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PubMed2003

Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1.

Integrins are cell surface receptors for several microbial pathogens including echovirus 1 (EV1), a picornavirus. Cryo-electron microscopy revealed that the functional domain (alpha(2)I) of human alpha(2)beta(1) integrin binds to a surface depression on the EV1 capsid. This three-dimensional structure of EV1 bound to alpha(2)I domain provides the first structural details of an integrin interacting with a picornavirus. The model indicates that alpha(2)beta(1) integrin cannot simultaneously bind both EV1 and the physiological ligand collagen. Compared with collagen binding to the alpha(2)I domain, the virus binds with a 10-fold higher affinity but in vitro uncoating of EV1 was not observed as a result of attachment of alpha(2)I. A molecular model, constructed on the basis of the EV1-integrin complex, shows that multiple alpha(2)beta(1) heterodimers can bind at adjacent sites around the virus 5-fold symmetry axes without steric hindrance. In agreement with this, virus attachment to alpha(2)beta(1) integrin on the cell surface was found to result in integrin clustering, which can give rise to signaling and facilitate the initiation of the viral entry process that takes place via caveolae-mediated endocytosis.

Capsid↗

PubMed2003

The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes.

Integrin-mediated adhesion plays an important role in B cell development and activation. Signaling initiated by antigens, chemokines, or phorbol esters can rapidly convert integrins to an activated adhesion-competent state. The binding of integrins to their ligands can then induce actin-dependent cell spreading, which can facilitate cell-cell adhesion or cell migration on extracellular matrices. The signaling pathways involved in integrin activation and post-adhesion events in B cells are not completely understood. We have previously shown that anti-Ig antibodies, the chemokine stromal cell-derived factor-1 (SDF-1; CXCL12), and phorbol esters activate the Rap1 and Rap2 GTPases in B cells and that Rap activation is essential for SDF-1-induced B cell migration (McLeod, S. J., Li, A. H. Y., Lee, R. L., Burgess, A. E., and Gold, M. R. (2002) J. Immunol. 169, 1365-1371; Christian, S. L., Lee, R. L., McLeod, S. J., Burgess, A. E., Li, A. H. Y., Dang-Lawson, M., Lin, K. B. L., and Gold, M. R. (2003) J. Biol. Chem. 278, 41756-41767). We show here that preventing Rap activation by expressing Rap-specific GTPase-activating protein II (RapGAPII) significantly decreased lymphocyte function-associated antigen-1- and alpha(4) integrin-dependent binding of murine B cell lines to purified adhesion molecules and to other cells. Conversely, augmenting Rap activation by expressing a constitutively active form of Rap2 enhanced B cell adhesion, showing for the first time that Rap2 can promote integrin activation. We also show that blocking Rap activation inhibited anti-Ig-induced cell spreading and phorbol ester-induced actin polymerization as well as anti-Ig- and SDF-1-induced phosphorylation of Pyk2, a tyrosine kinase involved in morphological changes and chemokine-induced B cell migration. Thus, the Rap GTPases regulate integrin-mediated B cell adhesion as well as processes that control B cell morphology and migration.

Actins↗

PubMed2004

Involvement of integrins and Src in insulin signaling toward autophagic proteolysis in rat liver.

Cell volume changes critically determine hepatic signal transduction and metabolism. Hepatocyte swelling by insulin contributes to p38(MAPK) activation leading to inhibition of autophagic proteolysis. Recently integrins were shown to sense hypoosmotic hepatocyte swelling. Here the role of integrins, Src, and focal adhesion kinase (FAK) in insulin signaling was investigated using the intact organ model of perfused rat liver. Insulin increases [Tyr(P)(418)]Src, [Tyr(P)(397)]FAK, and dual p38(MAPK) phosphorylation by about 2-fold. Infusion of the integrin-antagonizing hexapeptide GRGDSP or the Src inhibitor PP-2 prevented activation of Src and p38(MAPK) and, consequently, proteolysis inhibition by insulin. However, insulin-induced phosphorylation of IRbeta (Tyr(1158)) and protein kinase B (PKB, Ser(473)), as well as K(+)-uptake and cell swelling, was not reduced by the inhibitors. Both hypoosmotic swelling and insulin increase the plasma membrane levels of activated beta(1) integrin. Inhibition of insulin-induced swelling by furosemide largely abolished activation of beta(1) integrin and phosphorylation of Src, but not of PKB. Rapamycin does not affect either insulin-induced K(+)-retention and cell swelling or proteolysis inhibition, indicating that swelling-dependent proteolysis inhibition occurs independently from the mammalian target of rapamycin. The data suggest that sensing of cell swelling by integrins essentially contributes to insulin signaling, thereby defining a novel way of integrin involvement in growth factor signaling.

