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At least 19 recordsLinked to original sources
PubMed2006

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.

Amino Acid Sequence↗
PubMed1998

Use of a beta1 integrin-deficient human T cell to identify beta1 integrin cytoplasmic domain sequences critical for integrin function.

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. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that 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 could 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 did not occur if the carboxy-terminal five amino acids of the beta1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the beta1 integrin with the activating beta1 integrin-specific mAb TS2/16, although the effects were 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.

Amino Acid Sequence↗
PubMed2002

Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane.

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.

ADAM Proteins↗
PubMed1997

Alpha4 integrin binding interfaces on VCAM-1 and MAdCAM-1. Integrin binding footprints identify accessory binding sites that play a role in integrin specificity.

Integrins are a family of heterodimeric adhesion receptors that mediate cellular interactions with a range of matrix components and cell surface proteins. Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell ligand for two leukocyte integrins (alpha4beta1 and alpha4beta7). A related CAM, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is recognized by alpha4beta7 but is a poor ligand for alpha4beta1. Previous studies have revealed that all alpha4 integrin-ligand interactions are dependent on a key acidic ligand motif centered on the CAM domain 1 C-D loop region. By generating VCAM-1/MAdCAM-1 chimeras and testing recombinant proteins in cell adhesion assays we have found that alpha4beta1 binds to the MAdCAM-1 adhesion motif when present in VCAM-1, but not when the VCAM-1 motif was present in MAdCAM-1, suggesting that this region does not contain all of the information necessary to determine integrin binding specificity. To characterize integrin-CAM specificity further we measured alpha4beta1 and alpha4beta7 binding to a comprehensive set of mutant VCAM-1 constructs containing amino acid substitutions within the predicted integrin adhesion face. These data revealed the presence of key "regulatory residues" adjacent to integrin contact sites and an important difference in the "footprint" of alpha4beta1 and alpha4beta7 that was associated with an accessory binding site located in VCAM-1 Ig domain 2. The analogous region in MAdCAM-1 is markedly different in size and sequence and when mutated abolishes integrin binding activity.

Amino Acid Sequence↗
PubMedDate not supplied

Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity.

A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.

Antibodies, Monoclonal↗
PubMed1993

Inside-out integrin signaling in macrophages. Analysis of the role of the alpha 6A beta 1 and alpha 6B beta 1 integrin variants in laminin adhesion by cDNA expression in an alpha 6 integrin-deficient macrophage cell line.

Leukocytes use the alpha 6 beta 1 integrin to adhere to laminin based on mAb inhibition and affinity chromatography studies. This adhesion requires leukocyte stimulation with either PMA or specific cytokines, a process that has been termed "inside-out" integrin signaling. In the present study, the involvement of alpha 6 integrin structural variants in this regulated adhesion was examined using mouse macrophages. The two known alpha 6 structural variants, alpha 6A and alpha 6B, differ only in their cytoplasmic domain sequences. Using reverse transcriptase-polymerase chain reaction, we observed that macrophages express only the alpha 6A structural variant, in contrast to most cell types which express both alpha 6A and alpha 6B variants. The role of this integrin subunit in macrophage adhesion was assessed by cDNA transfection of P388D1 cells. We found that this mouse macrophage cell line does not adhere to laminin even in response to phorbol 12-myristate 13-acetate (PMA) stimulation, though it does adhere normally to fibronectin and tissue culture plastic. Subsequent analysis employing reverse transcriptase-polymerase chain reaction and immunoprecipitation of surface labeled cells revealed that this cell line expresses neither the alpha 6A nor alpha 6B integrin subunits. Stable transfection of either the chick or human alpha 6A cDNAs into P388D1 cells resulted in chimeric alpha 6A beta 1 surface expression. The alpha 6A transfectants exhibited inside-out integrin signaling because PMA stimulation markedly increased their ability to adhere to laminin but it did not increase alpha 6A beta 1 surface expression. Similar results were obtained after transfection of the human alpha 6B cDNA. Analysis of the human transfectants was facilitated by the generation of a monoclonal antibody, 2B7, that is specific for the human alpha 6 integrin subunit. These observations demonstrate that both alpha 6A beta 1 and alpha 6B beta 1 can be regulated by inside-out signaling pathways in macrophages, even though this cell type expresses only alpha 6A beta 1. The data presented also demonstrate clearly that the alpha 6A and alpha 6B cytoplasmic domains do not differ in their ability to be regulated by PMA.

