Expression of alpha 4 beta 7 integrin on eosinophils and modulation of alpha 4-integrin-mediated eosinophil adhesion via CD4.
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CD4 Antigens↗
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Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts.
Motile chick skeletal fibroblasts adhere to a laminin substrate by means of clustered beta 1 integrins. These integrin "macroaggregates" are similar to classic focal contacts but do not appear dark under interference-reflection microscopy. They contain alpha 5 integrin and are associated with extracellular fibronectin. To study their behavior during cell movement, time-lapse, low-light video microscopy was used to image integrins on living cells tagged with a fluorescent anti-beta 1 integrin antibody. Integrin macroaggregates remain fixed with respect to the substratum, despite the fact that they fluctuate in size, density, and shape over a period of minutes. Upon detachment of the cell rear, as much as 85% of the beta 1 integrin density of a macroaggregate remains behind on the substrate, along with both alpha 5 integrin and fibronectin. Release of the cell rear does not involve cleavage of the beta 1 integrin cytoplasmic domain from the remainder of the protein. These results indicate that cell motility does not require regulated detachment of integrin receptors from the substrate. On the other hand, cytoskeletal components and a variable fraction of the integrins are carried forward with the cell during detachment, suggesting that some type of cortical disassembly process does occur. Integrin macroaggregate structures are not recycled intact after detachment of the cell rear from the substrate. They do not persist on the cell surface, nor can they be seen to be engulfed by vesicles; yet, some of the individual integrins that make up these macroaggregates are eventually transported forward by both vesicular and cell-surface routes. Antibody-tagged integrins accumulate in dense patches at the lateral edges and dorsal surface of the cell, and move forward on the cell surface. The tagged integrins also enter cytoplasmic vesicles, which move forward within the cytoplasm. Macroaggregates generally form and grow at the cell front; however, application of fluorescent antibody causes integrins to disappear from the leading edge. Therefore, it has not been possible to directly visualize the recycling of the forward moving tagged integrins into new macroaggregates at the cell front. Surprisingly, under these conditions cells move normally despite the absence of any delivery of tagged integrin to the leading edge, indicating that recycling of integrins to the lamella is not required for apparently normal motility.
Animals↗
Regulation of vascular smooth muscle cell integrin expression by transforming growth factor beta1 and by platelet-derived growth factor-BB.
We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the alpha v beta 3 integrin complex was induced by both growth factors, although TGF-beta 1 appeared to be the more potent inducer. mRNA level of the beta 3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the alpha v integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the alpha v beta 3 integrin protein complex after growth factor stimulation was due to increased expression of the beta 3 integrin subunit mRNA. The TGF-beta 1 induced increase in beta 3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in beta 3 mRNA level. By contrast, PDGF-BB induced a more rapid increase in beta 3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable beta 3 integrin mRNA remained after 24 h. Interestingly, the PDGF-BB induced elevation of beta 3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the alpha 5 integrin subunit in response to growth factors was very similar to beta 3. However, in contrast to beta 3 and alpha 5, neither TGF-beta 1 nor PDGF-BB were able to alter the expression of the beta 1 integrin subunit in vascular SMC. However, in TGF-beta 1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the beta 1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-beta 1 treated fibroblasts. The alpha 1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with beta 1. Analysis of the alpha 1 integrin subunit mRNA level indicated that it was indeed induced by TGF-beta 1, but not by PDGF-BB, suggesting that the 190 kDa polypeptide may be the alpha 1 integrin subunit. These results indicate that TGF-beta 1 and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC.
Animals↗
Characterization of integrins in cultured human renal cortical tubule epithelial cells.