Animals↗

PubMed2004

Mechanistic insights from a refined three-dimensional model of integrin alphaIIbbeta3.

The integrin alpha(IIb)beta(3) plays an important role in platelet function, and abnormalities of this protein result in a serious bleeding disorder, known as Glanzmann thrombasthenia. Although crystallographic data exist for the related integrin alpha(V)beta(3), to date, there are no high resolution structures of integrin alpha(IIb)beta(3) available in the literature. Therefore, it is still unclear how specific elements of the alpha(IIb) subunit contribute to integrin alpha(IIb)beta(3) function. Here we describe a refined model of the alpha(IIb) N-terminal portion of integrin alpha(IIb)beta(3) obtained by using the alpha(V)beta(3) template combined with a new method for predicting the conformations of the unique alpha(IIb) loop regions comprising residues 71-85, 114-125, and 148-164. The refined model was probed based on a structural prediction that differentiates it from standard homology models: specifically, that Lys-118 of alpha(IIb) contacts Glu-171 of beta(3). To test this hypothesis experimentally, the mutant integrin chains alpha(IIb) K118C and beta(3) E171C were cotransfected into HEK 293 cells. We show that the cells expressed the mutants alpha(IIb)beta(3) on their surface as a disulfide-linked dimer, supporting the close proximity between alpha(IIb) Lys-118 and beta(3) Glu-171 predicted from the refined model. This validated model provides a specific structural context for the analysis and interpretation of structure-function relations of integrin alpha(IIb)beta(3). In addition, it suggests mechanistic hypotheses pertaining to both naturally occurring mutations responsible for Glanzmann thrombasthenia and to point mutations that affect ligand binding.

Alanine↗

PubMed2005

The P2Y2 nucleotide receptor interacts with alphav integrins to activate Go and induce cell migration.

Extracellular ATP and UTP induce chemotaxis, or directed cell migration, by stimulating the G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R). Previously, we found that an arginine-glycine-aspartic acid (RGD) integrin binding domain in the P2Y(2)R enables this receptor to interact selectively with alpha(v)beta(3) and alpha(V)beta(5) integrins, an interaction that is prevented by mutation of the RGD sequence to arginine-glycine-glutamic acid (RGE) (Erb, L., Liu, J., Ockerhausen, J., Kong, Q., Garrad, R. C., Griffin, K., Neal, C., Krugh, B., Santiago-Perez, L. I., Gonzalez, F. A., Gresham, H. D., Turner, J. T., and Weisman, G. A. (2001) J. Cell Biol. 153, 491-501). This RGD domain also was found to be necessary for coupling the P2Y(2)R to G(o)- but not G(q)-mediated intracellular calcium mobilization, leading us to investigate the role of P2Y(2)R interaction with integrins in nucleotide-induced chemotaxis. Here we show that mutation of the RGD sequence to RGE in the human P2Y(2)R expressed in 1321N1 astrocytoma cells completely prevented UTP-induced chemotaxis as well as activation of G(o), Rac, and Vav2, a guanine nucleotide exchange factor for Rac. UTP also increased expression of vitronectin, an extracellular matrix protein that is a ligand for alpha(v)beta(3)/beta(5) integrins, in cells expressing the wild-type but not the RGE mutant P2Y(2)R. P2Y(2)R-mediated chemotaxis, Rac and Vav2 activation, and vitronectin up-regulation were inhibited by pretreatment of the cells with anti-alpha(v)beta(5) integrin antibodies, alpha(v) integrin antisense oligonucleotides, or the G(i/o) inhibitor, pertussis toxin. Thus, the RGD-dependent interaction between the P2Y(2)R and alpha(v) integrins is necessary for the P2Y(2)R to activate G(o) and to initiate G(o)-mediated signaling events leading to chemotaxis.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2006

A region in urokinase plasminogen receptor domain III controlling a functional association with alpha5beta1 integrin and tumor growth.

