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- Barczyk, M., Carracedo, S. & Gullberg, D. Cell Tissue Res (2010) 339: 269. doi:10.1007/s00441-009-0834-6
Integrins are cell adhesion receptors that are evolutionary old and that play important roles during developmental and pathological processes. The integrin family is composed of 24 αβ heterodimeric members that mediate the attachment of cells to the extracellular matrix (ECM) but that also take part in specialized cell-cell interactions. Only a subset of integrins (8 out of 24) recognizes the RGD sequence in the native ligands. In some ECM molecules, such as collagen and certain laminin isoforms, the RGD sequences are exposed upon denaturation or proteolytic cleavage, allowing cells to bind these ligands by using RGD-binding receptors. Proteolytic cleavage of ECM proteins might also generate fragments with novel biological activity such as endostatin, tumstatin, and endorepellin. Nine integrin chains contain an αI domain, including the collagen-binding integrins α1β1, α2β1, α10β1, and α11β1. The collagen-binding integrins recognize the triple-helical GFOGER sequence in the major collagens, but their ability to recognize these sequences in vivo is dependent on the fibrillar status and accessibility of the interactive domains in the fibrillar collagens. The current review summarizes some basic facts about the integrin family including a historical perspective, their structure, and their ligand-binding properties.
KeywordsIntegrins History Ligands α and β subunits RGD GFOGER Collagen
Integrins are cell adhesion receptors that are evolutionary old (Johnson et al. 2009). Despite their long history, they have only been characterized at the molecular level for approximately 25 years. During this period, a large number of articles on the ever-increasing intricacies of integrin action has been published. To summarize this large amount of data on integrins “at a glance” is thus almost an impossible task. In the current review, we have however attempted to provide some of the basic facts about integrins. Recent excellent reviews on various aspects of integrins structure and function will be referred to in the text.
Characteristics of human integrin α subunits.Data are presented for the human integrin α chains and have been retrieved from original data submitted to the NCBI database (http://www.ncbi.nlm.nih.gov/sites/entrez) and original publications. For ligand specificity, see references in text (ICAM intercellular adhesion molecule, VCAM vascular cell adhesion molecule, VEGF vascular endothelial growth factor)
Integrin α subunits
Nine of the integrin α chains contain an I domain, also called the A domain, which is a domain of approximately 200 amino acids, inserted between blades 2 and 3 in the β-propeller (Larson et al. 1989). The αI first appeared in chordate integrins, and is thus absent in invertebrates but is present in vertebrates (Johnson et al. 2009). The αI domain is present in the β2 integrin subgroup of integrins, in the collagen-binding integrins belonging to the β1 subfamily (α1, α2, α10, and α11), and the αE integrin chain forming the αEβ7 heterodimer. The I domain assumes a Rossman fold with five β-sheets surrounded by seven α helices. Ligand binding occurs via a coordinating Mg2+ ion in the so-called metal-ion-dependent adhesion site (MIDAS) motif (Lee et al. 1995). The αI domains with the capacity to interact with collagens, in addition, contains a so-called αC helix (Emsley et al. 1997), which has been suggested to play a role in collagen binding.
Relatively little is known about proteins interacting with the α-chain cytoplasmic tails.
An area between the hybrid domain in the β subunit and a surface in the β-propeller of the α subunit seems to be crucial for the integrin heterodimerization that occurs intracellularly prior to transport to the cell surface (Humphries 2000). Presumably, the specificity of chain selection lies in those sequences adjacent to these interacting surfaces. Generally, an excess of β subunits exists in the cell, and the amount of α subunit determines the amount of receptor that will go to the cell surface (Santala and Heino 1991). Free α and β subunits do thus not exist at the cell surface.
Integrin β subunit
The β subunit contains a plexin-sempahorin-integrin (PSI) domain, a hybrid domain, a βI domain (Lee et al. 1995), and four cysteine-rich epidermal growth factor (EGF) repeats. The βI domain contains an Mg2+ coordinating MIDAS and a site adjacent to MIDAS (ADMIDAS) binding an inhibitory Ca2+ ion. This ADMIDAS site binds the Mn2+ ion leading to a conformational change resulting in an active form of the integrin (Humphries et al. 2003).
Some key proteins seem to be essential for binding and, in doing so, break salt bridges formed with the α subunit that keeps the integrin in the inactive conformation. A detailed study has recently clarified the interacting regions in the αIIb and β3 subunit transmembrane domains, suggesting a model for conformation-mediated changes over the membrane (Lau et al. 2009). Talin 1–2 and kindlins 1–3 seem to act synergistically to activate integrins by binding to integrin β subunit tails (Larjava et al. 2008; Senetar et al. 2007; Tadokoro et al. 2003), whereas filamin A negatively regulates activation (Kiema et al. 2006). Migfilin is another molecular switch that, by blocking the integrin-binding region of filamins (Ithychanda et al. 2009), can regulate integrin activation. More recently, the integrin-linked kinases (ILK) (Honda et al. 2009) and focal adhesion kinase (FAK) (Michael et al. 2009) enzymes taking part in outside-in signaling have also been shown to affect integrin activation via inside-out signaling.