Amino Acid Sequence↗
PubMed1998

Beta3-integrins rather than beta1-integrins dominate integrin-matrix interactions involved in postinjury smooth muscle cell migration.

BACKGROUND: Smooth muscle cell (SMC) migration is a vital component in the response of the arterial wall to revascularization injury. Cell surface integrin-extracellular matrix interactions are essential for cell migration. SMCs express both beta1- and beta3-integrins. In this study, we examined the relative functional roles of beta1- and beta3-integrin-matrix interactions in postinjury SMC migration. METHODS AND RESULTS: Flow cytometry and fluorescence microscopy of migrating SMCs immunostained with anti-beta1 and anti-alpha(v)beta3/5 antibodies (Abs) revealed expression of both beta1- and beta3-integrins, with beta1 observed as linear streaks and beta3 found in focal contacts. In a scrape-wound migration assay, anti-beta1 Abs (92.0+/-10.7% of control, P=.1) and 0.5 mmol/L linear RGD (105+/-5% of control, P=.2) did not alter SMC migration at 48 hours after injury. Beta3-blockade, however, via Abs (anti-beta3/5 35.7+/-4.5% of control, anti-beta3 61+/-12% of control, both P<.001) and cyclic RGD (0.5 mmol/L) (12+/-10% of control, P<.001) decreased migration. Neither beta1- nor beta3-inhibition altered postinjury [3H]thymidine incorporation. In the rat carotid injury model, local adventitial polymer-based delivery of radiolabeled linear or cyclic RGD led to uptake and retention of label, for both peptides, over a 72-hour period after injury. Local arterial wall beta1-blockade via polymer-based delivery of linear RGD had no effect on SMC migration at 4.5 days (11.5+/-3.2 versus 12.8 SMCs per x600 field [control], P=.6) or on neointimal thickening at 14 days (I/M area ratio, 0.664+/-0.328 versus 1.179+/-0.324 [control], P=.6) after injury. In contrast, local beta3-blockade via cRGD limited migration (0.8+/-0.8 versus 12.8+/-4.4 SMCs per x600 field [control], P<.01) and thickening (I/M area ratio, 0.004+/-0.008 versus 1.179+/-0.324 [control], P<.01). CONCLUSIONS: In postinjury migrating SMCs, beta3- rather than beta1-integrin-matrix interactions are of greater functional significance in adhesive processes essential for SMC migration in vitro and in vivo. Blockade of dominant SMC integrin (beta3)-matrix interactions may be a valuable approach for limiting injury-induced SMC migration and late arterial renarrowing.

Animals↗
PubMed1999

Cloning and characterization of a novel beta integrin-related cDNA coding for the protein TIED ("ten beta integrin EGF-like repeat domains") that maps to chromosome band 13q33: A divergent stand-alone integrin stalk structure.

Herein we describe the cDNA sequence of a novel human gene, ITGBL1, encoding a beta integrin-related protein termed TIED [for ten beta integrin epidermal growth factor (EGF)-like repeat domains]. Overlapping cDNA clones from fetal lung, HUVEC, and osteoblast cDNA libraries encode a sequence comprising a typical signal peptide, followed by a hydrophilic 471-amino-acid domain containing 10 tandem EGF-like repeats strikingly similar to those found in the cysteine-rich "stalk-like" structure of integrin beta subunits. The EGF-like repeats of TIED and beta integrins are unique in that they alternate in homology and possess two additional cysteines (eight in total) whose positions differ from those in the other eight-cysteine EGF-like domains of laminin, fibrillin, and the latent TGF-beta binding proteins. TIED mRNA transcripts of 2.8 kb were detected in aorta, thymus, and osteogenic sarcoma cells. The ITGBL1 gene was mapped to human chromosome 13, band 13q33. We suggest that ITGBL1 may be linked in some way with the evolution of the integrin beta subunits.

3' Untranslated Regions↗
PubMed1996

Domain-specific interactions between entactin and neutrophil integrins. G2 domain ligation of integrin alpha3beta1 and E domain ligation of the leukocyte response integrin signal for different responses.

