Talins and kindlins: partners in integrin-mediated adhesion

Abstract

Integrin receptors provide a dynamic, tightly-regulated link between the extracellular matrix (or cellular counter-receptors) and intracellular cytoskeletal and signalling networks, enabling cells to sense and respond to their chemical and physical environment. Talins and kindlins, two families of FERM-domain proteins, bind the cytoplasmic tail of integrins, recruit cytoskeletal and signalling proteins involved in mechanotransduction and synergize to activate integrin binding to extracellular ligands. New data reveal the domain structure of full-length talin, provide insights into talin-mediated integrin activation and show that RIAM recruits talin to the plasma membrane, whereas vinculin stabilizes talin in cell-matrix junctions. How kindlins act is less well-defined, but disease-causing mutations show that kindlins are also essential for integrin activation, adhesion, cell spreading and signalling.

FIG 1. Domain organisation and structural model of full-length talin

A: The domain organisation of talin1. The N-terminal talin head, which is comprised of an atypical talin FERM domain containing F0, F1, F2, and F3 domains, is joined by an unstructured linker of ~80 residues to the flexible talin rod. The rod is made up of 62 α-helices (numbered blue cylinders) that are organised into thirteen 4- or 5-helix bundles (R1–R13), with a single helical dimerisation domain (DD) at the C-terminus. Domain boundaries and the interaction sites for talin-binding proteins are indicated (IBS; integrin binding site). Helices that bind vinculin are in blue. Talin2 is predicted to have the same domain structure. B: Structural model of talin assembled from the crystal and NMR structures of the various domains. The position of the calpain2 cleavage sites are indicated. The R1 and R2 domains interact via an extensive hydrophobic interface34, and a long common helix joins R11 and R12 (not shown). Otherwise, helical bundles are joined by short linkers (not shown since their structures were not determined). Because the N- and C-termini of the three 4-helix bundles (R2R3R4) are positioned at the same end of the bundle, this region will be more compact than the long succession of 5-helix bundles linked via their N- and C-termini.

FIG 2. Mechanism of talin-mediated integrin activation

A: The sequences of two representative integrin β-tails, those of integrin β1D and integrin β3, are shown. The two NPxY-like motifs are underlined, and the conserved Asp723 (numbered using the β3 sequence) that forms a salt bridge with R995 in the αIIb tail, and leads to a low affinity state, is highlighted in red. Talin binds to the indicated region of β-tails via its F3 domain. B: The complex between talin2 F3 (yellow) and the β1D tail (red) using β1D numbering to indicate key residues. The membrane-proximal (MP) and membrane-distal (MD) regions of the complex are indicated. The first NPxY-like region in the β1D tail is indicated by Y783. The second NPxY-like region in the β1D tail was not seen in the X-ray structure, but is modelled here (indicated by Y795) to show that it is very exposed, and has the potential to bind kindlins. C: The NMR structure of the transmembrane segments of the αIIbβ3 integrin is shown on the left. This NMR structure and the structure of the talin2 F2F3 domains bound to the β1D-integrin tail were used to form the composite structure in the centre. The structure on the right was obtained after 100ns of molecular dynamics simulation in the presence of a membrane bilayer. Formation of favorable electrostatic interactions between talin and the membrane causes rotation of the talin–integirn β tail complex (centre and right structures), increasing the tilt of the β TM helix. This leads to separation of the α and β TM regions, and hence to integrin activation. The translucent rectangle indicates the position of the cytoplasmic face of the membrane bilayer. D: The structure of the autoinhibitory complex between the talin1 F3 domain (yellow) and the R9 domain of the talin1 rod (green). The talin1 F3 domain is shown in approximately the same orientation as in B. Note how binding of the talin rod to the talin F3 domain occludes the F3 binding site for the membrane-proximal portion of the integrin β tail, effectively preventing integrin binding and activation.

