myospheroid
Integrin 'inside-out' activation Activation of the ligand binding function of integrin heterodimers requires transmission of an 'inside-out' signal from the integrin small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alphaIIß3 has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the ß cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin (See Drosophila Talin), both of which are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the ß cytoplasmic tails restrains the integrin in a resting
state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation (Vinogradova, 2002).
Upon binding extracellular ligands, integrins transduce signals to the cytoplasm (outside-in signaling), which induces cascades of intracellular signaling events, including protein phosphorylation and cytoskeletal reorganization. However, ligand binding to integrins is not simply controlled by ligand availability but also through 'inside-out' signaling: cellular stimulation clusters integrins or alters conformation to increase their avidity or affinity for ligands. The prototypic example of such integrin activation via inside-out signaling occurs with alphaIIbß3. Platelets express alphaIIbß3 on their surface, but the receptor engages fibrinogen only if the cells have been stimulated with an agonist that induces the appropriate inside-out signal. Such inside-out regulation of alphaIIbß3 affinity allows for rapid platelet aggregation to prevent excess bleeding at the same time preventing uncontrolled receptor occupancy, resulting in thrombosis (Vinogradova, 2002).
As the trigger point of inside-out signaling, the integrin cytoplasmic face has been the focus of intense investigations. These studies have revealed that (1) while intact integrin can remain latent both in unstimulated cells and in a purified state, deletion of the cytoplasmic and transmembrane region activates the receptor; (2) point mutations in the membrane-proximal regions of the cytoplasmic tails or deletion of either can result in constitutive activation of the receptor; (3) replacement of the cytoplasmic-transmembrane regions by heterodimeric coiled-coil peptides or an artificial linkage of the tails inactivates the receptor, and breakage of the coiled-coil or clasp activates the receptor, and (4) overexpression of certain intracellular proteins that bind to the cytoplasmic tails, including the cytoskeletal protein talin, which binds to the ß cytoplasmic tail, can result in integrin activation. These data suggest that a direct interaction between the alpha/ß cytoplasmic tails might maintain the receptor in a latent state. However, vigorous biochemical/biophysical studies aimed at examining such interaction have yielded contradictory results. While certain studies have suggested such interactions, recent NMR studies have failed to detect a complex. The most recent NMR study reported interaction between truncated versions of the alphaIIb and ß3 cytoplasmic tail peptides, but structural analyses revealed two different complexes of unknown physiological relevance. Thus, the current view of the cytoplasmic face and its regulation on the integrin inside-out signaling remains unclear; this is a major impediment to an understanding of integrin structure-function relationships (Vinogradova, 2002 and references therein).
In this study, the successful structure determination of the intact integrin alphaIIbß3 cytoplasmic face using NMR spectroscopy is reported. The structure reveals that the alphaIIb and ß3 cytoplasmic tails do, indeed, interact; they engage in a weak handshake within their membrane-proximal regions. This handshake is unclasped by 'activating' mutations in the binding interface and by a known integrin activator, the talin head domain. The results provide a structural basis for how an integrin cytoplasmic face regulates inside-out activation (Vinogradova, 2002).
How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been a mystery. This has been addressed with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVß3 and alphaIIbß3 integrins. A highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn2+ results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and ß subunits enhances Mn2+-induced unbending and ligand binding (Takagi, 2002).
Integrins exist in at least three conformational states: a bent conformer, an extended conformer with a closed headpiece, and an extended conformer with an open headpiece. These studies unequivocally establish that the bent conformation exists in solution and on cell surfaces, and represents the highly physiologically relevant low-affinity conformation of integrins. The bent conformer is seen in Ca2+/Mg2+ and Ca2+, is stabilized by Ca2+, strongly destabilized by cyclo-RGDfV, and less strongly destabilized by Mn2+. In the presence of a C-terminal clasp, a higher proportion of alphaVß3 molecules is present in the V-shaped bent conformation in Ca2+. The bent alphaVß3 conformer does not bind the biological ligands fibrinogen or vitronectin. On the cell surface, alphaVß3 and alphaIIbß3 cannot be stimulated to bind their biological ligand fibrinogen with high affinity when locked in the bent conformation with a disulfide bond. Activation of ligand binding by the locked bent integrins, but not by wild-type integrins, requires disulfide reduction. Although the bent conformer does not detectably bind biological ligands, it clearly can bind high-affinity ligand-mimetic peptides as demonstrated by a co-crystal structure. The bent conformer is therefore referred to as a low-affinity rather than an inactive conformation (Takagi, 2002).
In Mn2+, the bent conformer and the two extended conformers with the open and closed headpiece are all present as shown with EM. Physiochemical measurements have demonstrated a conformational equilibrium in Mn2+, with equilibration between the bent and extended conformers occurring on a timescale much more rapid than 13 min. In Mn2+, alphaVß3 binds to fibrinogen and vitronectin, demonstrating that one or both of the extended conformers has high affinity for ligand. A higher percentage of molecules in the extended conformation is found for unclasped than clasped alphaVß3 in Mn2+, correlating with the finding that unclasped alphaVß3 has higher affinity for vitronectin and fibrinogen (Takagi, 2002).
In the presence of cyclo-RGDfV, the equilibrium shifts almost entirely to the extended conformer of alphaVß3 with the open headpiece. The same results were seen in Ca2+ or Mn2+ and with clasped or unclasped alphaVß3. Cyclo-RGDfV was used at concentrations far above its KD, which will drive the conformational equilibrium to the conformer with the highest affinity for ligand. This identifies the extended conformer with the open headpiece as the conformation with highest affinity for ligand (Takagi, 2002).
The extended conformer with the closed headpiece represents an intermediate conformation, because it shares the closed headpiece with the bent conformer, and shares the lack of a tailpiece-headpiece interface with the extended conformer with the open headpiece. The presence of all three species of conformers in Mn2+, and only the bent conformer in Ca2+ alone and only the extended, open conformer in cyclo-RGDfV also suggests that the extended, closed conformer is an intermediate in the conformational equilibrium between the low and high affinity forms. Because the equilbria of conformational change and ligand binding are thermodynamically linked, it is reasonable to assume that the extended, closed conformer has an intermediate affinity for ligand (Takagi, 2002).
Many anti-integrin mAbs have been reported that bind preferentially to the active and/or ligand-occupied form of integrins. Recently, a combined NMR structure and model of ß2 integrin I-EGF modules 2 and 3 localized epitopes of mAb that report integrin activation and induce integrin activation, and residues that interact with the alpha subunit to restrain activation. These residues are buried in the bent conformation, but exposed in an extended conformation. Therefore, a switchblade-like opening of the bent conformation upon integrin activation is postulated. The studies presented here confirm and markedly extend the switchblade model. The identification of the bent alphaVß3 conformer as the low affinity, resting state of integrins on the cell surface, and the shift of the conformational equilibrium toward the extended conformer by activation and ligand binding explain many previous results on LIBS and activation-dependent epitopes, including the presence of the vast majority of these epitopes on the ß subunit. In the bent alphaVß3 structure, the ß subunit is innermost in the bend and contributes about 70% of the solvent accessible surface area that is buried in the headpiece-tailpiece interface. A minority of alphaVß3 particles in Ca2+ had a V-shaped conformation with a wider separation between the headpiece and tailpiece. This provides direct evidence for 'breathing' at this interface. Upon temporary exposure of the headpiece-tailpiece interface during breathing movements, binding of an antibody to the inner side of the ß tail would keep the interface wedged open, prevent rebending of the receptor, and stabilize the extended conformation and hence ligand binding. It is interesting that antibodies that map further from the bend can bind to resting integrins and activate them, whereas a mAb that binds only after prior integrin activation binds close to the bend, where less opening would occur during breathing (Takagi, 2002).
The structural rearrangements demonstrated here after binding of cyclo-RGDfV to alphaVß3 define a pathway for communication from the ligand binding site in the headpiece to the membrane proximal segments of the alpha and ß legs; i.e., the extracellular portion of the integrin outside-in signaling pathway. The cyclo-RGDfV peptide differs only 4-fold in IC50 and in one methyl group from its N-methyl-Val derivative, cyclo-RGDf-mV. When soaked into alphaVß3 crystals along with Mn2+, cyclo-RGDf-mV binds to the bent conformation of alphaVß3, with its Asp sidechain coordinating the Mn2+ in the MIDAS of the ß3 I-like domain. By contrast, binding to alphaVß3 in solution results in a dramatic quaternary rearrangement to an extended conformation with an open headpiece. The lack of major rearrangements in the crystal structure is readily explained by the extensive lattice contacts stabilizing the bent conformation. Thus, in a first step, peptide binds to the low affinity, bent alphaVß3 conformer, which has a closed headpiece; and in a second step, in the absence of crystal lattice constraints, binding causes a dramatic quaternary rearrangement to the high affinity, extended conformer with an open headpiece (Takagi, 2002).