Extracellular matrix proteins activate neutrophils to up-regulate many physiologic functions that are necessary at sites of tissue injury. To elucidate the ligand-receptor interactions that mediate these functions, we examined neutrophil activation by the basement membrane protein, entactin. Entactin is structurally and functionally organized into distinct domains; therefore, we utilized glutathione S-transferase -fusion proteins encompassing its four major domains, G1, G2, E, and G3, to assess interactions between entactin and neutrophil integrin receptors. We show that the E domain, which contains the single RGD sequence of entactin, is sufficient for ligation of the beta3-like integrin, leukocyte response integrin, and signaling for chemotaxis. Moreover, the G2 domain signals for stimulation of Fc receptor-mediated phagocytosis via ligation of alpha3beta1. This receptor-ligand interaction was revealed only after stimulation of neutrophil by immune complexes or phorbol esters. Interestingly, the E domain does not enhance phagocytosis, and the G2 domain is not chemotactic. Furthermore, cleavage of entactin with the matrix metalloproteinase, matrilysin, liberates peptides that retain E domain-mediated chemotaxis and G2 domain-mediated enhancement of phagocytosis. These studies indicate that multiple domains of entactin have the ability to ligate individual integrins expressed by neutrophils and to activate distinct functions.

Antigen-Antibody Complex↗
PubMed1999

CD44 stimulation induces integrin-mediated adhesion of colon cancer cell lines to endothelial cells by up-regulation of integrins and c-Met and activation of integrins.

For cancer metastasis, tumor cells present in the circulation must first adhere to the endothelium. Integrins lymphocyte function-associated antigen (LFA) 1 and very late antigen 4 play a central role in leukocyte adhesion to the endothelium and subsequent migration into tissues. The majority of tumor cells derived from solid cancers including colorectal cancer do not express suitable adhesion receptors, LFA-1 and very late antigen 4. We investigated the mechanisms of adhesion and transendothelial migration of cancer cells using colorectal carcinoma cell lines. Our results showed the following novel features of CD44 on the cells: (a) colon cancer cells express high levels of CD44; (b) stimulation of cancer cells by CD44 cross-linking or fragmented hyaluronan markedly induces the expression of LFA-1s, some of which reveal an activation epitope on the cells; (c) CD44 cross-linking induces F-actin polymerization in the cell cortex; (d) fragmented hyaluronan induces up-regulation of the activation epitope of LFA-1, which is mediated through protein kinase C; (e) stimulation of CD44 augments the LFA-1-mediated adhesion of cancer cells to endothelial cells and intercellular adhesion molecule 1-transfected cells and facilitates transendothelial migration; (f) stimulation of CD44 also induces expression of the hepatocyte growth factor (HGF) receptor c-Met on cancer cells; and (g) HGF further amplifies the LFA-1-mediated adhesion of cells prestimulated by CD44-derived signaling. Our results indicated that stimulation by CD44 induces "outside-in signaling," which consists of a direct pathway via CD44 and an alternate pathway through the induction of c-Met expression via HGF. Such stimuli augment the expression and trigger the function of integrins via "inside-out signaling" in colon cancer cells, which leads to amplification of integrin-mediated adhesion to the vessel wall and subsequent transendothelial migration.

Actins↗
PubMed2001

Expression and cellular distribution of alpha(v)integrins in beta(1)integrin-deficient embryonic stem cell-derived cardiac cells.

