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PubMed2003

Beta1 integrin/focal adhesion kinase-mediated signaling induces intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation.

We have assessed characteristics of primary human osteoblasts, shedding light on signaling mediated by beta1 integrin. beta1 integrins are major receptors for these matrix glycoproteins. 1) Integrins beta1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphav were highly expressed on primary osteoblasts. 2) Engagement of beta1 integrins on osteoblasts by cross-linking with specific antibody or ligand matrices, such as fibronectin or collagen, augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on the surface. 3) Up-regulation of ICAM-1 and RANKL on osteoblasts by beta1 stimulation was completely abrogated by pretreatment with herbimycin A and genistein, tyrosine kinase inhibitors, or transfection of dominant negative truncations of focal adhesion kinase (FAK). 4) Engagement of beta1 integrins on osteoblasts induced tartrate-resistant acid phosphatase-positive multinuclear cell formation in the coculture system of osteoblasts and peripheral monocytes. 5) Up-regulation of tartrate-resistant acid phosphatase-positive multinuclear cell formation by beta1 stimulation was completely abrogated by transfection of dominant negative truncations of FAK. Our results indicate that beta1 integrin-dependent adhesion of osteoblasts to bone matrices induces ICAM-1 and RANKL expression and osteoclast formation via tyrosine kinase, especially FAK. We here propose that beta1 integrin/FAK-mediated signaling on osteoblasts could be involved in ICAM-1- and RANKL-dependent osteoclast maturation.

Antibodies, Monoclonal↗
PubMed2001

Clomiphene citrate does not affect the secretion of alpha3, alphaV and beta1 integrin molecules during the implantation window in patients with unexplained infertility.

BACKGROUND: The expression of integrin molecules on the endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation. A prospective, controlled study was carried out to investigate the effect of clomiphene citrate (CC) on secretions of beta1, alpha3 and alphaV integrin molecules in the endometrium of patients with unexplained infertility during the implantation window. METHODS: A total of 40 endometrial samples was evaluated in both spontaneous (n = 13) and ensuing clomiphene-treated cycles (100 mg on days 5-9) and also from fertile women serving as controls (n = 14) during postovulatory 7th or 8th day of menstrual cycle. A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities. Endometrial thickness and serum oestradiol and progesterone concentrations were also measured on the day of sampling. RESULTS: Staining of alpha(v) but not beta1 and alpha3 integrins was significantly less intense in infertile cases than fertile control cases (1.42 +/- 0.12 versus 2.21 +/- 0.13 respectively, P = 0.012) and this was not restored to normal concentrations with treatment. CONCLUSIONS: Our study indicated that cc treatment significantly decreased the endometrial thickness and increased oestradiol and progesterone concentrations. However, secretion of alpha(v), beta1 and alpha3 integrin molecules, which might play a role in implantation, was not affected.

Antigens, CD↗
PubMed1997

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Antibodies, Monoclonal↗
PubMed1998

The role of alpha2 beta1 and alpha3 beta1 integrin receptors in the initial anchoring of MDA-MB-231 human breast cancer cells to cortical bone matrix.

Molecular mechanisms involved in the rapid attachment of the human breast cancer cell line MDA-MD-231 to cortical bone matrix were studied. The attachment of MDA-MD-231 cells to cortical bone disks could be blocked by 75% when cells were pretreated with a monoclonal antibody to the beta1-subunit of the integrin family. Monoclonal antibodies against the alpha2, alpha3, and alpha5 integrin subunits inhibited the attachment by 76, 26, and 8 % respectively. Collagenase type I and collagen type I antibody blocked the cell attachment by 45 and 50 % whereas pretreatment of the cells with soluble collagen type I blocked the attachment by 85 %. Our study with a panel of cancer cell lines further showed a close correlation between alpha2 beta1 and alpha3 beta1 integrin receptor expression and the capability to attach to cortical bone. These receptors appear to be the key receptors utilized by cancer cells for the initial attachment to cortical bone, and this could facilitate the localization of alpha2 beta1 and alpha3 beta1 expressing cancer cells to the skeleton.

Animals↗
PubMed1998

Tyrosine phosphorylation of 40 kDa proteins in osteoblastic cells after mechanical stimulation of beta1-integrins.

