rod contributes to an autoinhibited form of the molecule. Here,
we report the solution structure of the primary F3 binding domain within the
C-terminal region of the talin rod and use intermolecular nuclear Overhauser
effects to determine the structure of the complex. The rod domain (residues
1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into
a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3
(residues 316–326) interacts with a cluster of acidic residues in the
middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain
competes with β3-integrin tails for binding to F3, and the structure of
the complex suggests that the rod is also likely to sterically inhibit binding
of the FERM domain to the membrane.The cytoskeletal protein talin has emerged as a key player, both in
regulating the affinity of the integrin family of cell adhesion molecules for
ligand (1) and in coupling
integrins to the actin cytoskeleton
(2). Thus, depletion of talin
results in defects in integrin activation
(3), integrin signaling through
focal adhesion kinase, the maintenance of cell spreading, and the assembly of
focal adhesions in cultured cells
(4). In the whole organism,
studies on the single talin gene in worms
(5) and flies
(6) show that talin is
essential for a variety of integrin-mediated events that are crucial for
normal embryonic development. In vertebrates, there are two talin
genes, and mice carrying a talin1 null allele fail to complete
gastrulation (7).
Tissue-specific inactivation of talin1 results in an inability to activate
integrins in platelets (8,
9), defects in the
membrane-cytoskeletal interface in megakaryocytes
(10), and disruption of the
myotendinous junction in skeletal muscle
(11). In contrast, mice
homozygous for a talin2 gene trap allele have no phenotype, although
the allele may be hypomorphic
(12).Recent structural studies have provided substantial insights into the
molecular basis of talin action. Talin is composed of an N-terminal globular
head (∼50 kDa) linked to an extended flexible rod (∼220 kDa). The
talin head contains a
FERM2 domain (made up
of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0
(2). Studies by Wegener et
al. (30) have shown how
the F3 FERM subdomain, which has a phosphotyrosine binding domain fold,
interacts with both the canonical NPXY motif and the
membrane-proximal helical region of the cytoplasmic tails of integrin
β-subunits (13). The
latter interaction apparently activates the integrin by disrupting the salt
bridge between the integrin α- and β-subunit tails that normally
keeps integrins locked in a low affinity state. The observation that the F0
region is also important in integrin activation
(14) may be explained by our
recent finding that F0 binds, albeit with low affinity,
Rap1-GTP,3 a known
activator of integrins (15,
16). The talin rod is made up
of a series of amphipathic α-helical bundles
(17–20)
and contains a second integrin binding site (IBS2)
(21), numerous binding sites
for the cytoskeletal protein vinculin
(22), at least two actin
binding sites (23), and a
C-terminal helix that is required for assembly of talin dimers
(20,
24).Both biochemical (25) and
cellular studies (16) suggest
that the integrin binding sites in full-length talin are masked, and both
phosphatidylinositol 4,5-bisphosphate (PIP2) and Rap1 have been implicated in
exposing these sites. It is well established that some members of the FERM
domain family of proteins are regulated by a head-tail interaction
(26); gel filtration,
sedimentation velocity, and electron microscopy studies all show that talin is
globular in low salt buffers, although it is more elongated (∼60 nm in
length) in high salt (27). By
contrast, the talin rod liberated from full-length talin by calpain-II
cleavage is elongated in both buffers, indicating that the head is required
for talin to adopt a more compact state. Direct evidence for an interaction
between the talin head and rod has recently emerged from NMR studies by Goksoy
et al. (28), who
demonstrated binding of 15N-labeled talin F3 to a talin rod
fragment spanning residues 1654–2344, an interaction that was confirmed
by surface plasmon resonance (Kd = 0.57 μm)
(28). Chemical shift data also
showed that this segment of the talin rod partially masked the binding site in
F3 for the membraneproximal helix of the β3-integrin tail
(28), directly implicating the
talin head-rod interaction in regulating the integrin binding activity of
talin. Goksoy et al.