Inside-out activation of integrins occurs as a result of breakage of interactions between the membrane proximal regions of the alpha and ß subunits. The talin head domain can directly activate integrins by binding to the ß subunit cytoplasmic domain. In the crystal structure, the most C-terminal, membrane proximal residues of alphaV and ß3 are very close to one another, as appropriate for a low affinity conformation with interacting membrane proximal segments. The C-terminal clasp stabilizes the bent conformation relative to the extended conformation, and stabilizes the extended conformation with a closed headpiece relative to the extended conformation with an open headpiece. These findings support the model that parting movement by the membrane proximal segments of the alpha and ß subunits is a mechanism for integrin activation in inside-out signaling. In the alphaVß3 crystal structure, the lower legs of the alpha and ß subunits, i.e., the portions below the bend at the genu, have substantial interactions with one another, and in turn the lower ß leg has a prominent role in the headpiece-tailpiece interface. Therefore, destabilizing the conformation of the lower ß leg by breakage of its interaction with the lower alpha leg would also destabilize the headpiece-tailpiece interface and favor a switchblade-like opening of this interface. This provides a mechanism for communicating conformational change from the membrane to the headpiece (Takagi, 2002).
Growth factors, integrins, and the extracellular matrix (ECM) are known to play key roles in epidermal wound healing, although the interplay between these proteins is not fully understood. Growth factor macrophage stimulating protein (MSP)- and its receptor Ron-mediated PI3K activation in keratinocytes induces phosphorylation of both Ron and alpha6beta4 integrin at specific 14-3-3 (see Drosophila 14-3-3zeta) binding sites. Consequently, a Ron/alpha6beta4 complex formed via 14-3-3 binding displaces alpha6beta4 from its location at hemidesmosomes (structures supporting cell adhesion) and relocalizes it to lamellipodia. Concomitant activation of alpha3beta1 and keratinocyte spreading/migration on laminin-5 occurs. Further, MSP-dependent beta4 tyrosine phosphorylation evokes p38 and NF-kappaB signaling required for keratinocyte wound closure. Based on these results, a mechanism is proposed based on MSP-Ron-dependent phosphorylation and 14-3-3 association, whereby the function of alpha6beta4 switches from a mechanical adhesive device into a signaling component, and might be critically involved in human epidermal wound healing (Santoro, 2003).
The complex interplay between the extracellular matrix (ECM), growth factors, and integrins is crucial for many biological processes, including skin wound healing. Wound healing is characterized by a number of overlapping phases including inflammation, reepithelization, granulation tissue formation, and tissue remodeling. The reepithelization process is tightly regulated by specific classes of integrin receptors and interacting extracellular matrix molecules. In particular, α3β1 and α6β4 integrins, as well as laminin-5, play a key role in keratinocyte migration. Laminin-5 is the primary ligand of adult epidermal basement membrane (BM) and is secreted by wounded keratinocytes in the provisional wound bed to promote migration. Laminin-5 is synthesized as a precursor heterotrimeric protein (α3,β3,γ2) that undergoes processing of α3 and γ2 subunits after being secreted. The domain interacting with integrin resides within the α3 C-terminal large globular domain, while the region for deposition in the BM resides in the γ2 subunit. Current models propose that laminin-5 mediates keratinocyte adhesion and migration via α6β4 and α3β1 integrins at distinct sites. α6β4 can be found in hemidesmosomes (HDs), which are structures linking ECM to keratin intermediate filaments thereby supporting cell adhesion. α3β1 is present in focal contacts and links ECM to the actin cytoskeleton, regulating cell spreading and migration. The interplay between α3β1 and α6β4 and different domains of laminin-5 is crucial at the wound edges because it regulates leading keratinocytes by (1) disassembling HDs, (2) remodeling matrix interactions with increased deposition of laminin-5 and metalloproteases (MMPs), and (3) recruiting α3β1 at focal contacts to mediate migration over the provisional matrix (Santoro, 2003 and references therein).
Macrophage stimulating protein (MSP) is the ligand of the Ron tyrosine kinase receptor. It is a biologically inactive soluble plasma factor activated at extravascular sites by specific serine proteases. The Ron receptor is selectively expressed by epithelia, including keratinocytes, and by hematopoietic cells. MSP leads to receptor trans-autophosphorylation and activation of several signaling pathways. MSP and Ron modulate keratinocyte functions such as proliferation, survival, and migration, and, recently, a role in epidermal wound healing has been suggested (Santoro, 2003 and references therein).
MSP-mediated PI3K pathway activation induces Ron serine phosphorylation at residue 1394 as well as α6β4 phosphorylation in the connecting sequence to generate 14-3-3 binding sites on both molecules. Thus, dimeric 14-3-3 proteins mediate the MSP-dependent formation of a Ron/α6β4 complex that in turn induces disassembly of HDs and α6β4 relocation at lamellipodia. Further, α3β1 integrin activation and keratinocyte spreading/migration on laminin-5 takes place. All these findings suggest a role for Ron and 14-3-3 in epidermal reepithelization processes (Santoro, 2003).
Activation (affinity regulation) of integrin adhesion receptors controls cell migration and extracellular matrix assembly. Talin connects integrins with actin filaments and influences integrin affinity by binding to the integrins' short cytoplasmic beta-tail. The principal beta-tail binding site in talin is a FERM domain, comprised of three subdomains (F1, F2, and F3). Previous studies of integrin alphaIIbbeta3 have shown that both F2 and F3 bind the beta3 tail, but only F3, or the F2-F3 domain pair, induces activation. Talin-induced perturbations of beta3 NMR resonances were examined to explore integrin activation mechanisms. F3 and F2-F3, but not F2, distinctly perturbed the membrane-proximal region of the beta3 tail. All domains also perturbed more distal regions of the beta3 tail that appear to form the major interaction surface, since the beta3(Y747A) mutation suppressed those effects. These results suggest that perturbation of the beta3 tail membrane-proximal region is associated with talin-mediated integrin activation (Ulmer, 2003).
Talin is an essential component of focal adhesions that couples beta-integrin cytodomains to F-actin and provides a scaffold for signaling proteins. Recently, the integrin beta3 cytodomain and phosphatidylinositol phosphate (PIP) kinase type 1gamma (a phosphatidylinositol 4,5-bisphosphate-synthesizing enzyme) were shown to bind to the talin FERM domain (subdomain F3). The PIP kinase-binding site was characterized by NMR using a 15N-labeled talin F2F3 polypeptide. A PIP kinase peptide containing the minimal talin-binding site formed a 1:1 complex with F2F3, causing a substantial number of chemical shift changes. In particular, two of the three Arg residues (Arg339 and Arg358), four of eight Ile residues, and one of seven Val residues in F3 were affected. Although a R339A mutation did not affect the exchange kinetics, R358A or R358K mutations markedly weakened binding. The Kd for the interaction determined by Trp fluorescence was 6 microm, and the R358A mutation increased the Kd to 35 microm. Comparison of these results with those of the crystal structure of a beta3-integrin cytodomain talin F2F3 chimera shows that both PIP kinase and integrins bind to the same surface of the talin F3 subdomain. Indeed, binding of talin present in rat brain extracts to a glutathione S-transferase integrin beta1-cytodomain polypeptide was inhibited by the PIP kinase peptide. The results suggest that ternary complex formation with a single talin FERM domain is unlikely, although both integrins and PIP kinase may bind simultaneously to the talin anti-parallel dimer (Barsukov, 2003).
The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin beta subunits. This study reports the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing alpha(IIb)beta(3), linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with beta(3)-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(3) cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. The in vivo relevance of this interaction was demonstrated by fluorescence resonance energy transfer between beta(3)-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed mapping out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, a cell biology approach showed that this integrin-binding site within the talin rod domain is important for beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation (Tremuth, 2004).
The cytoplasmic protein talin is an essential part of the integrin-cytoskeleton link. This study has characterized the interaction between integrin and two conserved regions of talin, the N-terminal 'head' domain and the C-terminus, which includes the I/LWEQ domain, within the living organism. Green-fluorescent-protein-tagged head and C-terminal domains are recruited to integrin adhesion sites. Both required integrins for recruitment, but the C-terminal domain also requires endogenous talin, showing it was not recruited directly by integrins. Chimeric transmembrane proteins containing the cytoplasmic domain of the integrin β subunit were used to examine the integrin-talin head interaction. Monomeric chimeric proteins did not recruit talin head, whereas dimeric chimeras efficiently recruited it and caused a strong inhibition of integrin-mediated adhesion. These chimeras recruited surprisingly few integrin-associated proteins, indicating that recruitment of talin does not initiate a cascade of recruitment. Mutagenesis of the integrin cytoplasmic domain, within the chimera, showed the dominant-negative inhibition is not due to talin sequestration alone and that additional interactions are required (Tanentzapf, 2006a).
For most integrin heterodimers, the majority of interactions with cytoplasmic proteins are made by the 47-residue cytoplasmic domain of the ß subunit. This short peptide has been subjected to extensive study, but there is no clear model of how it functions. Recent work by a number of groups has highlighted the importance of the large cytoskeletal linker talin, which was the first protein to be identified that binds to the ß tail. This study focused on the interaction between two domains of talin and the cytoplasmic domain of the ß subunit, using the Drosophila embryo. This has provided new insights into the early steps in the linkage between integrins and the cytoskeleton (Tanentzapf, 2006a).