We have characterized the integrins present on cultured tubule epithelial cells from human renal cortexes, enriched for proximal cells, using fluorescence microscopy, immunoprecipitation, and cell adhesion assays. By immunofluorescence, the alpha 3-integrin subunit stained most intensely and was present on all cells predominantly at cell-cell contacts. The alpha 6-subunit was present on all cells in a pattern consistent with extracellular matrix contacts. The alpha 5-subunit was present on most cells in a cell-matrix contact pattern; alpha V-subunit was weakly positive and occasionally seen in cell-matrix contacts. The alpha 2-subunit was present on clusters of distal tubule cells, predominantly at cell-cell contacts. Immunoprecipitation revealed the predominant integrin to be alpha 3 beta 1 with some alpha 2 beta 1, presumably contributed by distal cells. The alpha 5 beta 1-, alpha 6 beta 1-, alpha 6 beta 4-, and alpha V beta 3-integrins, as well as trace amounts of alpha 1 beta 1-integrins, were also present. The alpha 4 beta 1-integrin was not detected. Initial attachment to fibronectin was mediated by alpha V beta 3- and alpha 5 beta 1-integrins; initial attachment to laminin was mediated by the alpha 6 beta 1- and alpha 3 beta 1- integrins and, in some preparations, by an unidentified integrin; and initial attachment to collagen type IV was mediated by alpha V beta 3-integrin and an unidentified beta 1-integrin. After extensively immunodepleting membrane extracts with anti-alpha 1, -alpha 2, -alpha 3, -alpha 4, -alpha 5, -alpha 6, and -alpha V antibodies, an anti-beta 1 antibody still precipitated an integrin. Its electrophoretic mobility differs from the laminin-binding alpha 7 beta 1-integrin. Thus we have identified many of the integrins on cortical tubule cells and their role in mediating initial attachment to extracellular matrix. However, the cell adhesion assays and immunoprecipitations suggest the presence of an unidentified beta 1-integrin that may mediate renal tubule cell attachment to laminin and collagen.
Cell Adhesion↗
Increase in suprabasilar integrin adhesion molecule expression in human epidermal neoplasms accompanies increased proliferation occurring with immortalization and tumor progression.
In a previous prospective study of 80 patients with squamous cell carcinoma of the upper aerodigestive tract, a progressive increase in expression of the integrin cell adhesion molecule alpha 6 beta 4 in suprabasilar cell layers of the tumor parenchyma was associated with an increase in early recurrence after therapy. In this study, we determined the relationship of the altered expression pattern of the integrin to changes occurring during benign, invasive, or metastatic stages of tumor development. Suprabasilar expression of integrin alpha 6 beta 4 appeared with neoplastic transformation in benign squamous papillomas, but homogeneous expression occurred more frequently in the parenchyma of primary and metastatic squamous cell carcinomas. The variation in the extent of suprabasilar integrin expression among the tumors corresponded to the variation in the population undergoing proliferation as determined by two independent markers of proliferation. Integrin expression was quantified in primary, HPV 16 DNA-immortalized, and v-ki-ras oncogene-transformed keratinocytes, and the pattern of expression was compared with cell cycle progression. Primary keratinocyte lines showed a bimodal distribution of integrin expression, with one population showing decreased integrin expression, cell size, and a block of cell cycle progression consistent with differentiation, whereas another population exhibited high integrin expression and full progression through the cell cycle, consistent with proliferation. HPV-immortalized and v-ki-ras-transformed cell lines undergoing continuous proliferation exhibited uniformly strong integrin expression, which was similar in intensity to that observed in the proliferating population of normal keratinocytes. Similar increases in expression of two additional integrins, alpha 2 beta 1 and alpha 3 beta 1, occurred along with integrin alpha 6 beta 4 in tissue specimens and cell lines derived from neoplasms. Thus, epidermal neoplasms display an increase in a population of cells exhibiting constitutive expression of a repertoire of integrins, which is similar to that found transiently in the acute phase of epidermal wound healing, a physiological response in which hyperproliferation, retention of multiple layers of proliferating cells, and migration occur. The association of a progressive increase in suprabasilar expression of these integrins with early tumor recurrence and advanced neoplasia suggests that constitutive expression and function of the same repertoire of integrins may be advantageous, rather than sufficient, for tumor progression.
Antigens, Surface↗
Integrins in human cells and tumors.