(28) subdivided the F3 binding
site in this rod fragment into two sites with higher affinity
(Kd ∼3.6 μm; residues 1654–1848)
and lower affinity (Kd ∼78 μm; residues
1984–2344). Here, we define the rod domain boundaries and determine the
NMR structure of residues 1655–1822, a five-helix bundle. We further
show that this domain binds F3 predominantly via surface-exposed residues on
helix 4, with an affinity similar to the high affinity site reported by Goksoy
et al. (28). We also
report the structure of the complex between F3 and the rod domain and show
that the latter masks the known binding site in F3 for the β3-integrin
tail and is expected to inhibit the association of the talin FERM domain with
the membrane. 相似文献
6.
7.
FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for studying protein folding mechanisms and for testing of the accuracy of computational models. The suitability of this protein to describe the folding of naturally occurring α/β proteins has recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Using molecular dynamics simulations with the AMBER protein force field (ff96) coupled with the implicit solvent model (IGB = 5), we shed new light into the nature of this transition and resolve the experimental controversies. We show that the melting transition corresponds to the melting of the protein as a whole, and not solely to the helix-coil transition. The breadth of the folding transition arises from the spread in the melting temperatures (from ∼325 K to ∼302 K) of the individual transitions: formation of the hydrophobic core, β-hairpin and tertiary fold, with the helix formed earlier. Our simulations initiated from an extended chain accurately predict the native structure, provide a reasonable estimate of the transition barrier height, and explicitly demonstrate the existence of multiple pathways and multiple transition states for folding. Our exhaustive sampling enables us to assess the quality of the Amber ff96/igb5 combination and reveals that while this force field can predict the correct native fold, it nonetheless overstabilizes the α-helix portion of the protein (Tm = ∼387K) as well as the denatured structures. 相似文献
8.
Gingras AR Vogel KP Steinhoff HJ Ziegler WH Patel B Emsley J Critchley DR Roberts GC Barsukov IL 《Biochemistry》2006,45(6):1805-1817
Talin is a key protein involved in linking integrins to the actin cytoskeleton. The long flexible talin rod domain contains a number of binding sites for vinculin, a cytoskeletal protein important in stabilizing integrin-mediated cell-matrix junctions. Here we report the solution structure of a talin rod polypeptide (residues 1843-1973) which contains a single vinculin binding site (VBS; residues 1944-1969). Like other talin rod polypeptides, it consists of a helical bundle, in this case a four-helix bundle with a right-handed topology. The residues in the VBS important for vinculin binding were identified by studying the binding of a series of VBS-related peptides to the vinculin Vd1 domain. The key binding determinants are buried in the interior of the helical bundle, suggesting that a substantial structural change in the talin polypeptide is required for vinculin binding. Direct evidence for this was obtained by NMR and EPR spectroscopy. [1H,15N]-HSQC spectra of the talin fragment indicate that vinculin binding caused approximately two-thirds of the protein to adopt a flexible random coil. For EPR spectroscopy, nitroxide spin labels were attached to the talin polypeptide via appropriately located cysteine residues. Measurements of inter-nitroxide distances in doubly spin-labeled protein showed clearly that the helical bundle is disrupted and the mobility of the helices, except for the VBS helix, is markedly increased. Binding of vinculin to talin is thus a clear example of the unusual phenomenon of protein unfolding being required for protein/protein interaction. 相似文献
9.
Talin is a large cytoskeletal protein (2541 amino acid residues) which plays a key role in integrin-mediated events that are crucial for cell adhesion, migration, proliferation and survival. This review summarises recent work on the structure of talin and on some of the structurally better defined interactions with other proteins. The N-terminal talin head (approx. 50 kDa) consists of an atypical FERM domain linked to a long flexible rod (approx. 220 kDa) made up of a series of amphipathic helical bundle domains. The F3 FERM subdomain in the head binds the cytoplasmic tail of integrins, but this interaction can be inhibited by an interaction of F3 with a helical bundle in the talin rod, the so-called “autoinhibited form” of the molecule. The talin rod contains a second integrin-binding site, at least two actin-binding sites and a large number of binding sites for vinculin, which is important in reinforcing the initial integrin–actin link mediated by talin. The vinculin binding sites are defined by hydrophobic residues buried within helical bundles, and these must unfold to allow vinculin binding. Recent experiments suggest that this unfolding may be mediated by mechanical force exerted on the talin molecule by actomyosin contraction. 相似文献
10.