Using live imaging within the intact animal, in vivo evidence is provided to support a direct interaction between the head domain of talin and the cytoplasmic tail of the ß subunit. This interaction requires an 'alteration' to the cytoplasmic domain of integrin because only one of three chimeric transmembrane proteins containing this domain was able to recruit the talin head. Since the active chimera (integrin ß tail is placed on a heterologous transmembrane protein) is derived from a constitutively active receptor tyrosine kinase, it seems likely that its ability to constitutively dimerize or oligomerize accounts for its special activities, leading to it being referred to as diß. Previous work showed that diß was constitutively active in sending integrin signals that regulate gene expression, and this study shows that it also acts as a dominant negative protein on integrin-mediated adhesion at muscle attachment sites. The dominant negative phenotype of diß consists of a detachment between integrins and the cytoskeleton, similar to the phenotype seen in the absence of talin. This suggests an explanation for the dominant negative activity of diß: it sequesters talin away from the endogenous integrins. Consistent with this, diß recruited talin and TalinH-GFP, but not other proteins required for integrin adhesion, such as PINCH, ILK and tensin. In addition, this latter finding further supports the direct interaction between integrins and talin, and shows that recruitment of talin is not sufficient to trigger the assembly of the whole complex of proteins that contribute to the link between integrins and the cytoskeleton (Tanentzapf, 2006a).
Based on these observations, it is possible to propose a simple model where talin is the only protein recruited directly by integrins and the dominant negative activity of diß is solely due to talin sequestration. This was tested by generating point mutations in the ß subunit of the cytoplasmic domain within the diß chimera and by assaying their ability to cause muscle detachment and recruit TalinH-GFP, surmising that, if this model was correct there should be a clear correlation between the two. However, this proved not to be the case. Mutations throughout the length of the integrin tail caused a loss of dominant negative activity, but only those in the half closest to the membrane could not recruit TalinH-GFP. This leads a to hypothesis that a factor X binds to the second half of the integrin cytoplasmic domain and contributes to the dominant negative effect (Tanentzapf, 2006a).
Based on previous in vitro studies it has been proposed that the head domain of talin acts as the preferred site for integrin binding. The talin head domain, which in vertebrates can be found endogenously due to cleavage by the calcium dependent protease calpain, has also been shown to mediate integrin activation. This study has shown that the talin head can localize to the muscle attachments in an integrin-dependent manner and that expression of dimeric integrin cytoplasmic tail chimeras is sufficient to recruit it to the cell cortex. Experiments in myoblasts and the direct correlation between levels of integrin and talinH-GFP recruitment strongly suggest that, in vivo recruitment of talinH-GFP by the integrin cytoplasmic tail occurs independently of any accessory factors and, therefore, is most probably direct. The finding that talinH-GFP is the only protein tested that was found around the entire cortex of muscles containing diß or excess integrin also supports a direct interaction between them. There might be a difference between the membrane domains on the lateral sides of the muscles versus the ends, which blocks recruitment of additional integrin associated proteins to the lateral sides (Tanentzapf, 2006a).
Further support for a direct interaction between the ßPS cytoplasmic domain and the talin head domain in vivo comes from mutational analysis. Those residues essential to recruit talinH-GFP in vivo are within the same regions where the cytoplasmic domain binds to talin in vitro. Thus, the first NPxY motif of the ß integrin cytoplasmic tail is crucial for binding to the talin head domain both in vivo and in vitro. Both approaches show that talin binding requires more than just this region. For example, peptides that correspond to a small region covering the NPLY motif of ß3 integrin and the preceding eight amino acid residues are unable to bind to talin by themselves in comparison to peptides covering the membrane proximal region of the tail. This leads to the suggestion that the NPLY domain by itself can mediate talin binding in the context of the full-length cytoplasmic tail but that the membrane proximal region is sufficient by itself. NMR studies showed that, the region of the integrin cytoplasmic tail that is perturbed upon binding of the talin head includes residues in the membrane proximal region and this is abolished when NPxY is mutated. Peptides only containing the region that includes the NPxY region showed a surprisingly low binding affinity for the talin head, about 100 times less than the affinity for PIP4, 5-kinase. In vivo results support the idea that talin binding requires interaction with multiple regions of the integrin cytoplasmic tail either simultaneously or sequentially (Tanentzapf, 2006a).
In contrast to the talin head, the C-terminal domain of talin behaves quite differently. It localizes to sites of adhesion and is also specifically and strongly recruited to the developing Z-lines. Like the talinH-GFP, the C-terminal region requires integrins for its localization but, by contrast, it is not recruited by overexpression of integrins or diß, nor is it recruited more efficiently when the levels of endogenous talin are reduced, instead it requires talin for its localisation. This is not too surprising because GFP-talinC does not include the recently defined C-terminal integrin-binding domain (Tanentzapf, 2006a).
Since GFP-talinC contains the I/LWEQ domain, which is thought to have actin-binding activity, its failure to colocalise with actin filaments is surprising. In contrast to other fusion proteins of GFP to actin-binding domains tested, the talin C-term does not decorate actin filaments in the developing muscle. Instead, it decorates the Z-lines and muscle ends, with a distribution that is very similar to that of the N-terminal domain of tensin, which also binds actin in vitro. Recently it was reported that the actin-binding site is cryptic, supporting the current findings. Some possible candidates for the recruitment of this domain seem unlikely, such as vinculin and talin itself. GFP-talinC does not include any of the identified vinculin-binding sites. The talinC domain does contain a recently identified homodimerization site, but if it is recruited solely by dimerizing with endogenous talin, such strong Z-line relative to muscle-end staining would not be expected, because talin is much more enriched at the muscle ends. In addition, it would be expected to be recruited by talin associated with diß, which it is not. Therefore, the identity of the molecules that mediate the localization of GFP-talinC remains a topic for further study (Tanentzapf, 2006a).
Different chimeric proteins containing the ßPS cytoplasmic domain did not have an equal ability to recruit talinH-GFP to the cell membrane and, thus, the extracellular domain of the chimera influences intracellular interactions. One clear difference is that, using extracellular domains from a receptor tyrosine kinase that has been mutated so that it signals independently of ligand-binding resulted in recruitment of talinH-GFP. The strength of the constitutive signalling produced by these mutant receptors correlated with their ability to recruit talinH-GFP as chimeric proteins. These findings point to dimerisation or oligomerisation as a key trigger for talin recruitment. This fits with the recent discovery of homotypic interactions between the transmembrane domains of integrin subunits, leading to a model where ligand-bound integrin heterodimers exist in a complex-cluster on the cell surface. There, the transmembrane and cytoplasmic domains of the ß subunit form trimers and those of the alpha subunits form dimers in the membrane, whereas outside the cell alphaß heterodimers bind to ligand. The increased recruitment of talinH-GFP to the dimers or trimers of the ß cytoplasmic domain could occur by cooperative binding, which would require some interaction between different talin head domains, or the oligomers could stabilise a conformation of the ß cytoplasmic domain that binds tightly to the talin head. This latter model gives an alternate explanation: the chimeras with the mutant forms of the receptor tyrosine kinase could induce the ßPS cytoplasmic domain to adopt a different conformation compared with the CD2 chimera, which bind to talin more tightly. At present, tools are not available to unambiguously distinguish between these different possibilities, but oligomerization is favored as the working model (Tanentzapf, 2006a).
The formation of ß subunit oligomers might also explain why overexpression of the ßPS integrin subunit is dominant negative on integrin adhesion – similar to that produced by diß. Formation of oligomers can be suppressed by co-overexpressing the alphaPS2 subunit, indicating that it is not overexpression of integrin heterodimers that is the problem, but free ß subunits. One explanation is that free ßPS subunits form – just like diß – homodimers or homotrimers at low levels, which can be transported to the plasma membrane where they recruit cytoplasmic proteins but cannot mediate extracellular adhesion. This might also explain why the dependence on dimerization for dominant negative activity that was observed has not been reported in other systems, because even a low level of dimerization may induce dominant negative activity (Tanentzapf, 2006a).
Two models have been proposed that explain the dominant negative effect of chimeric proteins containing the integrin ß subunit cytoplasmic tail: a feedback model, involving excess signalling, and a competition model, involving sequestration of key cytoplasmic proteins required for endogenous integrin function. The results suggest that sequestration of talin accounts for some but not all of the dominant negative effect. Talin is one of the few integrin-associated proteins tested that is recruited by diß and its overexpression partially suppressed the dominant negative activity. Mutants of diß that have lost talinH-GFP binding also have lost dominant negative activity. However, mutations in the C-terminal part of the ß tail still recruited talin yet also lost dominant activity, suggesting involvement of another protein. It is therefore hypothesized that, binding of a factor X to the C-terminus is also required for the dominant negative effect. The requirement for two factors received some support from the finding that coexpression of certain pairs of diß mutants partially restored dominant negative activity; the thinking is that the heterodimers formed can now recruit both talin and factor X (Tanentzapf, 2006a).
It hard to explain how diß can produce a dominant negative effect only when sequestering two proteins, because it would be expected that recruiting a single protein has some effect, and recruiting both has an additive effect. Instead very strong synergy is seen, that is easier to explain if factor X has a signalling role. For example, one speculative model is that factor X is a kinase that phosphorylates and inactivates talin when it is bound at the adjacent site on the ß cytoplasmic tail. Exchange of the inactivated talin would then lead to a gradual inactivation of the cytoplasmic pool of talin, which could be partially alleviated by increasing the amount of talin by overexpression. In the case of the endogenous integrins, there would have to be a mechanism to inactivate this inhibition. This could be achieved by one of the integrin-associated proteins that is recruited by the endogenous integrins but not by the diß-inactivating factor X or by removing the inhibitory phosphate from talin. The inactivation of the inhibition by endogenous integrins must not be efficient enough to counter the negative effect of diß. Thus, in the end the best model combines both sequestration and excessive signalling (Tanentzapf, 2006a).
