A schematic of the interactions involved in integrin activation, including the role of kindlins ...
The X-ray crystal structure of the kindlin-1 PH domain in the foreground, and a representative ...
The adhesion of cells to their environments is mediated by specific protein-protein and protein-proteoglycan interactions at the plasma membrane. Chief among the direct points of contact are those centred on integrins, heterodimeric bi-directional signalling molecules which exist in a variety of states of activation. Integrin activation can be outside-in, mediated by proteins such as fibronectin and fibrillin, or inside-out, mediated principally by talin. However, it has recently been shown that talin is not sufficient for full integrin activation which requires also kindlin proteins. Kindlins assist talin in integrin activation, binding to their beta-subunit tail. However, there are no structures of even large fragments of kindlins and the ways in which they work on integrins – e.g. in sequence with or alongside talin, and how that is modulated by phosphorylation or other factors altering protein activity – are largely opaque.
One of the reasons why relatively little insight has been obtained into kindlin structure and function is because they have proved very difficult to express at high levels for structural and biochemical studies. We have, however, managed to establish expression of all three mammalian isoforms of the kindlins at high levels. We are the only lab in the world to have achieved this. We have also cloned and expressed a series of fragments, among which we have solved the structure of the pleckstrin homology (PH) domain region of kindlin-1 (Yates et al., submitted). We have also studied the lipid-binding tropism of the PH domain and its evolutionary relationship to similar domains found in a wide range of signalling and membrane-associated proteins (Yates et al., submitted). In addition to the PH domain, kindlins have (like talin) F0, F1, F2 and F3 subdomains within a FERM domain family fold. The F3 domain is involved in direct binding to integrin beta-tails and in a separate study we have used NMR to measure the interaction between specific tail residues and kindlin-3, alongside the PH domain-mediated lipid tropism of kindlin-3 and its overall conformation.
In this graduate studentship you would express and purify large fragments and full-length versions of kindlins 1-3. You would generate crystals of the purified proteins and solve their structures by X-ray crystallaography. You would also study the interactions between kindlins and their binding partners using biophysical methods and direct structural approaches such as NMR and co-crystallisation. This studentship would provide an excellent opportunity to develop skills in protein expression, purification and structure determination and to contribute to a major area of biomedical science. Kindlins are directly involved in cancers and connective tissue disorders and any structural advances made in their study will be of major impact.
Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Project reference number: 299
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Robert JC Gilbert | Structural Biology | Oxford University | UK | gilbert@strubi.ox.ac.uk |
2009. The Kindlin protein family: new members to the club of focal adhesion proteins. Trends Cell Biol., 19 (10), pp. 504-13. Read abstract | Read more
Kindlins are a group of proteins that have recently attracted attention for their ability to bind and activate integrins. Moreover, they have also been linked to inherited and acquired human diseases including Kindler syndrome, leukocyte adhesion deficiency, and cancer. Although most studies have focused on kindlins as key regulatory components of cell-extracellular matrix junctions such as focal adhesions, preliminary data suggest the involvement of additional cellular compartments in mediating their functions, particularly at cell-cell contacts and the nucleus. Investigating the many roles of kindlins is likely to expand and sharpen our view on the versatility of integrin-mediated cell adhesion, the nuclear function of focal adhesion proteins, and the crosstalk between cell-cell and cell-matrix adhesions in health and disease. Hide abstract
2006. The Kindlins: subcellular localization and expression during murine development. Exp. Cell Res., 312 (16), pp. 3142-51. Read abstract | Read more
The three Kindlins are a novel family of focal adhesion proteins. The Kindlin-1 (URP1) gene is mutated in Kindler syndrome, the first skin blistering disease affecting actin attachment in basal keratinocytes. Kindlin-2 (Mig-2), the best studied member of this family, binds ILK and Migfilin, which links Kindlin-2 to the actin cytoskeleton. Kindlin-3 is expressed in hematopoietic cells. Here we describe the genomic organization, gene expression and subcellular localization of murine Kindlins-1 to -3. In situ hybridizations showed that Kindlin-1 is preferentially expressed in epithelia, and Kindlin-2 in striated and smooth muscle cells. Kindlins-1 and -2 are both expressed in the epidermis. While both localize to integrin-mediated adhesion sites in cultured keratinocytes Kindlin-2, but not Kindlin-1, colocalizes with E-cadherin to cell-cell contacts in differentiated keratinocytes. Using a Kindlin-3-specific antiserum and an EGFP-tagged Kindlin-3 construct, we could show that Kindlin-3 is present in the F-actin surrounding ring structure of podosomes, which are specialized adhesion structures of hematopoietic cells. Hide abstract
2009. The structure of the N-terminus of kindlin-1: a domain important for alphaiibbeta3 integrin activation. J. Mol. Biol., 394 (5), pp. 944-56. Read abstract | Read more
The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the beta-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced alphaIIbbeta3 integrin activation and for the localization of kindlin-1 to focal adhesions. Hide abstract
2009. The tail of integrins, talin, and kindlins. Science, 324 (5929), pp. 895-9. Read abstract | Read more
Integrins are transmembrane cell-adhesion molecules that carry signals from the outside to the inside of the cell and vice versa. Like other cell surface receptors, integrins signal in response to ligand binding; however, events within the cell can also regulate the affinity of integrins for ligands. This feature is important in physiological situations such as those in blood, in which cells are always in close proximity to their ligands, yet cell-ligand interactions occur only after integrin activation in response to specific external cues. This review focuses on the mechanisms whereby two key proteins, talin and the kindlins, regulate integrin activation by binding the tails of integrin-beta subunits. Hide abstract