Nicholas J. Anthis Jacob R. Haling Camilla L. Oxley Massimiliano Memo Kate L. Wegener Chinten J. Lim Mark H. Ginsberg Iain D. Campbell 《The Journal of biological chemistry》2009,284(52):36700-36710
Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the β integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated 15N-labeled β3, β1A, and β7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of β3, β1A, and β7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of β integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins. 相似文献
11.
Diego Caballero Jukka Määttä Alice Qinhua Zhou Maria Sammalkorpi Corey S. O'Hern Lynne Regan 《Protein science : a publication of the Protein Society》2014,23(7):970-980
A fundamental question in protein science is what is the intrinsic propensity for an amino acid to be in an α-helix, β-sheet, or other backbone dihedral angle (-ψ) conformation. This question has been hotly debated for many years because including all protein crystal structures from the protein database, increases the probabilities for α-helical structures, while experiments on small peptides observe that β-sheet-like conformations predominate. We perform molecular dynamics (MD) simulations of a hard-sphere model for Ala dipeptide mimetics that includes steric interactions between nonbonded atoms and bond length and angle constraints with the goal of evaluating the role of steric interactions in determining protein backbone conformational preferences. We find four key results. For the hard-sphere MD simulations, we show that (1) β-sheet structures are roughly three and half times more probable than α-helical structures, (2) transitions between α-helix and β-sheet structures only occur when the backbone bond angle τ (N–Cα–C) is greater than 110°, and (3) the probability distribution of τ for Ala conformations in the “bridge” region of-ψ space is shifted to larger angles compared to other regions. In contrast, (4) the distributions obtained from Amber and CHARMM MD simulations in the bridge regions are broader and have increased τ compared to those for hard sphere simulations and from high-resolution protein crystal structures. Our results emphasize the importance of hard-sphere interactions and local stereochemical constraints that yield strong correlations between -ψ conformations and τ. 相似文献
12.
Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. 相似文献
13.
Nebojsa Murisic Vincent Hakim Ioannis?G. Kevrekidis Stanislav?Y. Shvartsman Basile Audoly 《Biophysical journal》2015,108(1):154-162
Metamorphic proteins, including proteins with high levels of sequence identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% sequence identity adopt the same fold. Which topologies can be bridged by these highly identical sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high sequence identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases. 相似文献
14.
He J Scott JL Heroux A Roy S Lenoir M Overduin M Stahelin RV Kutateladze TG 《The Journal of biological chemistry》2011,286(21):18650-18657
Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 Å resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded β-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the β4 and β7 strands that form a lipid-binding pocket within the β-barrel. The ARF1-binding site is located on the outer side of the β-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1. 相似文献
15.
Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation. 相似文献
16.
The α-Helix 4 Residue, Asn135, Is Involved in the Oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis Toxins 下载免费PDF全文
The insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis are comprised of three structural domains. Domain I, a seven-helix bundle, is thought to penetrate the insect epithelial cell plasma membrane through a hairpin composed of α-helices 4 and 5, followed by the oligomerization of four hairpin monomers. The α-helix 4 has been proposed to line the lumen of the pore, whereas some residues in α-helix 5 have been shown to be responsible for oligomerization. Mutation of the Cry1Ac1 α-helix 4 amino acid Asn135 to Gln resulted in the loss of toxicity to Manduca sexta, yet binding was still observed. In this study, the equivalent mutation was made in the Cry1Ab5 toxin, and the properties of both wild-type and mutant toxin counterparts were analyzed. Both mutants appeared to bind to M. sexta membrane vesicles, but they were not able to form pores. The ability of both N135Q mutants to oligomerize was also disrupted, providing the first evidence that a residue in α-helix 4 can contribute to toxin oligomerization. 相似文献
17.