FIG 3. Regulators of talin and kindlin binding to integrins

A schematic of the integrin β tail showing conserved residues: uppercase for near-invariant residues, lowercase for conserved residues with all other residues marked by a dot. Binding sites for integrin activators (green) and inhibitors of integrin activation (red), based on structural and biophysical studies^,,,,, are indicated by the box that encompasses the protein in question. Talin and kindlin can bind the integrin β tail simultaneously, but binding sites for the proteins that inhibit their binding, and thus inhibit integrin activation, overlap, suggesting that only one can bind at a time. Src-family kinase (SFK)-mediated tyrosine phosphorylation of the membrane-proximal or membrane-distal NPxY motif can inhibit talin and kindlin binding, respectively, and enhance binding of the inhibitor Dok1^,,. Threonine phosphorylation at residues between the NPxY-motifs has the potential to activate or inhibit integrin activation — it suppresses binding of the integrin inhibitor filamin^, and generates a binding site for 14-3-3 poteins that inhibit integrin activation. α-actinin can both positively and negatively regulate integrin–talin interactions, depending on the β tail in question. Binding of other proteins, such as migfilin or KRIT1^,,, to the integrin inhibitors filamin and ICAP1, respectively, prevents these inhibitors from binding to integrins and hence favours integrin activation.

FIG 4. Kindlin: an integrin co-activator

A: A schematic representation of kindlin domain organization, showing where kindlin interaction partners bind. Kindlin is predicted to fold as an atypical FERM domain composed of 5 subdomains (F0–F3 plus a PH domain). Similar to the talin head (Fig 1), kindlin is thought to form an extended structure. β Integrin tails bind to the F3 subdomain while phospho-inositide membrane-binding sites have been identified in the kindlin F0 and PH domains, and in the large unstructured loop in F1 ^,,,. Binding sites for ILK and migfilin have yet to be definitively mapped. B: Possible orientation of kindlin domains based on x-ray scattering, the kindlin-1 PH domain crystal structure, and NMR structures of the kindlin-1 F0 domain and the talin FERM domain. Domains are coloured as in part A. C: Models for cooperation between talin and kindlin during integrin activation. Binding of kindlin to the β integrin tail may directly potentiate talin-mediated integrin activation (top panel), perhaps by binding both the integrin β tail and the membrane to cause optimal exposure of the talin-binding site in the β tail. Alternatively, kindlin binding to the integrin may displace inhibitors, facilitating talin binding and activation (upper middle panel). In addition, kindlin may recruit other activating or adaptor proteins that cooperate with talin to activate integrins (lower middle panel). Finally, Kindlin may directly or indirectly (via another kindlin binding protein, labelled with a question mark) induce clustering of talin-activated integrins to increase avidity (bottom panel). Note: The Z-band alternatively spliced PDZ-motif containing protein (Zasp) also co-operates with talin to activate α5β1 integrin, although It is unknown whether kindlins have any role in this process.

FIG 5. Talin changes partners during adhesion assembly and maturation

A: Mechanisms of talin recruitment to the lamellipodium. The model envisages that auto-inhibited talin in the cytosol can be recruited to PIP2-rich microdomains in the plasma membrane via several mechanisms. First, by binding RIAM, which is complexed to membrane localised Rap1A-GTP^,,, via the N-terminal region of the talin rod. Second, by exocyst complexes that contain integrins and PIP-kinase type1γ, which creates the PIP2-rich microdomains. Third, by FAK complexed to Rngef, a p190RRhoGEF with a PH domain that binds phosphoinositides. Both PIP2 and RIAM binding may contribute to talin activation. Positively charged residues on one surface of the talin head are then envisaged to interact with PIP2 (see Box 1). Note that only one subunit of talin is shown for simplicity. B: Integrin activation following talin activation. Ba PIP2 binding to the talin head increases its affinity for β-integrin tails, leading to integrin binding and activation. This triggers the assembly of nascent adhesions. RIAM also drives membrane protrusion, probably by binding VASP, which recruits the actin polymerisation machinery. Bb The talin rod then binds F-actin undergoing retrograde flow, and the force exerted on talin is envisaged to alter the conformation of the N-terminal part of the talin rod. This disrupts the RIAM binding site and increases vinculin binding, which reinforces the connection of talin to F-actin and causes dynamic focal contacts to form. The vinculin Vd1 domain binds talin, while its C-terminal tail binds F-actin; it may also cross-link talin to PIP2 in the membrane. Bc Further increases in force exerted on talin by acto-myosin contraction induce more extensive conformational changes in both the talin rod and the unstructured linker between the head and rod. This enhances vinculin binding and promotes the maturation of dynamic focal contacts into more stable focal adhesions that are associated with actin stress fibres. Note that vinculin exists in an auto-inhibited cytoplasmic form and the mechansims by which it is activated have yet to be defined, although force exerted by acto-myosin contraction is involved. A schematic diagram showing the relative positions of nascent adhesions, focal contacts and focal adhesions in a migrating cell, and linking them to the relevant state of integrin activation, is shown.