We have studied the distribution of the alpha- and beta-subunits of integrins in developing and adult human kidney as well as in selected other tissues and cultured cells. In cultured cells some of the integrin subunits (beta 1, alpha 1, alpha 2 and alpha 5) colocalize with talin at focal adhesions when plated on an appropriate ligand. Similarly, in tissues the polarization of beta 1-integrins in colocalization with talin appears to indicate adhesive complexes, as demonstrated in adult glomeruli. In human kidney, the alpha subunits of integrins were seen to be segment-specifically expressed already in fetal tissues. In glomeruli the integrin alpha 1 subunit characterized mesangial cells while the alpha 2 and alpha 3 subunits showed immunoreactivity in endothelial cells and podocytes, respectively. In renal tubuli, the alpha 6 subunit, complexed with the beta 1 subunit, showed a typical polarized distribution coaligning with the tubular basement membrane while the alpha 3 and alpha 2 subunits were expressed in distal tubular cells. These results suggested that in kidney the alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 integrins can function as basement membrane receptors. The alpha 5 subunit was nearly lacking in the kidney and it appears to be mainly expressed in some smooth muscle cells. In other tissues distinct patterns in the expression of integrins were found. Thus, in many glandular epithelial cells the alpha 3 beta 1 integrin appeared to function as a basement membrane receptor while in various stratified epithelia and in the breast such a polarized localization could be found for the alpha 6 beta 4 integrin. Finally, although presenting a clearly polarized distribution for beta 1 integrins, none of the alpha subunits could be found in cardiac or skeletal muscle cells and none of the integrins could be revealed in neuronal cells of human developing and adult cerebrum or cerebellum, although neurons in peripheral tissues contained abundantly the alpha 6 beta 1 integrin complex. In human tumors, the tumor cells, including also metastastatic tumors, generally presented the same integrins as their tissues of origin. In some poorly differentiated tumors both a population heterogeneity and even a lack of expression or a disorganization of basement membrane receptor integrins was obvious.
Animals↗
Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated.
We have used laser optical trapping and nanometer-level motion analysis to investigate the cytoskeletal associations and surface dynamics of beta 1 integrin, a cell-substrate adhesion molecule, on the dorsal surfaces of migrating fibroblast cells. A single-beam optical gradient trap (laser tweezers) was used to restrain polystyrene beads conjugated with anti-beta 1 integrin mAbs and place them at desired locations on the cell exterior. This technique was used to demonstrate a spatial difference in integrin-cytoskeleton interactions in migrating cells. We found a distinct increase in the stable attachment of beads, and subsequent rearward flow, on the lamellipodia of locomoting cells compared with the retracting portions. Complementary to the enhanced linkage of integrin at the cell lamellipodium, the membrane was more deformable at the rear versus the front of moving cells while nonmotile cells did not exhibit this asymmetry in membrane architecture. Video microscopy and nanometer-precision tracking routines were used to study the surface dynamics of integrin on the lamellipodia of migrating cells by monitoring the displacements of colloidal gold particles coated with anti-beta 1 integrin mAbs. Small gold aggregates were rapidly transported preferentially to the leading edge of the lamellipod where they resumed diffusion restricted along the edge. This fast transport was characterized by brief periods of directed movement ("jumps") having an instantaneous velocity of 37 +/- 15 microns/min (SD), separated by periods of diffusion. In contrast, larger aggregates of gold particles and the large latex beads underwent slow, steady rearward movement (0.85 +/- 0.44 micron/min) (SD) at a rate similar to that reported for other capping events and for migration of these cells. Cell lines containing mutated beta 1 integrins were used to show that the cytoplasmic domain is essential for an asymmetry in attachment of integrin to the underlying cytoskeletal network and is also necessary for rapid, intermittent transport. However, enhanced membrane deformability at the cell rear does not require integrin-cytoskeletal interactions. We also demonstrated that posttranslational modifications of integrin could potentially play a role in these phenomena. These results suggest a scheme for the role of dynamic integrin-mediated adhesive interactions in cell migration. Integrins are transported preferentially to the cell front where they form nascent adhesions. These adhesive structures grow in size and associate with the cytoskeleton that exerts a rearward force on them. Dorsal aggregates more rearward while those on the ventral side remain fixed to the substrate allowing the cell body to move forward. Detachment of the cell rear occurs by at least two modes: (a) weakened integrin-cytoskeleton interactions, potentially mediated by local modifications of linkage proteins, which lead to weakened cell-substratum interactions and (b) ripping of integrins and the highly deformable membrane from the cell body.
3T3 Cells↗
Pneumocystis carinii attachment increases expression of fibronectin-binding integrins on cultured lung cells.