Ho-Sup Lee Chinten James Lim Wilma Puzon-McLaughlin Sanford J. Shattil Mark H. Ginsberg 《The Journal of biological chemistry》2009,284(8):5119-5127
Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule
(RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to
promote talin-dependent integrin activation. Here, we show that MRL proteins
function as scaffolds that connect the membrane targeting sequences in Ras
GTPases to talin, thereby recruiting talin to the plasma membrane and
activating integrins. The MRL proteins bound directly to talin via short,
N-terminal sequences predicted to form amphipathic helices. RIAM-induced
integrin activation required both its capacity to bind to Rap1 and to talin.
Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the
talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A.
This minimized Rap-RIAM module was sufficient to target talin to the plasma
membrane and to mediate integrin activation, even in the absence of Rap1
activity. We identified a short talin binding sequence in Lamellipodin (Lpd),
another MRL protein; talin binding Lpd sequence joined to a Rap1
membrane-targeting sequence is sufficient to recruit talin and activate
integrins. These data establish the mechanism whereby MRL proteins interact
with both talin and Ras GTPases to activate integrins.Increased affinity (“activation”) of cellular integrins is
central to physiological events such as cell migration, assembly of the
extracellular matrix, the immune response, and hemostasis
(1). Each integrin comprises a
type I transmembrane α and β subunit, each of which has a large
extracellular domain, a single transmembrane domain, and a cytoplasmic domain
(tail). Talin binds to most integrin β cytoplasmic domains and the
binding of talin to the integrin β tail initiates integrin activation
(2–4).
A small, PTB-like domain of talin mediates activation via a two-site
interaction with integrin β tails
(5), and this PTB domain is
functionally masked in the intact talin molecule
(6). A central question in
integrin biology is how the talin-integrin interaction is regulated to control
integrin activation; recent work has implicated Ras GTPases as critical
signaling modules in this process
(7).Ras proteins are small monomeric GTPases that cycle between the GTP-bound
active form and the GDP-bound inactive form. Guanine nucleotide exchange
factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas
GTPase-activating proteins
(GAPs)3 enhance the
hydrolysis of Ras-bound GTP to GDP (for review, see Ref.
8). The Ras subfamily members
Rap1A and Rap1B stimulate integrin activation
(9,
10). For example, expression
of constitutively active Rap1 activates integrin αMβ2 in
macrophage, and inhibition of Rap1 abrogated integrin activation induced by
inflammatory agonists
(11–13).
Murine T-cells expressing constitutively active Rap1 manifest enhanced
integrin dependent cell adhesion
(14). In platelets, Rap1 is
rapidly activated by platelet agonists
(15,
16). A knock-out of Rap1B
(17) or of the Rap1GEF,
RasGRP2 (18), resulted in
impairment of αIIbβ3-dependent platelet aggregation, highlighting
the importance of Rap1 in platelet aggregation in vivo. Thus, Rap1
GTPases play important roles in the activation of several integrins in
multiple biological contexts.Several Rap1 effectors have been implicated in integrin activation
(19–21).
Rap1-GTP-interacting adaptor molecule (RIAM) is a Rap1 effector that is a
member of the MRL (Mig-10/RIAM/Lamellipodin) family of adaptor proteins
(20). RIAM contains Ras
association (RA) and pleckstrin homology (PH) domains and proline-rich
regions, which are defining features of the MRL protein family. In Jurkat
cells, RIAM overexpression induces β1 and β2 integrin-mediated cell
adhesion, and RIAM knockdown abolishes Rap1-dependent cell adhesion
(20), indicating RIAM is a
downstream regulator of Rap1-dependent signaling. RIAM regulates actin
dynamics as RIAM expression induces cell spreading; conversely, its depletion
reduces cellular F-actin content
(20). Whereas RIAM is greatly
enriched in hematopoietic cells, Lamellipodin (Lpd) is a paralogue present in
fibroblasts and other somatic cells
(22).Recently we used forward, reverse, and synthetic genetics to engineer and
order an integrin activation pathway in Chinese hamster ovary cells expressing
a prototype activable integrin, platelet αIIbβ3. We found that Rap1
induced formation of an “integrin activation complex” containing
RIAM and talin (23). Here, we
have established the mechanism whereby Ras GTPases cooperate with MRL family
proteins, RIAM and Lpd, to regulate integrin activation. We find that MRL
proteins function as scaffolds that connect the membrane targeting sequences
in Ras GTPases to talin, thereby recruiting talin to integrins at the plasma
membrane. 相似文献
18.