A number of candidates for factor X that interact with the relevant region of the ß subunit cytoplasmic domain have already been identified, including filamin, non-muscle myosin, and Src. Non-muscle myosin can be recruited by the CD2ßPS chimera. Src is activated by binding to the C-terminal region the of the ß3 integrin cytoplasmic tail, and this is enhanced by clustering and homo-oligomerization of the integrins. The role of Src in integrin function in Drosophila has yet to be elucidated. Phosphorylated FAK was recruited by diß, and overexpression of FAK causes muscle detachment. By contrast, sequestration of FAK is unlikely to cause a defect because the absence of FAK did not cause defects in integrin-mediated adhesion in Drosophila. Thus, there are candidate kinases that interact with this region of the ß tail and could send inhibitory signals (Tanentzapf, 2006a).
This study found that when the integrin ß tail is placed on a heterologous transmembrane protein it recruited only some integrin associated proteins. In particular, talin and FAK were recruited, but not ILK, PINCH or tensin. This suggests additional input, required to assemble the full complement of proteins that contribute to the integrin-cytoskeleton link. Either of the two domains missing from these chimeras could provide the input: the extracellular ligand-binding domain, composed of both α and ß subunits, or the α subunit cytoplasmic domain. Since an integrin heterodimer lacking the α subunit cytoplasmic domain can mediate muscle attachment, the extracellular ligand-binding domain is favored. This could either provide unique conformational changes to the ß tail, or it could allow tension to be placed on the integrin-cytoskeletal linkage (Tanentzapf, 2006a).
Transmembrane adhesion receptors, such as integrins, mediate cell adhesion by interacting with intracellular proteins that connect to the cytoskeleton. Talin, one such linker protein, is thought to have two roles: mediating inside-out activation of integrins, and connecting extracellular matrix (ECM)-bound integrins to the cytoskeleton1. Talin's amino-terminal head, which consists of a FERM domain, binds an NPxY motif within the cytoplasmic tail of most integrin β subunits. This is consistent with the role of FERM domains in recruiting other proteins to the plasma membrane. The role of the talin-head-NPxY interaction in integrin function was tested in Drosophila. Introduction of a mutation that perturbs this binding in vitro into the isolated talin head disrupts its recruitment by integrins in vivo. Surprisingly, when engineered into the full-length talin, this mutation does not disrupt talin recruitment by integrins nor its ability to connect integrins to the cytoskeleton. However, it reduces the ability of talin to strengthen integrin adhesion to the ECM, indicating that the function of the talin-head-NPxY interaction is solely to regulate integrin adhesion (Tanentzapf, 2006b).
A model to explain these findings is as follows. It starts with the natural equilibrium between low- and high-affinity integrin conformations. For talin to activate integrins, it follows that the talin head must bind low-affinity integrins, but additional integrin-binding sites in talin may only interact with high-affinity, ligand-bound integrins. It is envisioned that integrin adhesion starts with transient high-affinity integrins binding the adjacent ECM and recruiting talin via the predicted novel integrin binding site. The bound talin clusters other high-affinity integrins by an interaction that does not involve the head (R367), but could involve IBS2. This leaves the head domain of the bound talin free to bind new low-affinity integrin heterodimers that diffuse into proximity, activating them so that they bind the ECM and further strengthen the adhesive cluster. This model, therefore, provides a mechanism for integrins to be preferentially activated close to existing adhesive sites (Tanentzapf, 2006b).
This work has two major implications: first, when the ability of the talin head to interact with the integrin cytoplasmic tail is compromised, talin can still localize to sites of adhesion and carry out its function as a cytoplasmic linker. This implies that talin forms different kinds of links to integrin to mediate its different functions. Second, disrupting the talin-head-integrin interaction reduces the ability of talin to function, resulting in defective attachment of integrin to the ECM. The results support the view that this is due to reduced conversion of integrins into a high-affinity state, and therefore that regulation of integrin affinity has an important role during development, as it generates strong adhesion of cells to an insoluble ECM (Tanentzapf, 2006b).
Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin β subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. This study defined the structure of a complex between talin and the membrane-proximal integrin β3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. Structure-based mutagenesis was used to engineer talin and β3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins (Wegener, 2007).
This study has revealed how the talin F3 domain is uniquely designed to activate integrins. The talin F3 domain forms a well-defined complex with the helix-forming MP region of the β-integrin tail, and this interaction holds the key to the molecular recognition required for activation. Mutations in integrin or talin that inhibit this interaction in vitro also prevent integrin activation in cells, and mutants with intermediate functional effects in cells retain a partial ability to form the F3-MP interaction. These findings are consistent with studies that identified a variety of activating mutations within the MP region of integrins, thus establishing that this region is critical for stabilizing the low-affinity conformation (Wegener, 2007).
The unique feature of talin F3 that promotes interaction with the MP region of β-integrin appears to be the flexible loop between strands S1 and S2 that forms a hydrophobic pocket that accepts the side chains of F727 and F730 in the complex. The talin mutation L325R within this pocket abolishes binding to the MP region and thus hinders activation. Other PTB-domain-containing proteins (DOK [“IRS-like”], Shc [”Shc-like”], and NUMB [”Dab-like”] lack such a mobile loop and so are unlikely to interact with the MP region. Indeed, DOK binds and inhibits integrin activation and binding occurs to the MD site but not the MP site. Several point mutants in talin also transform the F3 domain from an activator into an inhibitor. This is consistent with competition between endogenous talin and various PTB domains, including mutated talin F3, for the MD site. The concept of inhibition of talin activation by competitive binding to the β tails by a variety of proteins, especially those with PTB domains, may be an important feature in the regulation of integrin activity. These conclusions also apply to PTB (F3) subdomains in other FERM-domain proteins. The FERM-domain structures of band 4.1, ezrin, radixin, and moesin all have very short loops between strands 1 and 2 with no hydrophobic residues that could bury the two integrin phenylalanines (Wegener, 2007).
Migrating cells extend protrusions to establish new adhesion sites at their leading edges. One of the driving forces for cell migration is the directional trafficking of cell-adhesion molecules such as integrins. The endocytic adaptor protein Numb is an important component of the machinery for directional integrin trafficking in migrating cells. In cultured mammalian cells, Numb binds to integrin-βs and localizes to clathrin-coated structures (CCSs) at the substratum-facing surface of the leading edge. Numb inhibition by RNAi impairs both integrin endocytosis and cell migration toward integrin substrates. Numb is regulated by phosphorylation since the protein is released from CCSs and no longer binds integrins when phosphorylated by atypical protein kinase C (aPKC). Because Numb interacts with the aPKC binding partner PAR-3, a model is proposed in which polarized Numb phosphorylation contributes to cell migration by directing integrin endocytosis to the leading edge (Nishimura, 2007).
Numb localizes at a part of CCSs and functions in integrin endocytosis as a cargo-selective adaptor. Integrin is thought to be recycled from the tail to the front of migrating cells by endocytosis. However, many focal adhesions or focal complexes formed at the cell front disassemble behind the F-actin-rich lamellipodia. Numb mainly accumulated behind lamellipodia, although a certain population of Numb still remained and colocalized with integrin at the trailing edge. In addition, localization of Numb among CCSs correlated with the position of integrin adhesions, supporting the role of Numb in integrin endocytosis. Talin is a key molecule that tethers integrin to components of focal adhesions and actin stress fibers and is critical for focal-adhesion disassembly. Mutation of a conserved tyrosine residue within the integrin-β3 intracellular domain abolished the binding of both talin and Numb, suggesting that talin and Numb cannot bind to integrin simultaneously. Consistent with these observations, interaction of Numb with talin could not be detected. In addition, the binding of Numb to integrin does not activate the integrin extracellular domain, whereas the binding of talin does. The overexpression or knockdown of Numb does not directly affect cell adhesion. Thus, it appears that Numb does not actively promote focal-adhesion disassembly, but rather recruits free integrins without the components of focal adhesions to the AP-2 complex for internalization. Preferential localization of Numb around focal adhesions at the substratum-facing surface would facilitate recruitment of integrin during focal adhesion disassembly (Nishimura, 2007).
Recent genetic screening isolated Numb as a mutant defective for peripheral glia migration along axons in Drosophila (Edenfeld, 2007). Migration defects of postmitotic neurons have been described in Numb-knockout mice, indicating that Numb regulates particular cell migration in vivo. However, the defects of Numb knockdown on integrin endocytosis and cell migration are less marked than those of AP-2 and clathrin knockdown, suggesting that another adaptor molecule(s) may function in integrin endocytosis. A good candidate is disabled-2 (Dab2), which has a similar domain structure as Numb and binds to both components of clathrin-mediated endocytosis and to integrin-β. Dab2 is expressed in HeLa cells and positively controls cell adhesion and spreading. In contrast to Numb, Dab2 appears to preferentially localize to the apical surface. Thus, Numb and Dab2 may coordinately function in integrin endocytosis in different subcellular compartments for cell motility (Nishimura, 2007).
