Table of contents

Mutational alteration of integrins

The analysis of C. elegans ina-1 alpha integrin mutants provides the first genetic evidence that migrating neurons require integrins. Mosaic analysis and expression studies show that ina-1 acts autonomously in cells to promote their migrations. Although axons generally extend to their normal targets in ina-1 mutants, bundling of axons into fascicles is defective, defining a previously unrecognized role for integrins. In addition to these neuronal phenotypes, ina-1 mutants also display many morphogenetic defects. The C. elegans INA-1 alpha integrin subunit associates with the PAT-3beta subunit in vivo, suggesting that these proteins function together in cell migration, axon fasciculation, and morphogenesis (Baum 1997).

Leukocytes and platelets require stimulation for optimal beta3 integrin receptor function, whereas beta3 function is constitutive in many other cells. The molecular mechanisms that enhance integrin function in stimulated hematopoietic cells are poorly understood. Phosphorylation of the beta3 cytoplasmic tail is a recently described but prevalent phenomenon, with unknown effects on alphavbeta3 function. Mutation of the beta3 cytoplasmic tail tyrosine 747 to phenylalanine (Y747F) prevents beta3 tyrosine phosphorylation in two cell lines. Whereas this mutation has no effect on alphavbeta3-mediated adhesion in a cell with constitutive beta3 function, it completely abolishes adhesion and clot retraction by a cell that requires stimulation for beta3 function. Ligand-induced conformational change as detected by LIBS-1 antibody occurs normally in Y747F mutant alphavbeta3. Thus, tyrosine 747 of beta3 is required for stimulation of alphavbeta3-mediated adhesion, probably due to its phosphorylation. Because the motif in beta3 required for tyrosine phosphorylation is shared by several integrin beta-chains, this may be a conserved mechanism for regulation of integrin-dependent adhesion (Blystone, 1997).

beta1A integrin subunits with point mutations of the cytoplasmic domain were expressed in fibroblasts derived from beta1-null stem cells. beta1A in which one or both of the tyrosines of the two NPXY motifs (Y783, Y795) were changed to phenylalanines form active alpha5beta1 and alpha6beta1 integrins that mediated cell adhesion and support assembly of fibronectin. Mutation of the proline in either motif (P781, P793) to an alanine or mutation of a threonine in the inter-motif sequence (T788) to a proline, results in poorly expressed, inactive beta1A. Y783,795F cells develop numerous fine focal contacts and exhibit motility on a surface. When compared with cells expressing wild-type beta1A or beta1A with the D759A activating mutation of a conserved membrane-proximal aspartate, Y783, 795F cells have impaired ability to transverse filters in chemotaxis assays. Analysis of cells expressing beta1A with single Tyr to Phe substitutions indicates that both Y783 and Y795 are important for directed migration. Actin-containing microfilaments of Y783,795F cells are shorter and more peripheral than microfilaments of cells expressing wild-type beta1A. These results indicate that change of the phenol side chains in the NPXY motifs to phenyl groups (which cannot be phosphorylated) has major effects on the organization of focal contacts and cytoskeleton and on directed cell motility (Sakai, 1998).

The cytoplasmic domain of the integrin beta4 subunit mediates both association with the hemidesmosomal cytoskeleton and recruitment of the signaling adaptor protein Shc. To examine the significance of these interactions during development, mice were generated carrying a targeted deletion of the beta4 cytoplasmic domain. Analysis of homozygous mutant mice indicates that the tail-less alpha6beta4 binds efficiently to laminin 5, but is unable to integrate with the cytoskeleton. Accordingly, these mice display extensive epidermal detachment at birth and die immmediately thereafter from a syndrome resembling the human disease junctional epidermolysis bullosa with pyloric atresia (PA-JEB). In addition, a significant proliferative defect is found. Specifically, there is a reduction in the number of precursor cells in the intestinal epithelium, which remains adherent to the basement membrane, and in intact areas of the skin, and post-mitotic enterocytes display increased levels of the cyclin-dependent kinase inhibitor p27(Kip). These findings indicate that the interactions mediated by the beta4 tail are crucial for stable adhesion of stratified epithelia to the basement membrane and for proper cell-cycle control in the proliferative compartments of both stratified and simple epithelia (Murgia, 1998).