Pneumocystis carinii is an extracellular pathogen which requires attachment to alveolar epithelial cells for growth and replication. Previous studies have demonstrated that the extracellular matrix protein fibronectin (Fn) facilitates attachment of P. carinii to lung cells. This study addresses the role of cell surface Fn receptors (integrins) as mediators of P. carinii attachment and demonstrates the effect of P. carinii attachment on integrin expression on cultured lung cells. To determine the role of Fn-binding integrins in P. carinii attachment, attachment of 51Cr-labelled P. carinii organisms to the lung epithelial cell line A549 was quantified in the presence or absence of anti-integrin antibodies. Antibodies to the alpha v and alpha 5 integrin subunits significantly inhibited P. carinii attachment, while addition of antibody to the alpha subunit of a non-Fn-binding integrin, alpha 2, did not affect P. carinii attachment. To further investigate the role of Fn-binding integrins in P. carinii attachment, the effect of P. carinii attachment on expression of the alpha v and alpha 5 integrin subunits was determined. A549 cells incubated with either P. carinii organisms or with the P. carinii major surface glycoprotein gp120 demonstrated a 3- to 10-fold increase in expression of the alpha 5 integrin subunit; however, neither P. carinii nor gp120 affected the expression of alpha v integrin. Furthermore, the effects of P. carinii on A549 cell alpha 5 integrin expression were attenuated by the addition of an anti-gp120 antibody which blocks P. carinii attachment to A549 cells. Therefore, P. carinii attachment to lung epithelial cells appears to be mediated by alpha v- and alpha 5-containing integrins expressed on the epithelial cell surface, and P. carinii attachment results in increased expression of the alpha 5 integrin subunit.
Animals↗
Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development.
A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA-1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA-1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1 integrin at all.
Aorta↗
Transforming growth factor-beta regulates collagen gel contraction by increasing alpha 2 beta 1 integrin expression in osteogenic cells.
The contraction of floating collagen gels is suggested to mimic the reorganization of collagenous matrix during development and tissue healing. Here, we have studied two osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG. Transforming growth factor-beta (TGF-beta), a putative regulator of bone fracture healing, increased collagen gel contraction by MG-63 and HOS-MNNG, but not by HOS cells. Our data show that TGF-beta-induced fibronectin synthesis is not sufficient for the process. Instead, anti-beta 1 integrin antibodies could prevent the contraction. There are three different integrin heterodimers that are known to mediate the cell-collagen interaction, namely alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1. In MG-63 cells TGF-beta increased the expression of alpha 2 beta 1 integrin and decreased the expression of alpha 3 beta 1 integrin, whereas alpha 1 beta 1 integrin is not expressed. HOS cells had no alpha 2 beta 1 integrin, neither did TGF-beta induce its expression. However, HOS-MNNG cells expressed more alpha 2 beta 1 integrin when treated with TGF-beta. Thus, we suggest that the mechanism of the enhanced collagen gel contraction by TGF-beta is the increased expression of alpha 2 beta 1 integrin heterodimer. To further test this hypothesis, we expressed a full-length alpha 2 integrin cDNA in HOS cells and in MG-63 cells. We obtained HOS cell clones that expressed alpha 2 beta 1 heterodimer, and the ability of these cells to contract collagen gels was greatly enhanced. Furthermore, the contraction by MG-63 cells transfected with alpha 2 integrin cDNA was enhanced, and the contraction by cells transfected with antisense oriented alpha 2 integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the increased alpha 2 integrin expression alone was sufficient for the enhanced contraction of collagen gels. Furthermore, the amount of alpha 2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels.
Antigens, CD↗
Integrin alpha 6 is a differentiation antigen of human granulosa cells.
