Specialized functional properties of the integrin alpha 4 cytoplasmic domain.

For functional studies of the integrin alpha 4 cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha 4 (called X4C4), 2) two chimeric forms of alpha 4 (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha 2 and alpha 5, respectively, and 3) alpha 4 with no cytoplasmic domain (X4C0). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha 2 or alpha 5 tails markedly enhanced VLA-4-dependent K562 cells spreading (X4C2 > X4C5 > X4C4 > X4C0), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 > X4C5 > X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 > X4C5 > X4C4 > X4C0). Conversely, the alpha 4 tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4C0), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha 4 cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha 4 tail are consistent with the role of alpha 4 integrins on highly motile lymphocytes, monocytes, and eosinophils.     

Ligand-dependent activation of integrin alpha vbeta 3

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(v)beta(3). In this study, we examined the activation of alpha(v)beta(3) resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected alpha(v)beta(3), adhesion to vitronectin required tyrosine phosphorylation of the beta(3) subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without beta(3)-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.     

A membrane-distal segment of the integrin alpha IIb cytoplasmic domain regulates integrin activation

Previous evidence suggests that interactions between integrin cytoplasmic domains regulate integrin activation. We have constructed and validated recombinant structural mimics of the heterodimeric alpha(IIb)beta(3) cytoplasmic domain. The mimics elicited polyclonal antibodies that recognize a combinatorial epitope(s) formed in mixtures of the alpha(IIb) and beta(3) cytoplasmic domains but not present in either isolated tail. This epitope(s) is present within intact alpha(IIb)beta(3), indicating that interaction between the tails can occur in the native integrin. Furthermore, the combinatorial epitope(s) is also formed by introducing the activation-blocking beta(3)(Y747A) mutation into the beta(3) tail. A membrane-distal heptapeptide sequence in the alpha(IIb) tail ((997)RPPLEED) is responsible for this effect on beta(3). Membrane-permeant palmitoylated peptides, containing this alpha(IIb) sequence, specifically blocked alpha(IIb)beta(3) activation in platelets. Thus, this region of the alpha(IIb) tail causes the beta(3) tail to resemble that of beta(3)(Y747A) and suppresses activation of the integrin.     

Phosphorylation of the integrin alpha 4 cytoplasmic domain regulates paxillin binding

alpha4 integrins are essential for embryogenesis, hematopoiesis, inflammation, and immune response possibly because alpha4 integrins have distinct signaling properties from other integrins. Specifically, the alpha4 cytoplasmic domain binds tightly to paxillin, a signaling adaptor protein, leading to increased cell migration and an altered cytoskeletal organization that results in reduced cell spreading. The alpha4 tail contains potential phosphorylation sites clustered in its core paxillin binding region. We now report that the alpha4 tail is phosphorylated in vitro and in vivo. Furthermore, Ser(988) is a major phosphorylation site. Using antibodies specific for Ser(988)-phosphorylated alpha4, we found the stoichiometry of alpha4 phosphorylation varied in different cells. However, >60% of alpha4 was phosphorylated in Jurkat T cells. Phosphorylation at Ser(988) blocked paxillin binding to the alpha4 tail. A phosphorylation-mimicking mutant of alpha4 (alpha4S988D) blocked paxillin binding and reversed the inhibitory effect of alpha4 on cell spreading. Consequently, alpha4 phosphorylation is a biochemical mechanism to modulate paxillin binding to alpha4 integrins with consequent regulation of alpha4 integrin-dependent cellular functions.     

Disruption of focal adhesions by integrin cytoplasmic domain-associated protein-1 alpha

Regulation of integrin affinity and clustering plays a key role in the control of cell adhesion and migration. The protein ICAP-1 alpha (integrin cytoplasmic domain-associated protein-1 alpha) binds to the cytoplasmic domain of the beta(1A) integrin and controls cell spreading on fibronectin. Here, we demonstrate that, despite its ability to interact with beta(1A) integrin, ICAP-1 alpha is not recruited in focal adhesions, whereas it is colocalized with the integrin at the ruffling edges of the cells. ICAP-1 alpha induced a rapid disruption of focal adhesions, which may result from the ability of ICAP-1 alpha to inhibit the association of beta(1A) integrin with talin, which is crucial for the assembly of these structures. ICAP-1 alpha-mediated dispersion of beta(1A) integrins is not observed with beta(1D) integrins that do not bind ICAP. This strongly suggests that ICAP-1 alpha action depends on a direct interaction between ICAP-1 alpha and the cytoplasmic domain of the beta(1) chains. Altogether, these results suggest that ICAP-1 alpha plays a key role in cell adhesion by acting as a negative regulator of beta(1) integrin avidity.     

Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.     

Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation. The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase C alpha (PKC alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha activity, leading to "superactivation" of the enzyme, apparently through an interaction with its catalytic domain. We now have performed yeast two-hybrid and in vitro binding assays to determine the interaction sites between 4V and PKC alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence (4V(YF): LGKKPIFKK) did not interact with PKC alpha, indicating that tyrosine 192 in the syndecan-4 cytoplasmic domain might be critical for this interaction. Further assays identified a novel interaction site in the C terminus of the catalytic domain of PKC alpha (amino acid sequence 513-672). This encompasses the autophosphorylation sites, which are implicated in activation and stability. Yeast two-hybrid data were confirmed by in vitro binding and coimmunoprecipitation assays. The interaction of syndecan-4 with PKC alpha appears unique since PKC delta and epsilon did not interact with 4V in yeast two-hybrid assays or coimmunoprecipitate with syndecan-4. Finally, overexpression of syndecan-4 in rat embryo fibroblast cells, but not expression of the YF mutant, increased PKC alpha localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKC alpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase.     

Paxillin binding to a conserved sequence motif in the alpha 4 integrin cytoplasmic domain

alpha(4)beta(1) integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other beta(1) integrins. Paxillin, a signaling adapter protein, binds tightly to the alpha(4) cytoplasmic domain and is implicated in alpha(4) integrin signaling. We now report the mapping of a paxillin-binding site in the alpha(4) cytoplasmic domain and an assessment of its role in the alpha(4) tail-specific integrin functions. By using truncation mutants and a peptide competition assay, we found that a region of 9 amino acid residues (Glu(983)-Tyr(991)) within the alpha(4) cytoplasmic domain contains a minimal sequence sufficient for paxillin binding. Alanine scanning of this region implicated Tyr(991) and Glu(983) as critical residues. The role of these residues was confirmed by introducing these Ala substitutions into the full-length alpha(4) tail sequence. Y991A or E983A substitution disrupted the interaction of alpha(4) integrins with paxillin. These same two point mutations reversed the effects of the alpha(4) tail on cell spreading. The key features of the identified paxillin-binding sequence are present in all alpha(4) integrins sequenced to date, including that from Xenopus laevis. The maintenance of this sequence motif suggests that paxillin binding is an evolutionarily conserved function of alpha(4) integrins.     

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.     

Specific integrin alpha and beta chain phosphorylations regulate LFA-1 activation through affinity-dependent and -independent mechanisms

Integrins are adhesion receptors that are crucial to the functions of multicellular organisms. Integrin-mediated adhesion is a complex process that involves both affinity regulation and cytoskeletal coupling, but the molecular mechanisms behind this process have remained incompletely understood. In this study, we report that the phosphorylation of each cytoplasmic domain of the leukocyte function-associated antigen-1 integrin mediates different modes of integrin activation. alpha Chain phosphorylation on Ser1140 is needed for conformational changes in the integrin after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1 (Rap1V12). In contrast, the beta chain Thr758 phosphorylation mediates selective binding to 14-3-3 proteins in response to inside-out activation through the T cell receptor, resulting in cytoskeletal rearrangements. Thus, site-specific phosphorylation of the integrin cytoplasmic domains is important for the dynamic regulation of these complex receptors in cells.     

The beta1 cytoplasmic domain regulates the laminin-binding specificity of the alpha7X1 integrin

During muscle development, the laminin-specific alpha7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the alpha7X1 or the alpha7X2 variant. The relative level of alpha7X1 and alpha7X2 is developmentally regulated. Similarly, the partner beta1 integrin cytoplasmic domain is converted from the beta1A to the beta1D splice variant. To determine whether beta1D modulates the activity of the alpha7 receptor, cells were transfected with alpha7X1 and beta1D cDNA. alpha7X1 coupled with beta1A failed to adhere to laminin-1, whereas cotransfectants expressing alpha7X1 and beta1D showed strong adhesion. Interestingly, alpha7X1 complexed with beta1A and beta1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that alpha7 function is regulated not only by X1/X2 in its extracellular domain but also by beta1 cytoplasmic splice variants. It is likely that expression of beta1D alters alpha7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of alpha7beta1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.     

Phosphorylation of the cytoplasmic domain of the integrin CD18 chain by protein kinase C isoforms in leukocytes.