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in beta1 integrin-mediated adhesion of T cells to fibronectin. Used in this study was a mutant of the Jurkat T cell line, designated A1. This mutant lacks protein and mRNA expression of the beta1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin can be restored upon transfection of a wild-type human beta1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation does not occur if the carboxy-terminal five amino acids of the beta1 tail are truncated or if either of two well-conserved NPXY motifs are deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrates a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduce adhesion induced by direct stimulation of the beta1 integrin with the activating beta1 integrin-specific mAb TS2/16, although the effects are not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the beta1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of beta1 integrin structure and function in human T cells (Romzek, 1998).

ß1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of ß1 integrin in skin, mice were generated with a keratinocyte-restricted deletion of the ß1 integrin gene using the cre-loxP system. Mutant mice develop severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles are removed by infiltrating macrophages. The epidermis of the back skin becomes hyperthickened, the basal keratinocytes show reduced expression of alpha6ß4 integrin, and the number of hemidesmosomes decreases. Basement membrane components are atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal-epidermal junction. In contrast, the integrity of the basement membrane surrounding the ß1-deficient hair follicle is not affected. Finally, the dermis becomes fibrotic. These results demonstrate an important role of ß1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiaton and proliferation, and in the formation and/or maintenance of hemidesmosomes (Brakebusch, 2000).

Embryonal stem (ES) cells that are homozygous null for the ß1 integrin subunit fail to differentiate into keratinocytes in vitro but do differentiate in teratomas and wild-type/ß1-null chimeric mice. The failure of ß1-null ES cells to differentiate in culture might be the result of defective extracellular matrix assembly or reduced sensitivity to soluble inducing factors. By culturing embryoid bodies on dead, de-epidermized human dermis (DED) it has been shown that epidermal basement membrane does not induce ß1-null ES cells to undergo keratinocyte differentiation and does not stimulate the differentiation of wild-type ES cells. Coculture with epidermal keratinocytes also has no effect. However, when human dermal fibroblasts are incorporated into DED, the number of epidermal cysts formed by wild-type ES cells increases dramatically, and small groups of keratin 14-positive cells differentiate from ß1-null ES cells. Fibroblast-conditioned medium stimulates differentiation of K14-positive cells in wild-type and ß1-null embryoid bodies. Of a range of growth factors tested, KGF, FGF10, and TGFalpha all stimulate differentiation of keratin 14-positive ß1-null cells, and KGF and FGF10 are produced by the fibroblasts used in coculture experiments. The effects of the growth factors on wild-type ES cells are much less pronounced, suggesting that the concentrations of inducing factors already present in the medium are not limiting for wild-type cells. It is concluded that the lack of ß1 integrins decreases the sensitivity of ES cells to soluble factors that induce keratinocyte differentiation (Bagutti, 2001).

Integrins are extracellular matrix receptors composed of alpha and ß subunits involved in cell adhesion, migration and signal transduction. The ß1 subunit has two isoforms, ß1A ubiquitously expressed and ß1D restricted to striated muscle. They are not functionally equivalent. Replacement of ß1A by ß1D (ß1D knock-in) in the mouse leads to midgestation background-dependent lethality. The ß1D knock-in line was crossed into a less penetrant genetic background. This led to an attenuation of the midgestation lethality and revealed a second period of lethality around the time of birth. Midgestation death was apparently not caused by failure in cell migration, but rather by abnormal placentation. The ß1D knock-in embryos that survived midgestation developed until birth, but exhibited severely reduced skeletal muscle mass. Quantification of myotube numbers showed that substitution of ß1A with ß1D impairs primary myogenesis with no direct effect on secondary myogenesis. Furthermore, long-term primary myotube survival was affected in ß1D knock-in embryos. Finally, overexpression of ß1D in C2C12 cells impairs myotube formation while overexpression of ß1A primarily affects myotube maturation. Together these results demonstrate distinct roles for ß1 integrins in primary versus secondary myogenesis and that the ß1A and ß1D variants are not functionally equivalent in this process (Cachaçol, 2003).