Highly expressed urokinase plasminogen activator receptor (uPAR) can interact with alpha5beta1 integrin leading to persistent ERK activation and tumorigenicity. Disrupting this interaction reduces ERK activity, forcing cancer cells into dormancy. We identified a site in uPAR domain III that is indispensable for these effects. A 9-mer peptide derived from a sequence in domain III (residues 240-248) binds purified alpha5beta1 integrin. Substituting a single amino acid (S245A) in this peptide, or in full-length soluble uPAR, impairs binding of the purified integrin. In the recently solved crystal structure of uPAR the Ser-245 is confined to the large external surface of the receptor, a location that is well separated from the central urokinase plasminogen binding cavity. The impact of this site on alpha5beta1 integrin-dependent cell functions was examined by comparing cells induced to express uPAR(wt) or the uPAR(S245A) mutant. Transfecting uPAR(wt) into cells with low endogenous levels of uPAR, inactive integrin, low ERK activity, and a dormant phenotype in vivo restores these functions and reinstates growth in vivo. In contrast, transfection of the same cells with uPAR(S245A) elicits only very small changes. Incubation of highly malignant cells with the wild-type, but not the S245A mutant peptide, disrupts the uPAR integrin interaction leading to down-regulation of ERK activity. The relevance of this binding site, and of the lateral uPAR-alpha5beta1 integrin interaction, to ERK pathway activation and tumor growth implicates it as a possible specific target for cancer therapy.

Alanine↗

PubMed2006

Interaction of integrin alpha(v)beta3 with nectin. Implication in cross-talk between cell-matrix and cell-cell junctions.

Cell-matrix and cell-cell junctions cross-talk together, and these two junctions cooperatively regulate cell movement, proliferation, adhesion, and polarization. However, the mechanism of this cross-talk remains unknown. An immunoglobulin-like cell-cell adhesion molecule nectin first trans-interacts with each other to form cell-cell adhesion and induces activation of Rap1, Cdc42, and Rac small G proteins through c-Src. Trans-interacting nectin then recruits another cell-cell adhesion molecule cadherin to the nectin-based cell-cell adhesion sites and forms adherens junctions (AJs). Here, we show that integrin alpha(v)beta3 functionally and physically associates with nectin. Integrin alpha(v)beta3 colocalized with nectin at the nectin-based cell-cell adhesion sites. The association of integrin alpha(v)beta3 with nectin was direct and was mediated through their extracellular regions. This interaction was necessary for the nectin-induced signaling. Focal adhesion kinase, which relays the integrin-initiated outside-in signals to the intracellular signaling molecules, was also involved in the nectin-induced signaling. During the formation of AJs, the high affinity form of integrin alpha(v)beta3 co-localized with nectin at the primordial cell-cell contact sites, and then after the establishment of AJs, this high affinity form of integrin alpha(v)beta3 was converted to the low affinity form, which continued to co-localize with nectin. Thus, integrin alpha(v)beta3 and nectin play pivotal roles in the cross-talk between cell-matrix and cell-cell junctions and the formation of cadherin-based AJs.

Animals↗

PubMed2006

Deletion of core fucosylation on alpha3beta1 integrin down-regulates its functions.

The core fucosylation (alpha1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently alpha1,6-fucosylation has been further reported to be very crucial by the study of alpha1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on alpha3beta1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in alpha3beta1 integrin-mediated events has been studied by using Fut8(+/+) and Fut8(-/-) embryonic fibroblasts, respectively. We found that the core fucosylation of alpha3beta1 integrin, the major receptor for laminin 5, was abundant in Fut8(+/+) cells but was totally abolished in Fut8(-/-) cells, which was associated with the deficient migration mediated by alpha3beta1 integrin in Fut8(-/-) cells. Moreover integrin-mediated cell signaling was reduced in Fut8(-/-) cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of alpha3beta1 integrin.

Animals↗

PubMed1999

Expression of integrin receptors in the human trabecular meshwork.