David S. Harburger Mohamed Bouaouina David A. Calderwood 《The Journal of biological chemistry》2009,284(17):11485-11497
Integrin activation, the rapid conversion of integrin adhesion receptors
from low to high affinity, occurs in response to intracellular signals that
act on the short cytoplasmic tails of integrin β subunits. Talin binding
to integrin β tails provides one key activation signal, but additional
factors are likely to cooperate with talin to regulate integrin activation.
The integrin β tail-binding proteins kindlin-2 and kindlin-3 were
recently identified as integrin co-activators. Here we report an analysis of
kindlin-1 and kindlin-2 interactions with β1 and β3 integrin tails
and describe the effect of kindlin expression on integrin activation. We
demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin
β tails in pulldown binding assays. Our mutational analysis shows that
the second conserved NXXY motif (Tyr795), a preceding
threonine-containing region (Thr788 and Thr789) of the
integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the
kindlin FERM domain (kindlin-1 Trp612 and kindlin-2
Trp615) are required for direct kindlin-integrin interactions.
Similar interactions were observed for integrin β3 tails. Using
fluorescence-activated cell sorting we further show that transient expression
of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of
endogenous α5β1 or stably expressed αIIbβ3 integrins.
This inhibition is not dependent on direct kindlin-integrin interactions
because mutant kindlins exhibiting impaired integrin binding activity
effectively inhibit integrin activation. Consistent with previous reports, we
find that when co-expressed with the talin head, kindlin-1 or -2 can activate
αIIbβ3. This effect is dependent on an intact integrin-binding site
in kindlin. Notably however, even when co-expressed with activating levels of
talin head, neither kindlin-1 or -2 can cooperate with talin to activate
β1 integrins; instead they strongly inhibit talin-mediated activation. We
suggest that kindlins are adaptor proteins that regulate integrin activation,
that kindlin expression levels determine their effects, and that kindlins may
exert integrin-specific effects.Integrins are a family of αβ heterodimeric transmembrane
receptors that mediate cell adhesion to extracellular matrix, cell surface, or
soluble protein ligands and modulate a variety of intracellular signaling
cascades. A key feature of integrins is their ability to dynamically regulate
their affinity for extracellular ligands. In a tightly regulated process
generally termed integrin activation, intracellular signals that impinge upon
the β subunit cytoplasmic tail induce conformational rearrangements in
the integrin extracellular domains, increasing the binding affinity for
extracellular ligands
(1-3).
Ligand-bound integrins then recruit additional signaling, adaptor, and
cytoskeletal proteins to the integrin cytoplasmic domains, providing
mechanical connections to the actin cytoskeleton and a link to a variety of
signal transduction pathways
(2-8).Recent years have seen significant advances in our understanding of
integrin activation. Notable among these is the identification of the actin-
and integrin-binding protein talin as a key integrin activator
(1,
9). The 50-kDa talin head
contains the principal integrin-binding site, and expression of the talin head
is sufficient to activate β1 and β3 integrins
(10,
11). The talin head contains a
FERM (four point one ezrin radixin
moesin) domain. FERM domains consist of trefoil arrangement of
three subdomains (F1, F2, and F3). The phosphotyrosine-binding domain-like F3
subdomain of the talin FERM directly binds a conserved NP(I/L)Y motif in
integrin β tails, and this interaction is necessary for integrin
activation in vitro and in vivo
(10,
12-19).