The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. Extracellular matrix (ECM) receptors of the ß1 integrin family regulate myoblast fusion. ß1-deficient myoblasts adhere to one another, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of ß1-deficient myoblasts, suggesting that ß1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, ß1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of ß1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers (Schwander, 2003).

Integrin-extracellular matrix interactions play important roles in the coordinated integration of external and internal cues that are essential for proper development. To study the role of beta1 integrin in the mammary gland, Itgbeta1flox/flox mice were crossed with WAPiCre transgenic mice, which led to specific ablation of beta1 integrin in luminal alveolar epithelial cells. In the beta1 integrin mutant mammary gland, individual alveoli were disorganized resulting from alterations in cell-basement membrane associations. Activity of focal adhesion kinase (FAK) was also decreased in mutant mammary glands. Luminal cell proliferation was strongly inhibited in beta1 integrin mutant glands, which correlated with a specific increase of p21 Cip1 expression. In a p21 Cip1 null background, there was a partial rescue of BrdU incorporation, providing in vivo evidence linking p21 Cip1 to the proliferative defect observed in beta1 integrin mutant glands. A connection between p21 Cip1 and beta1 integrin as well as FAK was also established in primary mammary cells. These results point to the essential role of beta1 integrin signaling in mammary epithelial cell proliferation (Li, 2005).

Alternative splicing of beta Integrin

Beta1 integrin is alternatively spliced in muscle into a form that reinforces the cytoskeleton-matrix link. Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells, leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility, as compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin (see Drosophila Talin). In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton-matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction (Belkin, 1997).

Integrins and Focal adhesion kinase (FAK)

A characteristic feature of certain integrins is their ability to modulate their affinity for extracellular ligands in response to intracellular signals, a process termed "activation" or inside-out signaling". A Ras/Raf-initiated MAP kinase activity suppresses mammalian integrin activation. Using a screen for suppressors of integrin activation, the small GTP-binding protein H-Ras, and its effector kinase, Raf-1 were identified as negative regulators of integrin activation. HRas inhibits the activation of integrins with three distinct alpha and beta subunit cytoplasmic domains. Suppression is not associated with integrin phosphorylation and is independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlates with activation of the ERK MAP kinase pathway. It is possible that the integrin suppression pathway forms a local negative feedback loop for the regulation of integrin function. Ras activation through integrins might occur via the formation of a complex of FAK, GRB-2 and SOS. Cells derived from FAK-deficient mice show enhanced focal adhesion formation, suggesting that these cells may have lost a negative regulator of integrin function. In addition, dominant negative Ras can enhance focal adhesion formation. Further evidence for the existence of a negative feedback loop comes from observations that integrin occupancy or the expression of isolated beta subunit cytoplasmic domains can suppress the function of other integrins. It is likely that a cytoplasmic substrate of a MAP kinase is involved in suppression (Hughes, 1997).

Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular signaling pathways including a cascade of tyrosine phosphorylations. In many cell types, the cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes tyrosine-phosphorylated in response to adhesion; however, the molecular mechanisms regulating integrin-triggered FAK phosphorylation are not understood. Previous studies have shown that the integrin beta1, beta3, and beta5 subunit cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an extracellular reporter. In the present study, beta3 cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin beta3 cytoplasmic domain that regulate its ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of FAK. Analysis of these mutants indicate that structural information in both the membrane-proximal and C-terminal segments of the beta3 cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal segment of the beta3 cytoplasmic domain, the highly conserved NPXY motif is found to be required for the beta3 cytoplasmic domain to trigger FAK phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the beta3 cytoplasmic domain is found to be neither required nor sufficient for this signaling event. The serine 752 to proline mutation, known to cause a variant of Glanzmann's thrombasthenia, inhibits the ability of the beta3 cytoplasmic domain to signal FAK phosphorylation, suggesting that a single mutation in the beta3 cytoplasmic domain can inhibit both "inside-out" and "outside-in" integrin signaling (Tahiliani, 1997).