We previously reported a new murine monoclonal antibody (mAb), OG-1, raised against human granulosa cells (GC) and showed that the OG-1 antigen is a cell surface differentiation-related molecule expressed on GC of follicles and on large luteal cells in the early to midluteal phase. In this study, we further characterized the OG-1 antigen. An immunohistochemical study showed that the OG-1 antigen was also expressed on endometrial glandular and stromal cells and on first trimester and term placental trophoblasts. The antigen purified from endometrium, chorionic villi, or placenta by immunoaffinity chromatography using the mAb OG-1 consisted of a 120-kilodalton (kDa) protein. Partial amino acid sequencing of the 120-kDa protein purified from placenta revealed that the 16 amino acids from the N-terminus were identical to those of the heavy chain of human integrin alpha 6, the molecular mass of which is reportedly 118-126 kDa. We examined the tissue distribution of integrins alpha 6, beta 1, and beta 4, because integrin alpha 6 forms heterodimers with integrins beta 1 or beta 4. Indirect immunofluorescence staining of various follicles and corpora lutea using two known antiintegrin alpha 6 monoclonal antibodies (450-30A1 and GoH3) showed that integrin alpha 6 was expressed on granulosa and large luteal cells in a profile similar to that of the OG-1 antigen. Integrin beta 1 was expressed on granulosa and large luteal cells, but integrin beta 4 was undetectable. Integrin beta 1 was also expressed on thecal and stromal cells. In term placentas, the OG-1 antigen and integrins alpha 6 and beta 4 were expressed on trophoblasts, whereas integrin beta 1 was expressed on villous capillary endothelia. These results indicate that the mAb OG-1 recognizes integrin alpha 6, and that integrin alpha 6 is a differentiation-related antigen of human GC, suggesting the involvement of integrins in human folliculogenesis and luteal formation.
Adult↗
The alpha 5 beta 1 integrin associates with a dystrophin-containing lattice during muscle development.
The organization of the alpha 5 beta 1 integrin on skeletal muscle was studied in culture and in sections from adult and embryonic tissue using monoclonal antibodies specific for the alpha 5 subunit. The alpha 5 beta 1 integrin showed changes in organization and in the molecules with which it colocalizes. On early myoblasts, possessing a fibroblast-like morphology, the alpha 5 integrin organization was indistinguishable from that on fibroblasts; it was expressed prominently and localized in numerous focal contacts around the cell periphery. In bipolar myoblasts and early myotubes, the alpha 5 integrin was expressed only weakly and localized in a small number of focal contact-like structures. As myogenesis proceeded there was an apparent increase in integrin expression and a change in organization. In addition to the focal contact-like structures that persist throughout myogenesis in vitro, a dense lattice-like structure of integrin appeared. Fibrillar fibronectin, talin, and non-muscle alpha-actinin did not colocalize with the alpha 5 beta 1 integrin in the lattice structure as they did in the focal contact-like structures. However, dystrophin, which displayed a diffuse distribution earlier, now colocalized with the alpha 5 beta 1 integrin in the punctate lattice. Coincident with the registration of myofibrils into visible sarcomeres, the prominent dense, lattice structure disappeared leaving the focal contact-like structures as the only regions of organized alpha 5 beta 1 integrin. Despite the presence of the beta 1 integrin in neuromuscular or myotendinous junctions in vivo and on myotubes in vitro, the alpha 5 beta 1 integrin was not present in either junction. These observations suggest that the alpha 5 beta 1 integrin is involved in the adhesion of muscle to the extracellular matrix, the organization of the dystrophin-containing lattice, and the organization of nascent myofibrils which emanate from the focal contact- and stress fiber-like structures in muscle. Other integrins appear to anchor myofibrils at the myotendinous and neuromuscular junctions.
Animals↗
Integrin on developing and adult skeletal muscle.
Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu
Animals↗
Homology modelling of integrin EF-hands. Evidence for widespread use of a conserved cation-binding site.
Integrin alpha-subunits contain three or four peptide sequences that are similar to the EF-hand, a 13-residue bivalent cation-binding motif found in calmodulin and parvalbumin. The integrin sequences differ from classical EF-hands in that they lack a co-ordinating residue at position 12. One hypothesis to explain integrin-ligand binding is that aspartate-containing recognition sequences in integrin ligands, which bind at or near to the EF-hand-like sequences, may take the place of the missing residue and co-ordinate directly to the bound cation. In this report, homology modelling of integrin EF-hand-like sequences has been performed using the X-ray structure of calmodulin as a template in order to assess the functional activity of the integrin sequences. In the calmodulin-integrin hybrid structures, integrin EF-hand-like sequences were able to retain cations whereas control sequences did not. Structural analyses demonstrated that the integrin sequences in the hybrid proteins closely resembled conventional EF-hands. The integrin sequences are therefore highly likely to bind Ca2+ ions in vivo, a prerequisite for the ligand-binding model. Database searching with a matrix derived from known integrin EF-hand-like sequences has been used to identify other proteins containing the integrin EF-hand-like motif. Annexin V (anchorin CII), atrial natriuretic peptide receptors and the 70 kDa heat-shock protein were identified by the matrix; the functions of these proteins are known from previous studies to be bivalent cation-dependent. These findings suggest that the integrin EF-hand-like sequence may be a more common motif than originally thought.