However, although abundant evidence supports the importance of talin binding
to integrin β tails during integrin activation, differences in
sensitivity of integrins to talin activation and submaximal activation by
overexpressed talin suggested that other activating factors may cooperate with
talin (10,
20). In an attempt to identify
and characterize potential co-activators, we investigated the kindlin family
of FERM domain-containing proteins.Kindlin family proteins
(21) were first characterized
in nematodes where the sole Caenorhabditis elegans kindlin, UNC-112,
was identified in an embryonic screen for defective motility and shown to be
essential for the assembly of proper cell-matrix adhesion structures, where it
normally co-localized with β integrin
(22-24).
UNC-112 is conserved across many species, because the nematode, fly, and human
homologs are ∼60% similar (∼41% identical) over their entire length
(24). Humans express three
known homologs of UNC-112: kindlin-1 (Kindlerin, URP1, and FERMT1), kindlin-2
(Mig2 and mig-2), and kindlin-3 (Mig2B and URP2)
(25-27).
Kindlin-1 and -2 are most closely related, sharing 60% identity and 74%
similarity, whereas kindlin-3 shares 53% identity and 69% similarity to
kindlin-1 and 49% identity and 67% similarity to kindlin-2
(28). The kindlin proteins all
contain a predicted Pleckstrin homology domain and a FERM domain that is most
closely related to the talin FERM domain, particularly within the
integrin-binding F3 subdomain
(29). Based on this sequence
similarity we proposed that kindlin FERM domains may directly bind integrin
β tails, and we previously showed that kindlin-1 could be pulled down
from cell lysates using recombinant integrin β1 and β3 tails and
that kindlin-1 co-localized with integrins in focal adhesions
(29). A similar localization
was reported for kindlin-2
(26,
30), and recent reports
provided clear evidence implicating kindlin-2 and kindlin-3 in regulation of
integrin activation
(31-33).
Here, we have used integrin pulldown assays to demonstrate direct binding of
full-length kindlin-1 to the cytoplasmic tails of β1A and β3
integrins and to identify key binding residues within the integrin tails and
the kindlin F3 subdomain. We confirm that these interactions are important for
recruiting kindlin-1 to focal adhesions and show that, contrary to
expectations, overexpressed kindlin-1 or -2 inhibit β1 and β3
integrin activation. Overexpressed kindlin-1 or -2 can, however, cooperate
with expressed talin head to activate β3 but not β1 integrins. We
therefore provide the first data suggesting that kindlin-1 and -2 effects on
integrin activation may show β subunit specificity. 相似文献
19.
N Mizuno J Varkey NC Kegulian BG Hegde N Cheng R Langen AC Steven 《The Journal of biological chemistry》2012,287(35):29301-29311
α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ∼50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150–500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70–100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation. 相似文献
20.
During ATP hydrolysis by F1-ATPase subunit γ rotates in a hydrophobic bearing, formed by the N-terminal ends of the stator subunits (αβ)3. If the penultimate residue at the α-helical C-terminal end of subunit γ is artificially cross-linked (via an engineered disulfide bridge) with the bearing, the rotary function of F1 persists. This observation has been tentatively interpreted by the unfolding of the α-helix and swiveling rotation in some dihedral angles between lower residues. Here, we screened the domain between rotor and bearing where an artificial disulfide bridge did not impair the rotary ATPase activity. We newly engineered three mutants with double cysteines farther away from the C-terminus of subunit γ, while the results of three further mutants were published before. We found ATPase and rotary activity for mutants with cross-links in the single α-helical, C-terminal portion of subunit γ (from γ285 to γ276 in E. coli), and virtually no activity when the cross-link was placed farther down, where the C-terminal α-helix meets its N-terminal counterpart to form a supposedly stable coiled coil. In conclusion, only the C-terminal singular α-helix is prone to unwinding and can form a swivel joint, whereas the coiled coil portion seems to resist the enzyme''s torque. 相似文献