Focal adhesion kinase (FAK) overexpression enhances ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src. Focal adhesion kinase associates with integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr-397 and Tyr-925 promotes the binding of Src family protein tyrosine kinases (PTKs) and Grb2, respectively. To investigate the mechanisms by which FAK, c-Src, and Grb2 function in Fibronectin-stimulated signaling events to ERK2, wild type and mutant forms of FAK were expressed in human 293 epithelial cells by transient transfection. FAK overexpression enhances FN-stimulated activation of ERK2 approximately 4-fold. This is blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN stimulation of ERK2 is Ras-dependent. FN-stimulated c-Src PTK activity is enhanced by wild type FAK expression, whereas FN-stimulated activation of ERK2 is blocked by expression of the c-Src binding site Phe-397 mutant of FAK. Expression of the Grb2 binding site Phe-925 mutant of FAK enhances activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK does not. Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhances p130(Cas) association with FAK, Shc tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation. FN-induced Grb2-Shc association is another pathway leading to activation of ERK2 via Ras. The inhibitory effects of Tyr-397 FAK expression show that FAK-mediated association and activation of c-Src is essential for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the formation of a FAK.c-Src complex, and several of these can lead to Ras-dependent ERK2 mitogen-activated protein kinase activation (Schlaepfer, 1997).

Src family kinases (SFKs) have been implicated as important regulators of ligand-induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. Mouse embryos were generated harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. While Src, Yes and Fyn are largely dispensable for platelet-derived growth factor (PDGF)-induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin-induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, is nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells display reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix-coupled receptors (Klinghoffer, 1999).

pp125FAK is a tyrosine kinase that appears to regulate the assembly of focal adhesions and thereby promotes cell spreading on the extracellular matrix. In some cells, the C terminus of pp125FAK is expressed as a separate protein, pp41/43FRNK. Overexpression of pp41/43FRNK inhibits tyrosine phosphorylation of pp125FAK and paxillin and, in addition, overexpression delays cell spreading and focal adhesion assembly. Thus, pp41/43FRNK functions as a negative inhibitor of adhesion signaling and provides a tool to dissect the mechanism by which pp125FAK promotes cell spreading. The inhibitory effects of pp41/43FRNK expression can be rescued by the co-overexpression of wild-type pp125FAK and partially rescued by catalytically inactive variants of pp125FAK. However, both coexpression of a pp125FAK mutant for the autophosphorylation site that fails to bind the SH2 domain of pp60c-Src, or of a mutant that fails to bind paxillin, fail to promotes cell spreading. In contrast, expression of pp41/43FRNK and pp60c-Src reconstitute cell spreading and tyrosine phosphorylation of paxillin but do so without inducing tyrosine phosphorylation of pp125FAK. These data provide additional support for a model whereby pp125FAK acts as a "switchable adaptor" that recruits pp60c-Src to phosphorylate paxillin, promoting cell spreading. In addition, these data point to tyrosine phosphorylation of paxillin as being a critical step in focal adhesion assembly (Richardson, 1997).

pp125 focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase transducing signals initiated by integrin engagement and G protein-coupled receptors, is highly expressed in brain. FAK from brain has a higher molecular weight and an increased autophosphorylation activity, as compared to FAK from other tissues. In addition to a 9-base insertion in the 3'-coding region, which defines FAK+, rat striatal FAK mRNAs contained several additional short exons, coding for peptides of 28, 6, and 7 residues, respectively (termed boxes 28, 6, and 7), surrounding the autophosphorylated Tyr-397. In transfected COS 7 cells, the presence of boxes 6 and 7 confer an increased overall tyrosine phosphorylation, a higher phosphorylation of Tyr-397 assessed with a phosphorylation state-specific antibody, and a more active autophosphorylation in immune precipitates. The presence of box 28 does not further alter these parameters. Two-dimensional phosphopeptide maps of hippocampal FAK are identical to those of FAK+6,7. The presence of the various exons does not alter the interaction of FAK with c-Src, n-Src, or Fyn. Thus, several splice isoforms of FAK are preferentially expressed in rat brain, some of which have an increased autophosphorylation activity, suggesting that FAK may have specific properties in neurons (Burgaya, 1997).