Amino Acid Sequence↗
Purification and characterization of mammalian integrins expressed by a rat neuronal cell line (PC12): evidence that they function as alpha/beta heterodimeric receptors for laminin and type IV collagen.
Cells of the rat neuronal line, PC12, adhere well to substrates coated with laminin and type IV collagen, but attach poorly to fibronectin. Adhesion and neurite extension in response to these extracellular matrix proteins are inhibited by Fab fragments of an antiserum (anti-ECMR) that recognizes PC12 cell surface integrin subunits of Mr 120,000, 140,000, and 180,000 (Tomaselli, K. J., C. H. Damsky, and L. F. Reichardt. 1987. J. Cell Biol. 105:2347-2358). Here we extend our study of integrin structure and function in PC12 cells using integrin subunit-specific antibodies prepared against synthetic peptides corresponding to the cytoplasmic domains of the human integrin beta 1 and the fibronectin receptor alpha (alpha FN) subunits. Anti-integrin beta 1 immunoprecipitated a 120-kD beta 1 subunit and two noncovalently associated integrin alpha subunits of 140 and 180 kD from detergent extracts of surface-labeled PC12 cells. Immunodepletion studies using anti-integrin beta 1 demonstrated that these two putative alpha/beta heterodimers are identical to those recognized by the adhesion-perturbing ECMR antiserum. Anti-alpha FN immunoprecipitated fibronectin receptor heterodimers in human and rat fibroblastic cells, but not in PC12 cells. Thus, low levels of expression of the integrin alpha FN subunit can explain the poor attachment of PC12 cells to FN. The PC12 cell integrins were purified using a combination of lectin and ECMR antibody affinity chromatography. The purified integrins: (a) completely neutralize the ability of the anti-ECMR serum to inhibit PC12 cell adhesion to laminin and collagen IV; (b) have hydrodynamic properties that are very similar to those of previously characterized integrin alpha/beta heterodimeric receptors for ECM proteins; and (c) can be incorporated into phosphatidylcholine vesicles that then bind specifically to substrates coated with laminin or collagen IV but not fibronectin. Thus, the ligand-binding specificity of the liposomes containing the purified PC12 integrins closely parallels the substrate-binding preference of intact PC12 cells. These results demonstrate that mammalian integrins purified from a neuronal cell line can, when incorporated into lipid vesicles, function as receptors for laminin and type IV collagen.
Animals↗
Integrin heterodimer and receptor complexity in avian and mammalian cells.
We report data showing that the integrin receptor complex in chickens contains several discrete heterodimers all sharing the beta 1-integrin subunit combined separately with different alpha-subunits. Using antisera to synthetic peptides based on cDNA sequences of chicken and human alpha-integrin subunits to analyze the integrin complement of avian and mammalian cells, we show that band 2 of the chicken integrin complex contains alpha-subunits related to both alpha 3- and alpha 5-subunits of human integrins. alpha 3 beta 1 and alpha 5 beta 1 have both previously been shown in human cells to be fibronectin receptors and alpha 3 beta 1 can also act as a receptor for laminin and collagen. We also provide evidence for the presence, in band 1 of the chicken integrin complex, of a third integrin alpha-subunit which is also alpha 5 related. This integrin subunit exists in a separate heterodimer complex with beta 1 and binds to fibronectin-affinity columns. These results provide explanations for published data showing that the avian integrin complex contains receptor activity for a variety of extracellular matrix proteins. We conclude that the chicken integrin complex comprises a set of beta 1-integrin heterodimers equivalent to the human VLA antigens and includes at least two fibronectin receptors. Finally, we show that chicken embryo fibroblasts also contain a beta 3-class integrin related to the RGD receptors defined in various human cells.