The CD11/CD18 (beta(2)) integrins are leukocyte-specific adhesion receptors, and their ability to bind ligands on other cells can be activated by extracellular stimuli. During cell activation, the CD18 chain is known to become phosphorylated on serine and functionally important threonine residues located in the intracellular C-terminal tail. Here, we identify catalytic domain fragments of protein kinase C (PKC) delta and PKCbetaI/II as the major protein kinases in leukocyte extracts that phosphorylate a peptide corresponding to the cytoplasmic tail of the integrin CD18 chain. The sites phosphorylated in vitro were identified as Ser-745 and Thr-758. PKCalpha and PKCeta also phosphorylated these residues, and PKCalpha additionally phosphorylated Thr-760. Ser-745, a novel site, was shown to become phosphorylated in T cells in response to phorbol ester stimulation. Ser-756, a residue not phosphorylated by PKC isoforms, also became phosphorylated in T cells after phorbol ester stimulation. When leukocyte extracts were subjected to affinity chromatography on agarose to which residues 751-761 of the CD18 chain phosphorylated at Thr-758 were bound covalently, the only proteins that bound specifically were identified as isoforms of 14-3-3 proteins. Thus, PKC-mediated phosphorylation of CD18 after cell stimulation could lead to the recruitment of 14-3-3 proteins to the activated integrin, which may play a role in regulating its adhesive state or ability to signal.     

Cooperative role of the membrane-proximal and -distal residues of the integrin beta3 cytoplasmic domain in regulation of talin-mediated alpha IIb beta3 activation

Integrin cytoplasmic tails regulate integrin activation that is required for high affinity binding with ligands. The interaction of the integrin beta subunit tail with a cytoplasmic protein, talin, largely contributes to integrin activation. Here we report the cooperative interaction of the beta3 membrane-proximal and -distal residues in regulation of talin-mediated alpha IIb beta3 activation. Because a chimeric integrin, alpha IIb beta3/beta1, in which the beta3 tail was replaced with the beta1 tail was constitutively active, we searched for the residues responsible for integrin activation among the residues that differed between the beta3 and beta1 tails. Single amino acid substitutions of Ile-719 and Glu-749 in the beta3 membrane-proximal and -distal regions, respectively, with the corresponding beta1 residues or alanine rendered alphaIIbbeta3 constitutively active. The I719M/E749S double mutant had the same ligand binding activity as alpha IIb beta3/beta1. These beta3 mutations also induced alphaVbeta3 activation. Conversely, substitution of Met-719 or Ser-749 in the beta1 tail with the corresponding beta3 tail residue (M719I or S749E) inhibited alpha IIb beta3/beta1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type alpha IIb beta3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced alpha IIb beta3 activation. These results suggest that the beta3 membrane-proximal and -distal residues cooperatively regulate talin-mediated alpha IIb beta3 activation.     

Sequences determining the cytoplasmic localization of a chemoreceptor domain.

The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure.     

Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.     

Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions

Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human alpha IIb beta 3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of alpha IIb did not interfere with the ability of alpha IIb beta 3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the alpha IIb cytoplasmic domain allowed indiscriminate recruitment of alpha IIb beta 3 to focal adhesions formed by other integrins. Truncation of the beta 3 subunit cytoplasmic domain abolished cell spreading mediated by alpha IIb beta 3 and also abrogated recruitment of alpha IIb beta 3 to focal adhesions. This truncation also dramatically impaired the ability of alpha IIb beta 3 to mediate the contraction of fibrin gels. In contrast, the beta 3 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of alpha IIb beta 3. Thus, the integrin beta 3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the beta 3 cytoplasmic domain is required for the transmission of intracellular contractile forces to fibrin gels. The alpha subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins.     

The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes.

The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.     

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.     

The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.

The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.     

A fragment of paxillin binds the alpha 4 integrin cytoplasmic domain (tail) and selectively inhibits alpha 4-mediated cell migration

The alpha(4) integrins play important roles in embryogenesis, hematopoiesis, cardiac development, and the immune responses. The alpha(4) integrin subunit is indispensable for these biological processes, possibly because the alpha(4) subunit regulates cellular functions differently from other integrin alpha subunits. We have previously reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule, and this interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion. We also have identified a conserved 9-amino acid region (Glu(983)-Tyr(991)) in the alpha(4) cytoplasmic domain that is sufficient for paxillin binding, and an alanine substitution at either Glu(983) or Tyr(991) within this region disrupted the alpha(4)-paxillin interaction and reversed the effects of the alpha(4) cytoplasmic domain on cell spreading and migration. In the current study, we have mapped the alpha(4)-binding site within paxillin using mutational analysis, and examined its effects on the alpha(4) tail-mediated functional responses. Here we report that sequences between residues Ala(176) and Asp(275) of paxillin are sufficient for binding to the alpha(4) tail. We found that the alpha(4) tail, paxillin, and FAT, the focal adhesion targeting domain of pp125(FAK), could form a ternary complex and that the alpha(4)-binding paxillin fragment, P(Ala(176)-Asp(275)), specifically blocked paxillin binding to the alpha(4) tail more efficiently than it blocked binding to FAT. Furthermore, when expressed in cells, this alpha(4)-binding paxillin fragment specifically inhibited the alpha(4) tail-stimulated cell migration. Thus, paxillin binding to the alpha(4) tail leads to enhanced cell migration and inhibition of the alpha(4)-paxillin interaction selectively blocks the alpha4-dependent cellular responses.