How does Numb localize at the substratum-facing surface and polarize toward the leading edge? Integrin adhesions could activate several intracellular signaling events and promote protein transport to adhesion sites by targeting microtubules and linking actin stress fibers. The actin cytoskeleton and/or adhesion itself are important for the preferential localization of Numb around adhesions. However, Numb still localized at the substratum-facing surface in the presence of cytochalasin-D, indicating that an additional mechanism may exist. Observations indicate that direct phosphorylation by aPKC may be a part of the regulatory mechanism underlying Numb localization at the substratum-facing surface. In addition, polarized localization of Numb toward the leading edge was lost upon aPKC knockdown. In support of these observations, asymmetric localization of Numb in Drosophila has been shown to be dependent on cortical actomyosin and the polarized localization/function of aPKC and PAR-3. Conclusive evidence will require isolation of the responsible motor(s) and anchor protein(s) for specific Numb localization (Nishimura, 2007).
Numb-full-3A, mutated at three phosphorylation sites, did not function as a constitutively active form that promotes integrin endocytosis and cell migration, but rather inhibited these processes. Similarly, both the phospho-mimic and nonphosphorylated form of μ2-adaptin, which is phosphorylated by AAK1, inhibit transferrin endocytosis, suggesting that clathrin-mediated endocytosis is tightly controlled by cycles of phosphorylation and dephosphorylation. Additional phosphorylation during endocytosis may be required for the dissociation of Numb from the binding proteins, integrin-β, and α-adaptin. Numb is indeed phosphorylated by several PKCs and CaMKs, and phosphatase inhibitor dramatically increases the phosphorylation level of Numb. Thus, local phosphorylation and dephosphorylation seems to allow Numb to localize defined CCSs around adhesion sites (Nishimura, 2007).
Trafficking of internalized integrin is regulated by growth factors and the extracellular matrix through several adaptors/kinases, including PI3-kinase, PKB/Akt, GSK3β, and PKCs. Several growth factors and adhesions indeed promote the recycling of integrins, leading to the upregulation of cell-surface expression, whereas treatment of cells with PDGF does not affect the internalization rate of integrin. The degree of colocalization and interaction of Numb with integrin-β1 was not significantly altered in HeLa cells before and after wounding. These data suggest that Numb functions constitutively in integrin endocytosis, although it is possibile that polarized migration promotes the internalization rate and amount of integrin endocytosis. It might be difficult to detect the changes in the interaction of Numb and integrin during migration due to the nature of rapid cycling of endocytosis and exocytosis and possibly due to the transient interaction. It has been reported that the inhibition of directional membrane trafficking causes membrane extension in all directions. Membrane trafficking controls the directionality of migrating cells. Taking into account the fact that Numb localization becomes polarized coincidently with directional migration, the subcellular region at which integrin is internalized and the subsequent coupling with the recycling processes could be important for efficient cell migration suitable for the particular environment (Nishimura, 2007).
Transmembrane helices of integrin alpha and beta subunits have been implicated in the regulation of integrin activity. Two mutations, glycine-708 to asparagine-708 (G708N)and methionine-701 to asparagine-701, in the transmembrane helix of the beta3 subunit enabled integrin alphaIIbbeta3 to constitutively bind soluble fibrinogen. Further characterization of the G708N mutant revealed that it induced alphaIIbbeta3 clustering and constitutive phosphorylation of focal adhesion kinase. This mutation also enhanced the tendency of the transmembrane helix to form homotrimers. These results suggest that homomeric associations involving transmembrane domains provide a driving force for integrin activation. They also suggest a structural basis for the coincidence of integrin activation and clustering (Li, 2003).
Other cytoplasmic interactions of integrins Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may
be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a
phenomenon termed integrin crosstalk. Ligation of the integrin alphavbeta3 inhibits both
phagocytosis and migration mediated by alpha5beta1 on the same cell, and the beta3 cytoplasmic tail is
necessary and sufficient for this regulation of alpha5beta1. Ligation of alpha5beta1 activates the calcium-
and calmodulin-dependent protein kinase II (see Drosophila CamKII). This activation is required for alpha5beta1-mediated
phagocytosis and migration. Simultaneous ligation of alphavbeta3 or expression of a chimeric molecule
with a free beta3 cytoplasmic tail prevents alpha5beta1-mediated activation of CamKII. Expression of a
constitutively active CamKII restores alpha5beta1 functions blocked by alphavbeta3-initiated integrin
crosstalk. Thus, alphavbeta3 inhibition of alpha5beta1 activation of CamKII is required for its role in
integrin crosstalk. Structure-function analysis of the beta3 cytoplasmic tail demonstrates a requirement for
Ser752 in beta3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and
Tyr759, implicating Ser752, but not beta3 tyrosine phosphorylation in initiation of the alphavbeta3 signal
for integrin crosstalk (Blystone, 1999).
Cell adhesion regulates the
kinase activity and subcellular localization of c-Abl (See Drosophila Abl oncogene). The subcellular localization of c-Abl was examined in C3H mouse fibroblasts during integrin alpha5beta1-dependent adhesion and spreading on fibronectin. When fibroblastic cells are
detached from the extracellular matrix, the kinase activity of both cytoplasmic and nuclear
c-Abl decreases, but there is no detectable alteration in the subcellular distribution.
Upon adhesion to the extracellular matrix protein fibronectin, a transient recruitment of
a subset of c-Abl to early focal contacts is observed coincident with the export of
c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated
within 5 min of adhesion, but the nuclear c-Abl is reactivated after 30 min, correlating
closely with its return to the nucleus and suggesting that the active nuclear c-Abl
originates in the cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the
cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear c-Abl is not
activated upon cell attachment. These results show that c-Abl activation requires cell
adhesion and that this tyrosine kinase can transmit integrin signals to the nucleus
where it may function to integrate adhesion and cell cycle signals. (Lewis, 1996).
The integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase that
can interact directly with the cytoplasmic domains of the beta1 and beta3 integrin subunits and whose
kinase activity is modulated by cell-extracellular matrix interactions. Overexpression of constitutively
active ILK results in loss of cell-cell adhesion, anchorage-independent growth, and tumorigenicity in
nude mice. Modest overexpression of ILK in intestinal epithelial cells as well as in
mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of
E-cadherin expression, translocation of beta-catenin to the nucleus, formation of a complex between
beta-catenin and the high mobility group transcription factor, LEF-1 (Drosophila homolog: Pangolin), and transcriptional activation by
this LEF-1/beta-catenin complex. LEF-1 protein expression is rapidly modulated by
cell detachment from the extracellular matrix, and LEF-1 protein levels are constitutively
up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by
activated H-ras or v-src oncogenes do not result in the activation of LEF-1/beta-catenin. The results
demonstrate that the oncogenic properties of ILK involve activation of the LEF-1/beta-catenin
signaling pathway, and also suggest ILK-mediated cross-talk between cell-matrix interactions and
cell-cell adhesion as well as components of the Wnt signaling pathway (Novak, 1998).
Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine-threonine protein kinase capable of interacting with the
cytoplasmic domains of integrin beta1, beta2, and beta3 subunits. Overexpression of ILK in epithelial cells disrupts
cell-extracellular matrix as well as cell-cell interactions; suppresses suspension-induced apoptosis (also called Anoikis), and
stimulates anchorage-independent cell cycle progression. In addition, ILK induces nuclear translocation of beta-catenin,
where the latter associates with a T cell factor/lymphocyte enhancer-binding factor 1 (TCF/LEF-1) to form an activated
transcription factor. ILK activity is rapidly, but transiently, stimulated upon the attachment of cells to
fibronectin, as well as by insulin, in a phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. Furthermore,
phosphatidylinositol(3,4,5)trisphosphate specifically stimulates the activity of ILK in vitro, and in addition, membrane
targeted constitutively active Pi(3)K activates ILK in vivo. ILK is an upstream effector of the
Pi(3)K-dependent regulation of both protein kinase B (PKB/AKT) and glycogen synthase kinase 3 (GSK-3). Specifically,
ILK can directly phosphorylate GSK-3 in vitro and when either stably or transiently overexpressed in cells, can inhibit GSK-3
activity, whereas the overexpression of kinase-deficient ILK enhances GSK-3 activity. In addition, kinase-active ILK can
phosphorylate PKB/AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of
PKB/AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB/AKT
activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor
mediated activation of PKB/AKT, and the inhibition of GSK-3 (Delcommenne 1998).
CD2 is a cell surface glycoprotein expressed on most T lymphocytes that is generally viewed as a cell
adhesion molecule and, in this capacity, contributes to T cell receptor (TCR) signaling. The CD2 molecule is one of several T lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated
cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta1-integrin-mediated
adhesion to fibronectin. The HL60 cell line was used to map critical residues within the CD2 cytoplasmic domain
involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed
for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated
in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich
sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of beta1 integrin activity.
CD2-induced increases in beta1 integrin activity can be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or
by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain
mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K
enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the
constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the
K-G-P-P-L-P motif to alanines prevents CD2-mediated activation of integrin function and PI 3-K activity but not
mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocks CD2-mediated
activation of MAP kinase but has no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2
critical for PI 3-K-dependent regulation of beta1 integrin adhesion by CD2. In addition, these studies suggest that
CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion (Kivens, 1998).