Conformational changes in integrins
The crystallization of a soluble integrin heteodimer has made clear that integrins can exist in a compact bent conformation (Xiong et al. 2002). Later research has shown that this conformation represents an inactive conformation (Nishida et al. 2006). Some data suggest that integrins “breathe” and change between different conformations with individual variations as to their degree of bending. Furthermore, the bent conformation does not always seem to be inactive, especially with regard to small ligands (Askari et al. 2009). More recently, mechanical tension has been demonstrated to consolidate integrin contact points by further stretching the conformation to stabilize the active conformation, thereby increasing affinity (Askari et al. 2009; Astrof et al. 2006; Friedland et al. 2009). For integrin α5β1, the mechanical tension induces α5β1 engagement with the synergy site in fibronectin, in turn leading to FAK phosphorylation (Friedland et al. 2009). A recent synergy site in nephronectin has been suggested mainly to exert its action by bringing the RGD site into a best-fit conformation with regard to high-affinity integrin binding (Sato et al. 2009).
In addition to the affinity modulation that occurs in various activation states, integrin clustering by multivalent ligands, and possibly also changes in membrane fluidity, cause avidity changes of integrin contacts (Carman and Springer 2003).
For integrins that require a tight control of activity, such as platelet integrin αIIβ3 and β2 integrins, precise activating mechanisms must exist (Luo et al. 2007). When kindlin-3 is mutated, integrin activation in leukocytes and platelets fails (Moser et al. 2008; Svensson et al. 2009), demonstrating the central role of kindlin-3 for integrin activation on these cell types.
The ligand-binding site forms in a region at the intersection of the integrin α-chain β-propeller and the βI domain, with the α chain being central in determining ligand specificity. Integrins with an αI domain bind ligands via the αI domain, but since this ligand-binding causes distinct conformational changes in the I domain, this in turn affects the conformation of the β subunit (Luo et al. 2007). For some αI domains, interactions with the β chain are even needed for the proper folding of the αI domain (Valdramidou et al. 2008). The list of integrin ligands is long (Humphries et al. 2006; Johnson et al. 2009) and includes the major constituents of the ECM.
Prototypic integrin ligands and recognition sequences
In many biochemistry and cell biology books, the RGD sequence is presented as the major integrin-binding sequence, but only a third of the integrins are known to bind this sequence (Table 1). RGD peptides can be used to assess the involvement of RGD-binding integrins in a certain event, but lack of effect of RGD peptides does not exclude integrin involvement in a process since two-thirds of integrins bind ligands RGD-independently.
Some isoforms of the basement membrane protein laminin contain an RGD sequence, but the sequence is not evolutionary conserved in these isoforms and is not recognized by the laminin-binding integrins. In laminin-111, the RGD site is masked in the native molecule (Aumailley et al. 1990; Schulze et al. 1996), whereas in laminin isoforms containing the α5 chain, it is exposed in the native molecule (Domogatskaya et al. 2008; Forsberg et al. 1994; Genersch et al. 2003; Sasaki and Timpl 2001). The full physiological importance of the αvβ1- and αvβ3-mediated binding to laminin-α5-containing laminins however remains unclear. As pointed out, the EHS-produced laminin-111 is a poor ligand for the laminin-binding integrins. Accumulating data suggest that a major integrin-binding site is present in laminin LG1-3 domains in the C-terminal part of the laminin heterotrimer (Ido et al. 2004; Kunneken et al. 2004). In addition to laminin α-chain-specifc sequences, C-terminal residues of both β- and γ-chains contribute/facilitate integrin binding (Ido et al. 2004; Kunneken et al. 2004). Splicing in the extracellular domain of integrin α7 affects it preference for certain laminin isoforms (Nishiuchi et al. 2003; Taniguchi et al. 2009; von der Mark et al. 2002; Table 1).
RGD sequences present in triple helical fibrillar collagen sequences are normally not available for fibronectin receptors in native fibrillar collagen (Davis 1992; Gullberg et al. 1990), only becoming available in denatured collagen I. Instead, the collagen-binding integrins recognize the triple-helical GFOGER sequence (Knight et al. 1998), or variants thereof, in native collagens. An assembled database on collagen-I-binding sites and mutations has enabled the construction of a model in which the GFOGER site is present in a cell-interactive domain that is suggested to be exposed once per microfibril unit (each microfibrillar unit is composed of five collagen triple helix monomers), allowing the clustering of integrins (Sweeney et al. 2008). However, a number of components in the fibrillar matrix might influence the availability of these sites. Interestingly, collagen IX, which is present in cartilage, has been shown to be a good ligand for all of the collagen-binding integrins (Kapyla et al. 2004), but in vivo, not all of the collagen-binding integrins are expressed in chondrocytes. In the case of α11β1 integrin, if cartilaginous tissue is not dissected free of perichondrium, a signal from α11 will be derived from α11 expression in the perichondrium (Tiger et al. 2001), which lacks collagen IX. The identity of the integrins that might physiologically be relevant as collagen IX receptors in various regions of cartilage thus remains to be defined.