Animals↗
The role of protein tyrosine phosphorylation in integrin-mediated gene induction in monocytes.
Integrin-mediated cell adhesion, or cross-linking of integrins using antibodies, often results in the enhanced tyrosine phosphorylation of certain intracellular proteins, suggesting that integrins may play a role in signal transduction processes. In fibroblasts, platelets, and carcinoma cells, a novel tyrosine kinase termed pp125FAK has been implicated in integrin-mediated tyrosine phosphorylation. In some cell types, integrin ligation or cell adhesion has also been shown to result in the increased expression of certain genes. Although it seems reasonable to hypothesize that integrin-mediated tyrosine phosphorylation and integrin-mediated gene induction are related, until now, there has been no direct evidence supporting this hypothesis. In the current report, we explore the relationship between integrin-mediated tyrosine phosphorylation and gene induction in human monocytes. We demonstrate that monocyte adherence to tissue culture dishes or to extracellular matrix proteins is followed by a rapid and profound increase in tyrosine phosphorylation, with the predominant phosphorylated component being a protein of 76 kD (pp76). Tyrosine phosphorylation of pp76 and other monocyte proteins can also be triggered by incubation of monocytes with antibodies to the integrin beta 1 subunit, or by F(ab')2 fragments of such antibodies, but not by F(ab) fragments. The ligation of beta 1 integrins with antibodies or F(ab')2 fragments also induces the expression of immediate-early (IE) genes such as IL-1 beta. When adhering monocytes are treated with the tyrosine kinase inhibitors genistein or herbimycin, both phosphorylation of pp76 and induction of IL-1 beta message are blocked in a dose-dependent fashion. Similarly, treatment with genistein or herbimycin can block tyrosine phosphorylation of pp76 and IL-1 beta message induction mediated by ligation of beta 1 integrin with antibodies. These observations suggest that protein tyrosine phosphorylation is an important aspect of integrin-mediated IE gene induction in monocytes. The cytoplasmic tyrosine kinase pp125FAK, although important in integrin signaling in other cell types, seems not to play a role in monocytes because this protein could not be detected in these cells.
Benzoquinones↗
Integrin, a transmembrane glycoprotein complex mediating cell-substratum adhesion.
A monoclonal antibody, CSAT, which inhibits the adhesion of chick cells to substrata coated with fibronectin, laminin and vitronectin, has been used to identify a cell surface receptor required for cell-substratum adhesion. This receptor, termed integrin, is found on the ventral surface of cells in close contact adhesion sites, at the periphery of adhesion plaques and beneath stress fibres. It is a heterodimer consisting of non-covalently linked alpha and beta subunits. Integrin binds directly to laminin, fibronectin and vitronectin with dissociation constants in the micromolar range. The binding of integrin to matrix molecules is sensitive to peptides carrying the cell-binding sequence Arg-Gly-Asp and requires heteromeric integrity. Integrin also binds directly to the cytoskeleton-associated protein talin. Thus, integrin has the properties of a transmembrane molecule capable of bringing extracellular matrix and cytoskeleton-associated molecules in proper juxtaposition to form adhesion structures. The integrin beta subunit is phosphorylated following Rous sarcoma virus transformation. Phosphorylation alters the ability of the receptor to bind extracellular matrix molecules as well as talin, suggesting a mechanism for the alteration of cellular adhesive and morphological properties following malignant transformation. A major phosphorylation site is on the cytoplasmic domain of the beta subunit. Synthetic peptides homologous with this region of integrin inhibit integrin-talin binding. The gene for the beta subunit of integrin has been sequenced. Its structure is consistent with the membrane-spanning properties of the receptor. Integrin is structurally and serologically related to adhesion receptors from mammalian tumour cells, fibroblasts, platelets and lymphocytes. It appears to be a member of a supergene family of receptors involved in cellular adhesive interactions. Antibody and peptide inhibition experiments have suggested a role for integrin and integrin-like molecules in cell migration, neurite extension, neural differentiation, histogenesis and embryonic development in Drosophila. Thus, integrin appears representative of a set of evolutionarily conserved, biologically important adhesive molecules.
Animals↗