CRKL is an SH2-SH3-SH3 adapter protein that is a major substrate of the BCR/ABL oncogene. The function of CRKL in normal cells
is unknown. In cells transformed by BCR/ABL, CRKL is associated with two focal adhesion proteins,
tensin and paxillin, suggesting that CRKL could be involved in integrin signaling. CRKL rapidly associates with tyrosine-phosphorylated proteins after cross-linking of beta1 integrins with fibronectin or
anti-beta1 integrin monoclonal antibodies. The major tyrosine-phosphorylated CRKL-binding protein in megakaryocytic MO7e cells
was identified as p120(CBL: see Drosophila Cbl), the cellular homolog of the v-Cbl oncoprotein. However, in the lymphoid H9 cell line, the major
tyrosine-phosphorylated CRKL-binding protein is p110(HEF1). In both cases, this binding is mediated by the CRKL SH2 domain.
Interestingly, although both MO7e and H9 cells express p120(CBL) and p110(HEF1), beta1 integrin cross-linking induces tyrosine
phosphorylation of p120(CBL) (but not p110[HEF1]) in MO7e cells and of p110(HEF1) (but not p120[CBL]) in H9 cells. In both cell
types, CRKL is constitutively complexed to C3G, SOS, and c-ABL through its SH3 domains; the stoichiometry of these
complexes does not change upon integrin ligation. Thus, in different cell types CRKL and its SH3-associated proteins may form
different multimeric complexes depending on whether p120(CBL) or p110(HEF1) is tyrosine-phosphorylated after integrin ligation.
The shift in association of CRKL and its SH3-associated proteins from p120(CBL) to p110(HEF1) could contribute to different
functional outcomes of "outside-in" integrin signaling in different cells (Sattler, 1997).
Mutant macrophages that are deficient in expression of Src-family kinases have been used to define
an integrin signaling pathway that is required for macrophage adhesion and migration. Following the
ligation of surface integrins by fibronectin, the p120c-cbl (Cbl) protein rapidly becomes tyrosine
phosphorylated and associates with the Src-family kinases Fgr and Lyn. In hck-/-fgr-/-lyn-/-
triple mutant cells, which are defective in spreading on fibronectin-coated surfaces in vitro and
show impaired migration in vivo, Cbl tyrosine phosphorylation is blocked; Cbl protein levels are
low; adhesion-dependent translocation of Cbl to the membrane is impaired, and Cbl-associated,
membrane-localized, phosphatidylinositol 3 (PI-3)-kinase activity is dramatically reduced. In
contrast, adhesion dependent activation of total cellular PI-3 kinase activity is normal in mutant cells,
demonstrating that it is the membrane-associated fraction of PI-3 kinase that is most critical in
regulating the actin cytoskeletal rearrangements that lead to cell spreading. Treatment of wild-type cells
with the Src-family-specific inhibitor PP1, or with Cbl antisense oligonucleotides or pharmacological
inhibitors of PI-3 kinase, blocks cell spreading on fibronectin surfaces. These data provide a
molecular description for the role of Src-family kinases Hck, Fgr and Lyn in beta1-integrin signal transduction in macrophages (Meng, 1998).
The central role of PI-3 kinase in initiating actin cytoskeletal rearrangements, membrane ruffling
and cell migration following integrin clustering has been demonstrated in several cell systems,
including neutrophils, fibroblasts, platelets and, most recently, carcinoma cells. In all these
systems, different integrin subunits are involved; however, in each case, integrin-mediated
adhesion leads to activation of PI-3 kinase and accumulation of D3 phosphoinositides. Treatment
of cells with PI-3 kinase-specific inhibitors leads to the blockade of cell spreading. In carcinoma
cells, activation of PI-3 kinase has been associated with a specific integrin-dependent function:
promotion of invasion in vitro. However, none of the previous studies recognized the recognition
that association with Cbl (or other adaptor proteins expressed in non-hematopoietic cells) and
cytoskeletal translocation of PI-3 kinase are both critical events leading to actin cytoskeletal
rearrangement. In this regard, it is the fortuitous observation in the Src-family kinase knockout
macrophages that total cellular activation of PI-3 kinase is normal, yet the association of PI-3
kinase with Cbl and translocation of PI-3 kinase activity are impaired. This allows for the inference
that the cytoskeletal localization of the PI-3 kinase-Cbl complex (not just overall activation of lipid
kinase activity) is the critical event in regulating integrin-induced cytoskeletal rearrangements. It is believed that the abnormal subcellular localization of PI-3 kinase in adherent hck-/-fgr-/-lyn-/-
macrophages is a cause of the abnormal cytoskeletal actin in these cells, rather than an effect of it.
This is based on the rationale that treatment of wild-type cells with Cbl antisense oligonucleotides
or PI-3 kinase inhibitors produces the same cellular phenotype of impaired Fn-induced cell
spreading observed in the mutant macrophages. In macrophages the downstream signaling pathways such as MAP
kinase or NF-kappaB activation that follow integrin clustering do not require the major
Src-family kinases expressed in these cells. FAK has also been shown not to be required for integrin-induced MAP kinase
activation in fibroblasts. Hence the possibility that dual and separable
integrin signaling pathways lead to actin cytoskeletal rearrangements, versus activation of the MAP
kinase cascade, may be a generalizable conclusion (Meng, 1998 and references).
pp72(syk) is essential for the development and function of several hematopoietic cells; it becomes
activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk
is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to
study Syk activation by the platelet integrin, alphaIIbbeta3. As in platelets, Syk undergoes tyrosine
phosphorylation and activation during CHO cell adhesion to alphaIIbbeta3 ligands, including fibrinogen.
This involved Syk autophosphorylation and the tyrosine kinase activity of Src; it exhibits two novel
features: Cell motility on extracellular-matrix (ECM) substrates
depends on the regulated generation of force against the
substrate through integrins. Integrin-mediated traction forces can be
selectively modulated by the tyrosine kinase Src. In
Src-deficient fibroblasts, cell spreading on the ECM
component vitronectin is inhibited, while the strengthening
of linkages between integrin vitronectin receptors and the
force-generating cytoskeleton in response to substrate
rigidity is dramatically increased. In contrast, Src deficiency
has no detectable effects on fibronectin-receptor function.
Finally, truncated Src (lacking the kinase domain)
co-localizes to focal-adhesion sites with alphav but
not with beta1 integrins. These data are consistent
with a selective, functional interaction between Src and the
vitronectin receptor. This interaction takes place at an integrin-cytoskeleton
interface to regulate cell spreading and migration (Felsenfeld, 1999).
The precise site of action of Src and its location in the signaling cascade that regulates vitronectin-receptor function remain unclear. The Src-family kinases Fyn and Src bind the focal-adhesion kinase (FAK), allowing an indirect interaction between these kinases and the cytoplasmic tail of the integrin beta-subunit. Fyn may also selectively associate with the fibronectin receptor through an indirect association with the integrin alpha5 subunit. However, these pathways have been identified on the basis of biochemical association and changes in kinase activity downstream of integrin activation ('outside-in' signaling), in contrast to the modulation of integrin function by Src described here (Felsenfeld, 1999).
These results indicate that Src may act at or near the cytoplasmic membrane. First, the vitronectin-dependent localization of Src to stable pools on the bottom of the cell and the co-localization of Src with alphav but not beta1 integrins is consistent with the selective association of Src and vitronectin receptor at the membrane. Moreover, Src co-precipitates with vitronectin receptor but not fibronectin receptor in whole-cell lysates, indicating a selective association that is consistent with the functional specificity shown in this study. Finally, force generation through the vitronectin receptor and fibronectin receptor is likely to involve many of the same proteins, including actin/myosin and structural components of the focal-adhesion complex such as vinculin, talin and others. Therefore, it is inferred that Src acts at or near the cytoplasmic tail of the vitronectin receptor, at the point at which the pathways transducing force through vitronectin and fibonectin receptors diverge. The ability of cells to recognize and respond to the rigidity of the cellular environment implies the existence of a force-sensing apparatus within the cell. These results indicate that Src forms a regulatory component of the complex that links integrins to the cytoskeleton (Felsenfeld, 1999).
With the exception of the divergent beta4 and beta8 chains, the integrin beta subunit
cytoplasmic domains are short and highly conserved sequences. Consensus motifs are
found among the different cytoplasmic beta chains. Experiments using chimeric
receptors demonstrate that the 47 amino acids of the beta1 subunit cytoplasmic
domain contain sufficient information to target integrins to adhesion plaques. Three
clusters of amino acids, named cyto-1, cyto-2 and cyto-3, seem to contribute to this
localization. Cyto-2 and cyto-3 exhibit NPXY motifs. At present, the exact function of
these motifs remains unknown but it is likely that these sequences are involved in
protein-protein interactions. Although NPXY motifs often act as internalization signals
at the cytoplasmic tail of membrane receptors, previous results have shown that the
two NPXY motifs are not responsible for the alpha5beta1 integrin endocytosis.
Within the integrin beta1 cytoplasmic
tail, the two NPXY motifs are required for the recruitment of the integrin in focal
adhesions. These two motifs appear to control (but do not
belong to) the talin-binding sites. The analysis of the phenotypes of NPXY
mutants reveals that the interaction of talin with the beta1 cytosolic domain is not
sufficient to target the integrins to focal adhesions (Vignoud, 1995).