beta(1)integrin-deficient (beta(1)-/-) ES cells showed increased differentiation of cardiac cells characterized by reduced adhesion and high beating frequency. Whereas in whole embryoid body outgrowths of beta(1)-/- cells maximum levels of alpha(v), beta(3)and beta(5)integrin mRNA were delayed and transiently upregulated, in cardiac clusters isolated from beta(1)-/- cells, only beta(3)integrin mRNA levels were enhanced in comparison to wild-type (wt) cells. To answer the question, whether alpha(v)and beta(3)integrins may compensate, at least partially, the loss of beta(1)integrin function during cardiac differentiation, the distribution of alpha(v)and beta(3)integrins in beta(1)-/- and wt pacemaker-like cardiac cells was analyzed. A different distribution of alpha(v)and beta(3)integrins in beta(1)-/- v wt cardiac cells was found. In wt cardiac cells, beta(1)integrin was localized in specialized subsarcolemmal regions, in particular, at focal contacts and costameres, but alpha(v)integrin was diffusely distributed. In contrast, in beta(1)-/- cardiac cells, alpha(v)integrin was preponderantly localized at cell membranes, focal contacts and costameres. beta(3)integrin displayed a diffuse pattern both in wt and in beta(1)-/- pacemaker-like cells at early differentiation stages, whereas at terminal stages, beta(3)was colocalized with sarcomeres in wt, but not in beta(1)-/- pacemaker-like cells. Quantitative immunofluorescence analysis revealed increased alpha(v)and beta(3)integrin levels in beta(1)-/- pacemaker-like cardiac cells. Our results led us to conclude that altered cellular distribution of alpha(v)integrin and upregulation of beta(3)integrin correlate with growth and survival of beta(1)-/- cardiac pacemaker-like cells at an early developmental state. However, alpha(v)and beta(3)integrins cannot functionally compensate the loss of beta(1)integrin during terminal differentiation of cardiac cells implicating that cardiomyocytes require specific beta(1)integrin functions for cardiac specialization.

Animals↗
PubMed2002

The integrin beta1 subunit cytoplasmic tail forms oligomers: a potential role in beta1 integrin clustering.

Integrins are alpha/beta heterodimeric cell surface receptors devoid of enzymatic activity. Signal transduction therefore requires the association of cytosolic and cytoskeletal proteins with the integrin subunit intracellular regions. This association is initiated upon ligand binding to the integrin receptor and includes clustering of the integrins and recruitment of focal adhesion-associated proteins. Whether integrin clustering is solely dependent on ligand binding to the integrin extracellular parts or involves also interactions between the intracellular tails of integrins is so far unknown. To investigate intracellular events in integrin clustering, we have used peptides corresponding to the integrin beta1 cytoplasmic region. Loading of cells with the peptides results in a decreased cell adhesion and in an inhibition of cell spreading in agreement with the previously reported dominant negative effect of the beta1 integrin cytoplasmic tail on integrin clustering. Direct protein-protein interaction studies by surface plasmon resonance demonstrate that integrin beta1 cytoplasmic peptides self-associate in contrast to integrin beta3 cytoplasmic tails. Size exclusion chromatography and SDS-PAGE analysis of the peptides further show that the integrin beta1 cytoplasmic parts form oligomers and that they assume alpha helical conformation to the extent of about 13% and that this fraction is increased upon aggregation. Thus self-association of the integrin beta1 subunit cytoplasmic regions may be central to beta1 integrin clustering.

Cell Adhesion↗
PubMed1998

Altered integrin expression and the malignant phenotype: the contribution of multiple integrated integrin receptors.

The integrins are a family of cell surface adhesion receptors that mediate adhesion to either components of the extracellular matrix or to other cells. The beta1 family of integrins represent the major class of cell substrate receptors with specificities primarily for collagens, laminins, and fibronectins. The role of the integrin family of cell surface adhesion receptors in normal mammary gland morphogenesis and the contributions of altered integrin receptor expression to the invasive and metastatic phenotype have been the primary focus of our lab, as well as a number of other laboratories. The alpha2beta1 integrin is expressed at high levels by normal differentiated epithelial cells including those of the normal breast. Using breast cancer as a model, we evaluated changes in integrin expression in malignancy. We and other investigators made the key observation that alpha2beta1 integrin expression is decreased in adenocarcinoma of the breast in a manner that correlates with the stage of differentiation. Studies of other adenocarcinomas have yielded similar results. When the alpha2beta1 integrin was reexpressed in a poorly differentiated mammary carcinoma that expressed no detectable alpha2 integrin subunit, a dramatic reversion of malignant phenotype to a differentiated epithelial phenotype was observed, indicating a critical role for alpha2beta1 expression in mammary gland differentiation. Other laboratories using monoclonal antibodies to competitively inhibit alpha2beta1 integrin adhesion or oncogenic transformation using c-erb2 have confirmed the important role of that alpha2beta1 integrin in mammary gland morphogenesis. Re-expression of the alpha2beta1 integrin also results in upregulation of both the alpha6 and beta4 integrin subunits. To determine the contribution of enhanced alpha6 and beta4 integrin expression to the abrogation of the malignant phenotype by alpha2beta1 integrin expression, we have now separately re-expressed the human alpha6 or beta4 integrin subunit in the breast cancer model.