PURPOSE: To examine the expression of integrin receptors in the human trabecular meshwork. METHODS: The expression of integrins in human tissues was visualized by immunohistochemical staining using integrin-specific antibodies. Immunoprecipitation was performed after biotin labeling of cell surface proteins. Reverse transcriptase-polymerase chain reaction was used to detect the presence of mRNA species for integrin subunits. RESULTS: Human trabecular meshwork tissues obtained from donors 2 to 65 years old stained positively for integrins alpha1, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, beta4 and beta5. The staining was observed mostly around the edges of trabecular beams in association with trabecular meshwork cells. The staining intensity did not appear to vary with donor age. In addition, immunoprecipitation of tissue extracts revealed the presence of integrin alpha2 and confirmed the absence of beta2. Results from polymerase chain reactions were consistent with these findings. CONCLUSIONS: A spectrum of integrin receptors that may have important roles in the cell-matrix interactions are demonstrated in the human trabecular meshwork. The repertoire identified in tissues is similar to that found in cultured cells except that the beta4 expression in tissues is lost in cultures.

Adult↗

PubMed2003

Integrin alphavbeta3 binding to human alpha5-laminins facilitates FGF-2- and VEGF-induced proliferation of human ECV304 carcinoma cells.

Human ECV304 cells respond reproducibly by tube formation to complex basement membrane matrices. Laminins are major glycoproteins of basement membranes. We therefore studied the ability of ECV304 cells to attach to defined laminin isoforms and to fibronectin, and identified the involved laminin receptors. The cells bound poorly to fibronectin, to some extent to laminin-1, whereas laminin-2/4 and -10/11 were strong adhesive substrates. Antibody perturbation assays showed that adhesion to laminin-1 was mediated by integrin alpha6beta1, and adhesion to laminin-2/4 by cooperative activity of integrins alpha3beta1 and alpha6beta1. Adhesion of ECV 304 cells to laminin-10/11 was mainly mediated by integrins alpha3beta1, with minor involvement of alpha6beta1/4 and alphavbeta3. Solid-phase binding assays confirmed that integrin alphavbeta3 binds human laminin-10/11 and -10, in an RGD-dependent fashion. Although integrin alphavbeta3 played a very minor role in cell adhesion to laminin-10/11, this interaction facilitated growth factor-induced proliferation of ECV304 cells. In response to FGF-2 or VEGF, the cells proliferated better when attached on laminin-10/11 than on laminin-1, -2/4, or gelatin. The proliferation induced by the joint application of laminin-10/11 and either one of the growth factors could be blocked by antibodies against integrin alphavbeta3. Fragments of several other basement membrane components are known to interact with alphavbeta3. The current data show that that integrin alphavbeta3 can bind intact alpha5-containing laminin trimers. Since the laminin alpha5 chain is broadly expressed in adult basement membranes, this interaction could be physiologically important. Our data suggest that this interaction is involved in the regulation of cellular responses to growth factors known to be involved in epithelial and endothelial development.

Cell Division↗

PubMed2004

Functional characterization of beta1-integrin-positive epidermal cell populations.

Epidermal keratinocytes are heterogeneous and can be divided into stem cells (strong beta1-integrin expression) with unlimited clonogenic potential, transient amplifying cells (weaker beta1-integrin expression) with restricted proliferative capacity and terminally differentiated cells (no beta1-integrin expression) that have lost the capacity to divide. We tested the hypothesis that cell kinetic characteristics of the epidermal subpopulations differ. Single cell suspensions from small human skin punch biopsies were sorted flow cytometrically into a beta1-integrin weakly positive (dim) and strongly positive (bright) subpopulation and the clonogenic potential was compared in cell culture experiments. Image analysis was used to determine growth characteristics of the colonies. We found that cell size in the beta1-integrin bright subpopulation increased when colonies aged, whereas this was constant in the dim subpopulation. The total number of colonies formed and the growth rate of the colonies were higher in the beta1-integrin dim cells than in the bright subpopulation. Experimental data from this study confirm the hypothesis that cell kinetic characteristics of beta1-integrin dim and bright cells are different. Combining flow cytometric sorting, cell culture and image analysis provides powerful means for phenotypical and functional characterization of epidermal subpopulations.

Adult↗

PubMedDate not supplied

Integrins and regulation of the microcirculation: from arterioles to molecular studies using atomic force microscopy.