The cytoplasmic domains of integrins are essential for cell adhesion. A novel protein, ICAP-1
(integrin cytoplasmic domain- associated protein-1) binds to the 1 integrin cytoplasmic domain. The interaction between
ICAP-1 and beta1 integrins is highly specific, as demonstrated by the lack of interaction between ICAP-1 and the cytoplasmic
domains of other beta integrins, and requires a conserved and functionally important NPXY sequence motif found in the
COOH-terminal region of the beta1 integrin cytoplasmic domain. Mutational studies reveal that Asn and Tyr of the NPXY
motif and a Val residue located NH2-terminal to this motif are critical for the ICAP-1 binding. Two isoforms of ICAP-1, a
200-amino acid protein (ICAP-1alpha) and a shorter 150-amino acid protein (ICAP-1beta), derived from alternatively
spliced mRNA, are expressed in most cells. ICAP-1alpha is a phosphoprotein; the extent of its phosphorylation is
regulated by a two-part cell-matrix interaction: (1) an enhancement of ICAP-1alpha phosphorylation is observed when cells are
plated on fibronectin-coated but not on nonspecific poly-L-lysine-coated surface. (2) The expression of a constitutively
activated RhoA protein, disrupting the cell-matrix interaction, results in dephosphorylation of ICAP-1alpha. The regulation of
ICAP-1alpha phosphorylation by the cell-matrix interaction suggests an important role of ICAP-1 during integrin-dependent
cell adhesion (Chang, 1997).
The integrin family of adhesion receptors are involved in cell growth, migration and tumour metastasis.
Integrins are heterodimeric proteins composed of an alpha and a beta subunit, each with a large
extracellular domain,
a single transmembrane domain, and a short cytoplasmic domain. The dynamic regulation of
integrin affinity for ligands in response to cellular signals is central to integrin function. This process is
energy dependent and is mediated through integrin cytoplasmic domains. However, the cellular
machinery regulating integrin affinity remains poorly understood. This paper describes a genetic strategy
to disentangle integrin signalling pathways. Dominant suppression occurs when overexpression of
isolated integrin beta1 cytoplasmic domains blocks integrin activation. Proteins involved in integrin
signaling were identified by their capacity to complement dominant suppression in an expression
cloning scheme. CD98, an early T-cell activation antigen that associates with functional integrins, is
found to regulate integrin activation. Furthermore, antibody-mediated crosslinking of CD98 stimulates
beta1 integrin-dependent cell adhesion. These data indicate that CD98 is involved in regulating integrin
affinity, and validate an unbiased genetic approach to analyzing integrin signaling pathways (Fenczik, 1997).
Adhesion of human primary skin fibroblasts and ECV304 endothelial cells either to immobilized matrix proteins or to anti-beta1 or anti-alphav integrin antibodies, stimulates
tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30
min after adhesion, and it occurs in the absence of receptor ligands. Similar results are observed with EGF receptor-transfected NIH-3T3 cells. Use of a
kinase-negative EGF receptor mutant demonstrates that the integrin-stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase
activity. Integrin-mediated EGF receptor activation leads to Erk-1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin
AG1478 and by expression of a dominant-negative EGF receptor mutant. EGF receptor and Erk-1/MAP kinase activation by integrins does not lead per se
to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular
matrix-mediated cell survival. Adhesion-dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing
this molecule above a threshold level [5x10(3) receptors per cell]. These results demonstrate that integrin-dependent EGF receptor activation is a novel
signaling mechanism involved in cell survival and proliferation in response to extracellular matrix (Moro, 1998).
Integrins play pivotal roles in supporting shear- and mechanical-stress-resistant cell adhesion and
migration. These functions require the integrity of the short beta subunit cytoplasmic domains, which
contain multiple, highly conserved tyrosine-based endocytic signals, typically found in receptors
undergoing regulated, clathrin-dependent endocytosis. It is hypothesized that these sequences may
control surface integrin dynamics in statically adherent and/or locomoting cells via regulated
internalization and polarized recycling of the receptors. By using site-directed mutagenesis and ectopic
expression of the alphaL/beta2 integrin in Chinese hamster ovary cells, it has been found that Y735 in the
membrane-proximal YRRF sequence is selectively required for recycling of spontaneously internalized
receptors to the cell surface and to growth factor-induced membrane ruffles. Disruption of this motif by
non-conservative substitutions has no effect on the receptor's adhesive function, but diverts internalized
integrins from a recycling compartment into a degradative pathway. Conversely, the non-conservative
F754A substitution in the membrane-proximal NPLF sequence abrogates ligand-dependent adhesion
and spreading without affecting receptor recycling. Both of these mutants display a severe impairment
in ligand-supported migration, suggesting the existence in integrin cytoplasmic domains of independent
signals regulating apparently unrelated functions that are required to sustain cell migration over specific
ligands (Fabbri, 1999).
The mechanism by which platelets regulate the function of integrin alphaIIbbeta3 (or GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may involve the
binding of proteins or other factors to integrin cytoplasmic domains. To identify candidate cytoplasmic domain binding proteins, a human fetal liver
cDNA library was screened in the yeast two-hybrid system, using the alphaIIb cytoplasmic domain as 'bait', and a novel 855-base pair clone was isolated. The open reading frame
encodes a novel 191-amino acid polypeptide (termed CIB for calcium- and integrin-binding protein) that appears to be specific for the cytoplasmic domain of
alphaIIb, since it does not interact with the alphav, alpha2, alpha5, beta1, or beta3 integrin cytoplasmic domains in the yeast two-hybrid system. This protein has
sequence homology to two known Ca2+-binding regulatory proteins; calcineurin B (58% similarity) and calmodulin (56% similarity), and has two EF-hand motifs
corresponding to the two C-terminal Ca2+ binding domains of these proteins. Moreover, recombinant CIB specifically binds 45Ca2+ in blot overlay assays. Using
reverse transcriptase-polymerase chain reaction and Western blot analysis, CIB mRNA and protein ( approximately 25 kDa), respectively, was detected in human
platelets. An enzyme-linked immunosorbent assay, using either immobilized recombinant CIB or monoclonal antibody-captured alphaIIbbeta3, indicates a
specific interaction between CIB and intact alphaIIbbeta3. These results suggest that CIB is a candidate regulatory molecule for integrin alphaIIbbeta3 (Naik, 1999).
Recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides have been used to study their in vitro complexation and ligand binding capacity by surface
plasmon resonance. alpha/beta heterodimerization occurs in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations are not required for
this association but stabilize the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation is impaired by the R995A substitution or the
KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bond
to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding
of CIB to intact alphaIIbbeta3 occurs preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB
with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site,
spontaneously exposed by the free alphaIIb peptide. Since CIB does not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevent activated alphaIIbbeta3
occupancy by PAC-1, it is concluded that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor
occupancy event (Vallar, 1999).
The alphaIIbbeta3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating
platelet aggregation. Fibrinogen binding to alphaIIbbeta3 subsequently induces a cascade of intracellular signaling events. The molecular mechanisms of this
bi-directional alphaIIbbeta3-mediated signaling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. A novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein), interacts specifically with the alphaIIb cytoplasmic
domain in the yeast two-hybrid system. Further analysis of numerous tissues and cell lines indicates that CIB mRNA and protein are widely expressed. In addition,
isothermal titration calorimetry indicates that CIB binds to an alphaIIb cytoplasmic-domain peptide in a Ca(2+)-dependent manner, with moderate affinity and 1:1 stoichiometry. In aggregated platelets, endogenous CIB and alphaIIbbeta3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel
manner, demonstrating that the cellular localization of CIB is regulated, potentially by alphaIIbbeta3. Thus CIB may contribute to integrin-related functions by
mechanisms involving Ca(2+)-modulated binding to the alphaIIb cytoplasmic domain and changes in intracellular distribution (Shock, 1999).
Integrin receptors play an important role during cell migration by mediating
linkages and transmitting forces between the extracellular matrix and the actin
cytoskeleton. The mechanisms by which these linkages are regulated and released
during migration are not well understood. Cell-permeable
inhibitors of the calcium-dependent protease calpain inhibit both beta1 and
beta3 integrin-mediated cell migration. Calpain inhibition specifically
stabilizes peripheral focal adhesions, increases adhesiveness, and decreases the
rate of cell detachment. Furthermore, these inhibitors alter the fate of
integrin receptors at the rear of the cell during migration. A Chinese hamster
ovary cell line expressing low levels of calpain I also shows reduced migration
rates with similar morphological changes, further implicating calpain in this
process. Taken together, the data suggest that calpain inhibition modulates cell
migration by stabilizing cytoskeletal linkages and decreasing the rate of
retraction of the cell's rear. Inhibiting calpain-mediated proteolysis may
therefore be a potential therapeutic approach to control pathological cell
migration such as tumor metastasis (Huttenlocher, 1997).
Integrin-induced adhesion leads to cytoskeletal reorganizations, cell migration,
spreading, proliferation, and differentiation. The details of the signaling
events that induce these changes in cell behavior are not well understood but
they appear to involve activation of Rho family members that activate signaling
molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases.