Breast↗
PubMed1999

Role of abnormal integrin-cytoskeletal interactions in impaired beta1 integrin function in chronic myelogenous leukemia hematopoietic progenitors.

Abnormal circulation and unregulated proliferation of chronic myelogenous leukemia (CML) progenitors is related, at least in part, to BCR/ABL induced abnormalities in beta1 integrin-mediated adhesion and signaling. The BCR/ABL oncogene has several potential interactions with cytoskeletal elements that are important for normal integrin signaling. In the present study, we evaluated whether abnormalities in beta1 integrin-cytoskeletal interactions were present in primary CML progenitors and contributed to defective integrin function. beta1 integrin-cytoskeletal interactions were studied in CML and normal CD34+ primary hematopoietic progenitors as well as BCR/ABL-transfected or mock-transfected M07e cells. In normal CD34+ progenitors, antibody-mediated cross-linking of beta1 integrins resulted in their redistribution into caps via a process requiring receptor-cytoskeletal interactions. CML CD34+ cells demonstrated significantly impaired beta1 integrin capping. This defect was related to the presence of the BCR/ABL gene, because capping also was impaired in BCR/ABL-transfected M07e cells. Defective receptor capping was not seen for non-integrin receptors. In addition, CML CD34- and M07eBCR/ABL cells also demonstrated increased actin polymerization and altered actin cytoskeletal organization. Further studies suggested that impaired beta1 integrin capping and defective integrin-mediated adhesion and proliferation inhibition in CML cells were related to abnormally enhanced integrin-cytoskeletal association and restricted receptor mobility. Finally, interferon alpha, which restores integrin-mediated adhesion and signaling in CML progenitors, also enhanced integrin capping in CD34+ cells. These studies suggest that p210BCR/ABL induces abnormal association of integrin receptors with the cytoskeleton and restricted receptor mobility and provide new insights into mechanisms underlying abnormal integrin function in CML progenitors.

Actins↗
PubMed1994

The size of the intracellular beta 1-integrin precursor pool regulates maturation of beta 1-integrin subunit and associated alpha-subunits.

A large pool of precursor beta 1-integrin subunits is frequently found intracellularly. During malignant transformation this pool often disappears. Concomitantly, integrin-mediated cell-adhesion functions are disturbed, even though no change in the number of beta 1-integrin receptors on the cell surface can be observed. Here, we have studied the role of an intracellular pre-beta 1-integrin pool by transfecting human MG-63 osteosarcoma cells with plasmid construction producing an antisense RNA for the beta 1-integrin subunit. Stable cell clones expressing beta 1-integrin antisense RNA were shown to have a reduced intracellular pool of pre-beta 1-integrin subunits. In the antisense-transfected cells, the synthesis of the beta 1-integrin chain was reduced by 65% compared with non-transfected or vector-transfected MG-63 cells. The decreased synthesis of the beta 1-integrin chain was associated with accelerated maturation of the beta 1-integrin chain (half-maturation time about 5 h in antisense-transfected cells compared with about 10.5 h in control cells), whereas maturation of the alpha-integrin chain slowed down. The amount of beta 1-integrins on the cell surface, however, remained unaltered. Cell clones with the largest decrease in the relative amount of the pre-beta 1-integrin subunit also showed altered integrin function. They were found to synthesize fibronectin, but were unable to assemble it into a fibronectin matrix on the cell surface. Thus we conclude that the repression of biosynthesis of the beta 1-integrin chain leads to alterations in receptor maturation and may be connected with altered receptor function.

Amino Acid Sequence↗
PubMed1991

Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins.