FIG 5. Talin changes partners during adhesion assembly and maturation

A: Mechanisms of talin recruitment to the lamellipodium. The model envisages that auto-inhibited talin in the cytosol can be recruited to PIP2-rich microdomains in the plasma membrane via several mechanisms. First, by binding RIAM, which is complexed to membrane localised Rap1A-GTP^,,, via the N-terminal region of the talin rod. Second, by exocyst complexes that contain integrins and PIP-kinase type1γ, which creates the PIP2-rich microdomains. Third, by FAK complexed to Rngef, a p190RRhoGEF with a PH domain that binds phosphoinositides. Both PIP2 and RIAM binding may contribute to talin activation. Positively charged residues on one surface of the talin head are then envisaged to interact with PIP2 (see Box 1). Note that only one subunit of talin is shown for simplicity. B: Integrin activation following talin activation. Ba PIP2 binding to the talin head increases its affinity for β-integrin tails, leading to integrin binding and activation. This triggers the assembly of nascent adhesions. RIAM also drives membrane protrusion, probably by binding VASP, which recruits the actin polymerisation machinery. Bb The talin rod then binds F-actin undergoing retrograde flow, and the force exerted on talin is envisaged to alter the conformation of the N-terminal part of the talin rod. This disrupts the RIAM binding site and increases vinculin binding, which reinforces the connection of talin to F-actin and causes dynamic focal contacts to form. The vinculin Vd1 domain binds talin, while its C-terminal tail binds F-actin; it may also cross-link talin to PIP2 in the membrane. Bc Further increases in force exerted on talin by acto-myosin contraction induce more extensive conformational changes in both the talin rod and the unstructured linker between the head and rod. This enhances vinculin binding and promotes the maturation of dynamic focal contacts into more stable focal adhesions that are associated with actin stress fibres. Note that vinculin exists in an auto-inhibited cytoplasmic form and the mechansims by which it is activated have yet to be defined, although force exerted by acto-myosin contraction is involved. A schematic diagram showing the relative positions of nascent adhesions, focal contacts and focal adhesions in a migrating cell, and linking them to the relevant state of integrin activation, is shown.

FIG 5. Talin changes partners during adhesion assembly and maturation

A: Mechanisms of talin recruitment to the lamellipodium. The model envisages that auto-inhibited talin in the cytosol can be recruited to PIP2-rich microdomains in the plasma membrane via several mechanisms. First, by binding RIAM, which is complexed to membrane localised Rap1A-GTP^,,, via the N-terminal region of the talin rod. Second, by exocyst complexes that contain integrins and PIP-kinase type1γ, which creates the PIP2-rich microdomains. Third, by FAK complexed to Rngef, a p190RRhoGEF with a PH domain that binds phosphoinositides. Both PIP2 and RIAM binding may contribute to talin activation. Positively charged residues on one surface of the talin head are then envisaged to interact with PIP2 (see Box 1). Note that only one subunit of talin is shown for simplicity. B: Integrin activation following talin activation. Ba PIP2 binding to the talin head increases its affinity for β-integrin tails, leading to integrin binding and activation. This triggers the assembly of nascent adhesions. RIAM also drives membrane protrusion, probably by binding VASP, which recruits the actin polymerisation machinery. Bb The talin rod then binds F-actin undergoing retrograde flow, and the force exerted on talin is envisaged to alter the conformation of the N-terminal part of the talin rod. This disrupts the RIAM binding site and increases vinculin binding, which reinforces the connection of talin to F-actin and causes dynamic focal contacts to form. The vinculin Vd1 domain binds talin, while its C-terminal tail binds F-actin; it may also cross-link talin to PIP2 in the membrane. Bc Further increases in force exerted on talin by acto-myosin contraction induce more extensive conformational changes in both the talin rod and the unstructured linker between the head and rod. This enhances vinculin binding and promotes the maturation of dynamic focal contacts into more stable focal adhesions that are associated with actin stress fibres. Note that vinculin exists in an auto-inhibited cytoplasmic form and the mechansims by which it is activated have yet to be defined, although force exerted by acto-myosin contraction is involved. A schematic diagram showing the relative positions of nascent adhesions, focal contacts and focal adhesions in a migrating cell, and linking them to the relevant state of integrin activation, is shown.