Integrins are an important class of receptors for extracellular matrix proteins that can mediate both force transmission, by virtue of their connections with the cell matrix and cytoskeleton; and signal transduction, resulting from the assemblages of signaling proteins that associate with focal contacts. Consequently, integrins have been proposed to be the mechanosensor in vascular smooth muscle and endothelial cells and to play a central role in mechanotransduction. In this regard, mechanical force is an important stimulus for many vascular functions, including contractile and relaxation processes,proliferation, migration, attachment, and cell phenotype determination. Collectively, these functions define physiological properties of the vasculature such as control of blood flow, capillary pressure,permeability, and peripheral vascular resistance, and play a role in pathophysiological processes like hypertension, diabetes, and arteriosclerosis. Our knowledge concerning how integrins sense and transduce physical forces into cellular signals and which integrins are involved is incomplete. Compared to other cell surface receptors, integrins have a relatively low affinity for their binding sites on the extracellular matrix and their affinity can be regulated. These characteristics of integrin-ligand interaction may facilitate dynamic processes such as cell migration, cell remodeling, and contractile activation in response to external forces. Important questions remain concerning the nature and origin of integrin-mediated signaling in the vascular wall.

Animals↗

PubMed1990

The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin.

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN.

Antibodies, Monoclonal↗

PubMed1991

The role of integrins in the maintenance of endothelial monolayer integrity.

This paper shows that, in confluent human umbilical vein endothelial cell (EC) monolayers, the integrin heterodimers alpha 2 beta 1 and alpha 5 beta 1, but not other members of the beta 1 subfamily, are located at cell-cell contact borders and not at cellular free edges. Also the alpha v chain, but not its most common partner beta 3, that is widely expressed in EC cell-matrix junctions, is found at cell-cell borders. In EC monolayers, the putative ligands of alpha 2 beta 1 and alpha 5 beta 1 receptors, i.e., laminin, collagen type IV, and fibronectin, are also organized in strands corresponding to cell-cell borders. The location of the above integrin receptors is not an artifact of in vitro culture since it has been noted also in explanted islets of the native umbilical vein endothelium. The integrins alpha 2 beta 1 and alpha 5 beta 1 play a role in the maintenance of endothelial monolayer continuity in vitro. Indeed, specific antibodies to alpha 2 beta 1, alpha 5 beta 1, and the synthetic peptide GRGDSP alter its continuity without any initial cell detachment. Moreover, antibodies to alpha 5 beta 1 increase the permeation of macromolecules across confluent EC monolayers. In contrast beta 3 antibodies were ineffective. It is suggested that the relocation of integrins to cell-cell borders is a feature of cells programmed to form polarized monolayers since integrins have a different distribution in nonpolar confluent dermal fibroblasts. The conclusion is that some members of the integrin superfamily collaborate with other intercellular molecules to form lateral junctions and to control both the monolayer integrity and the permeability properties of the vascular endothelial lining. This also suggest that integrins are adhesion molecules provided with a unique biochemical adaptability to different biological functions.

Actins↗

PubMed1993

Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels.

Electrical signals elicited by integrin interaction with ECM components and their role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN), this adhesion induced neurite outgrowth in N1 but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), N1 cells undergo a marked (approximately equal to 20 mV) hyperpolarization of the resting potential (Vrest) that occurred within the first 20 min after cell contact with ECM, and persisted for approximately 1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptor (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on Vrest of N7 cells, abolished in N1 cells the FN- or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the alpha V containing complexes. Both clones possessed Ca2+ and K+ delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K+ (KIR) channels. The latter governed the Vrest, and, unlike KDR channels, were blocked by Ba2+ and Cs+. By moving patched cells in contact with FN-coated beads, it was shown that KIR channel activation was responsible for the FN-mediated hyperpolarization of Vrest. Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G protein is interposed between integrins and KIR channels and that the activation of these channels is required for neuritogenesis. In fact, the block of KIR channels by Cs+ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the first two hours after N1 cell contact with FN.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1994

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library.

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.

Amino Acid Sequence↗

PubMed1995

Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains.

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

Actinin↗

PubMed1995

Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit.

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3-Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3-endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.

Amino Acid Sequence↗