Collagen IX lacks the prototypic collagen-binding integrin recognition sequence GFOGER, and the exact sequence awaits mapping. Certain variants of the GFOGER sequence might show specificity for the different collagen-binding integrins, as indicated by a recent finding showing that the bacterial collagen-like peptide, sscl, with the active binding site GLPGER, is preferred by α11β1-expressing cells over α2β1-expressing cells (Caswell et al. 2008).
Other integrin-binding motifs include the α4-integrin-binding sequence LDV (Clements et al. 1994; Komoriya et al. 1991), variants of which are also present in the adhesion molecules ICAMs and MadCAM recognized by β2 integrins and the α4β7 heterodimer, respectively (Wang and Springer 1998).
In addition to physiological ligands, integrin ligands generated by proteolysis are receiving increasing recognition. Endostatin (derived from collagen XVIII), endorepellin (derived from perlecan), and tumstatin derived from collagen α3 (IV) are the best-known examples (Bix and Iozzo 2005; Suhr et al. 2009; Table 1). In addition, integrins can bind snake toxins called disintegrins (Calvete et al. 2005; Swenson et al. 2007) and certain viruses (Stewart and Nemerow 2007) and bacteria (Hauck et al. 2006; Palumbo and Wang 2006). Some of these interactions occur outside the regular ligand-binding sites in the integrins and display distinct binding characteristics compared with the binding of physiological ligands.
Integrins as mechanical links
The first function established for integrins was their function as links between the ECM and the cytoskeleton. For a majority of integrins, the linkage is to the actin cytoskeleton (Geiger et al. 2009), whereas α6β4 connects to the intermediate filament system (Nievers et al. 1999). Recently, the intermediate filament protein vimentin has been shown to be dependent on β3 integrins for its recruitment to the cell surface (Bhattacharya et al. 2009), indicating that an intimate relationship exists between the various cytoskeletal networks and integrins.
Some of the components in this mechanical linkage, such as talin, play a dual role and also take part in activating integrins in an inside-out signaling mechanism (Tadokoro et al. 2003). A new dimension of integrins as mechanical links has come to the fore with the realization that integrins can act as mechanosensors and generate signals that affect cell physiology via complex intracellular signaling mechanisms including autocrine and paracrine mechanisms (Chen and O’Connor 2005; Linton et al. 2007; Millward-Sadler and Salter 2004; Millward-Sadler et al. 1999; Zhu et al. 2007). As mentioned above, mechanical tension can also increase integrin affinity.
Integrins as signaling receptors
Integrins are bi-directional signaling receptors involved in outside-in and inside-out signaling. The inside-out signaling mainly acts to bring the integrin into the active conformation. As previously mentioned, talins, kindlins, filamins, migfilin, FAK, but also ILK (Honda et al. 2009) can regulate integrin activation.
Upon ligand binding, integrins undergo conformation changes leading to outside-in signaling. This activates signaling events that are complex and cell-specific, depending on what other signaling receptors and signaling systems are available in the cell. The scope of this review does not include these events, but readers are referred to recent excellent reviews on this subject (Askari et al. 2009; Gahmberg et al. 2009; Larjava et al. 2008; Legate et al. 2009; Luo and Springer 2006).
Integrins are widely expressed, and every nucleated cell in the body possesses a specific integrin signature. Importantly, the regulation of integrins is dynamic and quickly changes once cells are taken out of their normal environment. A few integrins are more restricted than others to certain cell lineages, but the expression if often developmentally regulated.
Integrin signatures of some selected cell types. Each cell in an organism contains a specific integrin signature under certain conditions. The integrin repertoire is dynamic, changes with developmental age, and is strongly responsive to microenvironmental conditions. Sometimes, a specific integrin isoform is indicative of the cell type or the differentiation status (integrins given in bold are the predominating integrins present)
Phenotypes of unchallenged and challenged integrin mutant mice. The knockout data were collected from the Mouse Genome Informatics (MGI) database (http://www.informatics.jax.org/) and, whenever needed, updated with relevant Pubmed (http://www.ncbi.nlm.nih.gov/pubmed/) retrieved original references
We have just started to comprehend the way that integrins work in complex biological systems. A new age with more refined assays, methods, and tools is likely to meet the challenge of understanding the integrated role of integrins in these biological systems.
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