The integrin family of cell adhesion receptors mediates many of the interactions between cells and the extracellular matrix. Because the extracellular matrix has profound influences on cell behavior, it seems likely that integrins transduce biochemical signals across the cell membrane. The nature of these putative signals has, thus far, remained elusive. Antibody-mediated clustering of integrin receptors was used to mimic the integrin clustering process that occurs during formation of adhesive contacts. Human epidermal carcinoma (KB) cells were incubated with an anti-beta 1 integrin monoclonal antibody for 30 min on ice followed by incubation at 37 degrees C with anti-rat IgG. This treatment, which induced integrin clustering, stimulated the phosphorylation on tyrosine residues of a 115- to 130-kDa complex of proteins termed pp130. When integrins were clustered in the presence of the phosphatase inhibitor sodium orthovanadate, pp130 showed a substantial increase in phosphorylation compared to the case in which integrins were clustered in the absence of vanadate. Maximal pp130 phosphorylation was observed 10-20 min after initiation of integrin clustering in the absence of vanadate or after 5-10 min in its presence. These time courses roughly parallel the formation of integrin clusters on the cell surface as observed by fluorescence microscopy. pp130 phosphorylation depended on the amount of anti-integrin antibody present. Additionally, the tyrosine phosphorylation of pp130 showed specificity since it was stimulated by antibodies to the integrin alpha 3 and beta 1 subunits but not by antibodies to other integrin alpha subunits or to nonintegrin cell surface proteins. Immunoprecipitation experiments clearly demonstrated that pp130 is not itself a beta 1 integrin. It is postulated, therefore, that the integrin-stimulated tyrosine phosphorylation of pp130 may reflect part of an important signal transduction process between the extracellular matrix and the cell interior.

Antigen-Antibody Complex↗
PubMed1995

Inducible interaction of integrin alpha 2 beta 1 with calreticulin. Dependence on the activation state of the integrin.

We have previously demonstrated an interaction between the highly conserved KXGFFKR sequence of the integrin alpha-subunit cytoplasmic domains and calreticulin. Since this highly conserved sequence motif has been implicated in the regulation of the integrin affinity state, we wanted to determine whether the calreticulin-integrin interaction also depended on the integrin affinity state, and whether calreticulin occupation of integrin via the KXGFFKR motif was involved in the regulation of the ligand affinity state. We now demonstrate that anti-integrin antibody- or phorbol 12-myristate 13-acetate (PMA)-induced activation of the alpha 2 beta 1 integrin on Jurkat cells, as determined by stimulation of adhesion to collagen type I, resulted in an increased amount of calreticulin bound to this integrin. alpha 2 beta 1 activation with either anti-beta 1 or anti-alpha 2 monoclonal antibodies resulted in a greater amount of calreticulin coimmunoprecipitating with this integrin. Inactivation by neutralizing anti-integrin antibodies abrogated the calreticulin-integrin interaction. A correlation was also found between PMA-induced alpha 2 beta 1 activation and the amount of calreticulin bound to this integrin. Furthermore, pretreatment of streptolysin O-permeablized Jurkat cells with an anti-calreticulin antibody resulted in a significant and specific inhibition of the adhesion to collagen type I that could be induced by antibodies to alpha 2 beta 1 or by PMA. These data suggest that the active, high affinity form of alpha 2 beta 1 binds calreticulin and that calreticulin binding to the alpha 2 cytoplasmic domain may be required for stabilizing the high affinity state of this integrin. The data presented here also demonstrate, for the first time, an inducible interaction of an integrin with an intracellular protein that occurs via the alpha subunit of the integrin.

Amino Acid Sequence↗
PubMed1994

Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly.

The ability of single subunit chimeric receptors containing various integrin beta intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the beta 1, beta 3, beta 3B, or beta 5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the beta 3 and beta 5 chimeras mimicked endogenous ligand-occupied integrins and, like the beta 1 chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the beta 3B intracellular domain (a beta 3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intracellular mechanism. Furthermore, when expressed at higher levels, the beta 1 and beta 3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The beta 5 chimera was a less effective inhibitor, and the beta 3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected beta 1 chimera and the endogenous beta 1 subunit indicated that in 10 to 15 h assays, the beta 1 chimera can inhibit cell spreading when expressed at levels approximately equal to the endogenous beta 1 subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit alpha 5 beta 1 localization to fibrils and matrix assembly. Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that beta intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.

Amino Acid Sequence↗