Integrin alphaIIbbeta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either "inside-out" or "outside-in" signaling through alphaIIbbeta3 is the phosphorylation of focal adhesion kinase [pp125(FAK)] and the association of pp125(FAK) with cytoskeletal components. To examine the role of pp125(FAK) in these integrin-mediated events, pp125(FAK) phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of alphaIIbbeta3: alphaIIbbeta3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125(FAK) is altered, and alphaIIbbeta3(F727A/K729E/F730A), in which the putative binding site for alpha-actinin is altered. Whereas cells expressing alphaIIbbeta3(D723A/E726A) are able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing alphaIIbbeta3(F727A/K729E/F730A) adhere to fibrinogen, but have reduced focal adhesions and stress fibers. pp125(FAK) is recruited to focal adhesions in adherent cells expressing alphaIIbbeta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing alphaIIbbeta3(F727A/K729E/F730A), pp125(FAK) is phosphorylated despite reduced formation of focal adhesions and stress fibers. It is concluded that activation of pp125(FAK) can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation (Lyman, 1997).

Apoptotic cells undergo characteristic morphological changes that include detachment of cell attachment from the substratum and loss of cell-cell interactions. Attachment of cells to the extracellular matrix and to other cells is mediated by integrins. The interactions of integrins with the extracellular matrix activate focal adhesion kinase (FAK) and suppress apoptosis in diverse cell types. Members of the tumor necrosis family such as Fas and Apo-2L, also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induce apoptosis in both suspension and adherent cells through the activation of caspases. These caspases, when activated, cleave substrates that are important for the maintenance of nuclear and membrane integrity. FAK is sequentially cleaved into two different fragments early in Apo-2L-induced apoptosis. FAK cleavage is mediated by caspases, and FAK shows unique sensitivity to different caspases. These results suggest that disruption of FAK may contribute to the morphological changes observed in apoptotic suspension and adherent cells (Wen, 1997).

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. Expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein results in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation are also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, these components were examined after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity are inhibited by expression of PTEN, whereas EGF receptor autophosphorylation is unaffected. The ability of cells to spread at normal rates is partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation is enhanced as indicated by paxillin staining. The phosphatase domain of PTEN is essential for all of these functions, because PTEN with an inactive phosphatase domain does not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression does not affect c-Jun NH2-terminal kinase (JNK) or PDGF-stimulated Akt. These data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading (Gu, 1998).

There are contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation. Ectopic expression of the integrin alpha5 or alpha6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. The mechanisms by which changes in integrin alpha subunit ratios regulate this decision are addressed. Ectopic expression of truncated alpha5 or alpha6A indicate that the alpha5 cytoplasmic domain is permissive for the proliferative pathway, whereas the COOH-terminal 11 amino acids of alpha6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha5 and alpha6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta1 signaling. Ectopically expressed IL2R-alpha5 or IL2R-alpha6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta1A integrin subunit or IL2R-beta1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha5 or alpha6A ratios also significantly affects activation of beta1 integrin signaling pathways. Ectopic alpha5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (1) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta1A subunit and is modulated by the alpha subunit cytoplasmic domains; (2) perturbing alpha subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (3) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase (Sastry, 1999).

FAK independent and other signaling by integrins

Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggests the existence of a distinct signaling pathway regulated by Syk. Multiple proteins are activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin alphaIIbbeta3. The guanine nucleotide exchange factor Vav1 is inducibly phosphorylated in a Syk-dependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 trigger lamellipodia formation in fibrinogen-adherent cells; both Syk and Vav1 colocalize with alphaIIbbeta3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induce activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 is not dependent on actin polymerization. It is concluded that Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3'-kinase-regulated kinase Akt (Miranti, 1998).

Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix proteins and to one another in cell-cell interactions. Integrins also transmit outside-in signals that regulate functional responses of cells, and are known to influence gene expression by regulating transcription. Platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by outside-in signals. Using strategies that interrupt engagement of integrin alphaIIbbeta3 by fibrinogen and platelets deficient in this integrin, it was found that alphaIIbbeta3 regulates the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. Synthesis of Bcl-3, which occurs via a specialized translation control pathway regulated by mammalian target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of thrombin and when integrin alphaIIbbeta3 is engaged by a conformation-altering antibody against integrin alphaIIbbeta3. Thus, outside-in signals delivered by integrin alphaIIbbeta3 are required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin alpha2beta1 by collagen also triggers synthesis of Bcl-3. Thus, control of translation may be a general mechanism by which surface adhesion molecules regulate gene expression (Pabla, 1999).

Recycling of integrins

Components of intracellular signaling that mediate the stimulation-dependent recycling of integrins are being identified, but key transport effectors that are the ultimate downstream targets remain unknown. ACAP1, a GAP for ARF6, has been shown to function as a transport effector in the cargo sorting of transferrin receptor (TfR) that undergoes constitutive recycling. This study shows that ACAP1 also participates in the regulated recycling of integrin β1 to control cell migration. However, in contrast to TfR recycling, the role of ACAP1 in β1 recycling requires its phosphorylation by Akt, which is, in turn, regulated by a canonical signaling pathway. Disrupting the activities of either ACAP1 or Akt, or their assembly with endosomal β1, inhibits β1 recycling and cell migration. These findings advance an understanding of how integrin recycling is achieved during cell migration, and also address a basic issue of how intracellular signaling can interface with transport to achieve regulated recycling (Li, 2005).

The ARF family of small GTPases initiates intracellular transport by regulating the recruitment of coat proteins and other cargo-sorting adaptors from the cytosol to membrane. The GAPs for these small GTPases in the better-characterized transport pathways have been shown to function not only as negative upstream regulators of ARFs, but also as their effectors, by being components of coat complexes. An important implication of the cumulative findings on ACAP1 as a cargo-sorting device is that this role will be relevant for a broad range of cellular activities that are known to involve endocytic recycling. Besides cell migration, which itself underlies a wide range of physiologic and pathologic events, other important examples that require endocytic recycling include insulin-stimulated recycling of glucose transporters, cell polarity, cytokinesis, and phagocytosis. Thus, the future investigation of a potential role for ACAP1 in these examples will likely contribute to a better mechanistic understanding of how these events are achieved (Li, 2005),

Phosphorylation of integrins

Density-Enhanced Phosphatase-1 (DEP-1) (see Drosophila Ptp4E) de-phosphorylates various growth factor receptors and adhesion proteins to regulate cell proliferation, adhesion and migration. Moreover, dep-1/scc1 mutations have been detected in various types of human cancers, indicating a broad tumor suppressor activity. During C. elegans development, DEP-1 mediates binary cell fate decisions by negatively regulating EGFR signaling (see Drosophila EGFR signaling). Using a substrate-trapping DEP-1 mutant in a proteomics approach, this study identified the C. elegans β-integrin subunit PAT-3 (see Drosophila mys) as a specific DEP-1 substrate. DEP-1 selectively de-phosphorylates tyrosine 792 in the membrane-proximal NPXY motif to promote integrin activation via talin (see Drosophila rhea) recruitment. The non-phosphorylatable β-integrin mutant pat-3(Y792F) partially suppresses the hyperactive EGFR signaling phenotype caused by loss of dep-1 function. Thus, DEP-1 attenuates EGFR signaling in part by de-phosphorylating Y792 in the β-integrin cytoplasmic tail, besides the direct de-phosphorylation of the EGFR. Furthermore, in vivo FRAP analysis indicates that the αβ-integrin/talin complex attenuates EGFR signaling by restricting receptor mobility on the basolateral plasma membrane. The study proposes that DEP-1 regulates EGFR signaling via two parallel mechanisms, by direct receptor de-phosphorylation and by restricting receptor mobility through αβ-integrin activation (Walser, 2017).

Table of contents

myospheroid: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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