The result is the formation of focal complexes, focal adhesions, and bundles and
networks of actin filaments that allow the cell to spread. The present study
shows that mu-calpain is active in adherent cells, that it cleaves proteins
known to be present in focal complexes and focal adhesions, and that
overexpression of mu-calpain increases the cleavage of these proteins, induces
an overspread morphology and induces an increased number of stress fibers and
focal adhesions. Inhibition of calpain with membrane permeable inhibitors or by
expression of a dominant negative form of mu-calpain results in an inability of
cells to spread or to form focal adhesions, actin filament networks, or stress
fibers. Cells expressing constitutively active Rac1 can still form focal
complexes and actin filament networks (but not focal adhesions or stress fibers)
in the presence of calpain inhibitors; cells expressing constitutively active
RhoA can form focal adhesions and stress fibers. Taken together, these data
indicate that calpain plays an important role in regulating the formation of
focal adhesions and Rac- and Rho-induced cytoskeletal reorganizations and that
it does so by acting at sites upstream of both Rac1 and RhoA (Kulkarni, 1999).
Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell
behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal
adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. Clusters of integrin that form upstream of Rac activation have been identified. These clusters contain a Rac-binding protein(s) and appear to
be involved in Rac activation. The integrin clusters contain calpain and calpain-cleaved ß3 integrin, while the focal complexes and focal
adhesions (that form once Rac and Rho are activated) do not. Moreover, the integrin clusters are dependent on calpain for their
formation. In contrast, while Rac- and Rho-GTPases are dependent on calpain for their activation, formation of focal complexes and focal adhesions by
constitutively active Rac or Rho, respectively, occurs even when calpain inhibitors are present. Taken together, these data are consistent with a model in which
integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin,
and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s) (Bialkowska, 2000).
It is possible to speculate on the way in which an action of calpain induces the formation of integrin clusters. In the present study, the availability of antibodies that
selectively recognize the calpain-cleaved ß3-integrin subunit allowsthis protein to be used as a marker to show that a calpain-cleaved protein is
present in the clusters. However, the presence of cleaved integrin does not comment on whether or not cleavage of this protein is involved in formation of the clusters.
Many other cytoskeletal proteins and signaling molecules present in integrin complexes are known to be cleaved by calpain. The platelet is the cell in which the calpain-induced
cleavage of proteins involved in integrin signaling has been studied in the most detail. In the platelet, proteins that are cleaved include spectrin, actin-binding protein, dystrophin-related protein, protein kinase C, cortactin, tyrosine
phosphatase PTP-1B, and the ß3 integrin subunit. While several of these are cleaved in endothelial cells spreading on an integrin substrate, it is not possible to conclude which protein must be cleaved for spreading to occur. Likewise, it is not possible at this point to determine
which substrate must be cleaved for the integrin clusters identified in this study to form (Bialkowska, 2000).
Thy-1 is a glycosyl phosphatidylinositol (GPI)-anchored glycoprotein of the immunoglobulin superfamily (IgSF) expressed in various cell types, particularly those of the T cell lineage and the neuronal system. In neurons, Thy-1 expression is developmentally regulated, whereby both the initial appearance and ultimate distribution are controlled to ensure that Thy-1 is excluded from regions of axonal growth. Thy-1 expression is preferentially initiated toward the end of axon extension, consistent with the idea that it might participate in stabilizing existing neuronal connections and inhibiting future neurite outgrowth. Interestingly, Thy-1-deficient mice breed and behave normally, despite the fact that this protein is highly expressed in the adult brain. These mice show an impairment in long-term potentiation, which does not appear to affect spatial learning. Thy-1 appears to be involved in cell adhesion and activation. For instance, Thy-1 promotes the adhesion of thymocytes to thymic epithelia, the adhesion of CTL clones to L cells, T cell activation, and the adhesion of a Thy-1-transfected lymphoma to astrocytes. Furthermore, an astrocytic binding site for neuronal Thy-1 has been described, whereby the interactions between Thy-1 and the putative ligand modulate neurite outgrowth (Leyton, 2001).
Integrins often recognize short peptide segments containing an RGD motif, whereby the essential nature of the aspartic acid residue in this context was first identified in the central integrin binding domain of fibronectin. RGD peptides or related motifs are now known to be present in many cell-surface molecules, like L1, that interact with integrins. Interestingly, the alignment of the Thy-1 sequences from human, mouse, and rat led to the identification of a single RLD motif in a highly conserved sequence element. Since RLD is a binding
motif for integrins alphaVß3 and alphaMß2, the hypothesis that Thy-1 might indeed function as a heterophilic ligand for members of the integrin family was tested. In this context, alphaVß3 integrin appears to be the more likely candidate, since this molecule is present on the surface of astrocytes, while the expression of alphaMß2 is restricted to leukocytes (Leyton, 2001).
Engagement and clustering of integrin receptors directly initiate a variety of signal transduction events, including an increase in tyrosine phosphorylation of a subset of proteins, activation of serine-threonine kinases, and alterations in cellular phospholipid and calcium levels. These events are associated with the formation of focal adhesions, specialized sites of adhesion formed by many cells in culture that are known to be important in cell spreading and motility. Focal adhesions contain a variety of structural (e.g., talin, vinculin, and alpha-actinin), signaling (focal adhesion kinase [FAK] and Src-family kinases), and adaptor molecules (including paxillin, tensin, and p130Cas) and represent the intracellular sites in which tyrosine phosphorylation levels are highest (Leyton, 2001).
A ß3 integrin in astrocytes, most likely expressed as alphaVß3, is a receptor for Thy-1. Furthermore, Thy-1 binding to astrocytes induces cell-signaling events specifically linked to focal adhesion formation, promoting astrocyte attachment and spreading. The addition of Thy-1 to matrix-bound astrocytes induces recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increases tyrosine phosphorylation of proteins such as p130Cas (see CAS/CSE1 segregation protein) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone is sufficient to promote focal adhesion formation and phosphorylation on tyrosine. Hence, as for other IgSF molecules, Thy-1 interaction with ß3 integrin may elicit bidirectional signaling between neurons and astrocytes (Leyton, 2001).
The Nck-interacting kinase (NIK: Drosophila homolog Misshapen), a member of the STE20/germinal center kinase (GCK) family, has been identified as a partner for the beta1A integrin cytoplasmic domain. NIK is expressed in the nervous system and other tissues in mouse embryos and colocalizes with actin and beta1 integrin in cellular protrusions in transfected cells. To demonstrate the functional significance of this interaction, Caenorhabditis elegans was used, since it has only one beta (PAT-3) integrin chain, two alpha (INA-1 and PAT-2) integrin chains, and a well-conserved NIK ortholog (MIG-15). Using three methods, it has been shown that reducing mig-15 activity results in premature branching of commissures. A significant aggravation of this defect is observed when mig-15 activity is compromised in a weak ina-1 background. Neuronal-specific RNA interference against mig-15 or pat-3 leads to similar axonal defects, thus showing that both mig-15 and pat-3 act cell autonomously in neurons. A genetic interaction occurs between mig-15, ina-1, and genes that encode Rac GTPases. This study provides the first evidence that the kinase NIK and integrins interact in vitro and in vivo. This interaction is required for proper axonal navigation
in C. elegans (Poinat, 2002).
The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrin biological functions by binding to cytoplasmic proteins. Most integrin beta tails contain one or two NPXYF motifs that can form beta turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-alpha bind to integrin beta tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction the binding of a series of recombinant PTB domains to a panel of short integrin beta tails was examined. In addition to the known integrin-binding proteins, Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domains bind to integrin tails. Furthermore, Dok-1 physically associates with integrin alpha IIb beta 3. Mutations of the integrin beta tails confirm that these interactions are canonical PTB domain-ligand interactions: (1) the interactions were blocked by mutation of an NPXY motif in the integrin tail; (2) integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. This specificity, and a molecular model of an integrin beta tail-PTB domain interaction, was used to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in beta 7 and beta 3 tails. These data establish that short integrin beta tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism (Calderwood, 2003).
A direct interaction occurs between the β1 integrin cytoplasmic tail and Rab25, a GTPase that has been linked to tumor aggressiveness and metastasis. Rab25 promotes a mode of migration on 3D matrices that is characterized by the extension of long pseudopodia, and the association of the GTPase with α5β1 promotes localization of vesicles that deliver integrin to the plasma membrane at pseudopodial tips as well as the retention of a pool of cycling α5β1 at the cell front. Furthermore, Rab25-driven tumor-cell invasion into a 3D extracellular matrix environment is strongly dependent on ligation of fibronectin by α5β1 integrin and the capacity of Rab25 to interact with β1 integrin. These data indicate that Rab25 contributes to tumor progression by directing the localization of integrin-recycling vesicles and thereby enhancing the ability of tumor cells to invade the extracellular matrix (Caswell, 2007).
At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. This study reports that postsynaptic β3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting β3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of β3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of β3 integrins, and in turn, β3 integrins are required for synaptic scaling. These findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength (Cingolani, 2008).
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These studies
demonstrate that Syk is a proximal component in alphaIIbbeta3 signaling and is regulated as a
consequence of intimate functional relationships with the alphaIIbbeta3 cytoplasmic tails and with Src
or a closely related kinase. There are fundamental differences in the activation of Syk by
alphaIIbbeta3 and immune response receptors, suggesting a unique role for integrins in Syk function (Gao, 1997).
myospheroid:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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