Using a method for the mechanical stimulation of cells which was adapted from one developed by Wang and Ingber employing magnetic microbeads [Wang, N. D., D. E. Ingber: Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension. Biophys. J. 66, 2181-2189 (1994)], mechanical stress could be applied to specific receptors on the cell surface. To achieve this, ferromagnetic microbeads coated with different ligands were magnetized after adhesion to the cells. The beads were then 'twisted' using a second magnetic field oriented perpendicular to the magnetizing one. Contrary to most current methods, it was possible to confer the strain without deforming the cell as a whole, thus being able to observe the individual reactions of transmembrane receptors to mechanical stress. An increase in tyrosine phosphorylation of proteins migrating at approximately 40 kDa could be observed as a reaction to stress on the beta1-subunits of the integrin family, while stress to other transmembrane molecules like the transferrin or low density lipoprotein receptors with no connection to the cytoskeleton did not give this reaction. Fibroblastic cells showed, contrary to osteoblastic cells, no reaction to stress applied on transmembrane proteins.

Cell Adhesion↗
PubMed1998

Integrin activation by dithiothreitol or Mn2+ induces a ligand-occupied conformation and exposure of a novel NH2-terminal regulatory site on the beta1 integrin chain.

Integrins can be expressed in at least three functional states (i.e. latent, active, and ligand-occupied). However, the molecular bases for the transitions between these states are unknown. In the present study, changes in the accessibility of several beta1 epitopes (e.g. N29, B44, and B3B11) were used to probe activation-related conformational changes. Dithiothreitol or Mn2+ activation of integrin-mediated adhesion in the human B cell line, IM9, resulted in a marked increase in the exposure of the B44 epitope, while N29 expression levels were most sensitive to dithiothreitol treatment. These results contrasted with the epitope expression patterns of spontaneously adherent K562 cells, where N29 was almost fully accessible and B44 was low. Addition of a soluble ligand resulted in a marked increase in B44 levels, suggesting that this antibody detected a ligand-induced binding site. The N29 epitope was mapped to a cysteine-rich region near the NH2 terminus of the integrin chain, thus defining a novel regulatory site. These studies indicate that the activation of integrin function by different stimuli may involve related but nonidentical conformations. Both Mn2+ and dithiothreitol appear to induce localized conformational changes that mimic a ligand-occupied receptor. This differs from the "physiologically" activated integrins on K562 cells that display a marked increase in overall epitope accessibility without exposure of the ligand-induced binding site epitopes. The increased exposure of the N29 site on K562 cells may indicate a role for this region in the regulation of integrin function.

B-Lymphocytes↗
PubMed1998

Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation.

Several recent studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. The present study has addressed the role of the residues flanking the RGD sequence in regulating the recognition by disintegrin of the alphaIIb beta3 and alpha5beta1 complexes by construction of a panel of recombinant molecules of Elegantin (the platelet aggregation inhibitor from the venom of Trimerasurus elegans) expressing specific RGD sequence motifs. Wild-type Elegantin (ARGDNP) and several variants including Eleg. AM (ARGDMP), Eleg. PM (PRGDMP) and Eleg. PN (PRGDNP) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The inhibitory efficacies of the panel of Elegantin variants were analysed in platelet adhesion assays with substrates immobilized with fibrinogen and fibronectin. Elegantin molecules containing an Ala residue N-terminal to the RGD sequence (wild-type Elegantin and Eleg. AM) showed strong inhibitory activity towards alphaIIbbeta3-dependent platelet adhesion on fibronectin, whereas a Pro residue in this position (Eleg. PM and Kistrin, the inhibitor from the venom of Calloselasma rhodostoma) engendered lower activity. The decreased activity could not be attributed to a decrease in the affinity of the disintegrin for the alphaIIb beta3 complex because both Eleg. AM and Eleg. PM had similar Kd (app) values. In contrast, Elegantin molecules into which a Met residue was introduced in place of the Asn residue C-terminal to the RGD sequence showed 10-13-fold elevated inhibitory activity towards platelet adhesion on fibrinogen and this was maintained with either a Pro or Ala residue N-terminal to the RGD sequence. In experiments with the alpha5 beta1 complex on K562 cells, the inhibitory efficacies of the panel of Elegantin molecules were analysed under two different cation conditions. First, in the presence of Ca2+/Mg2+, K562 cell adhesion on fibronectin was inhibited equally well by Elegantin and Eleg. AM but inhibited poorly by Eleg. PM and Kistrin. In contrast with platelets, the decreased inhibitory efficacy of the PRGDMP disintegrins was due to poor recognition of the alpha5 beta1 complex. In the presence of Mn2+ cation, K562 cell adhesion on fibrinogen was observed in an alpha5 beta1-dependent manner. Under these conditions both PRGD and ARGD containing disintegrins were strong inhibitors of K562 cell adhesion on fibrinogen and this was due to a markedly improved recognition of the alpha5 beta1 complex by the PRGD molecules. These observations demonstrate the pivotal role of the amino acids flanking the RGD sequence for disintegrin recognition of integrin complexes and highlight the subtle nature by which integrin-ligand binding specificity can be modulated by both cation and adhesive motif.

Amino Acid Sequence↗
PubMed2000

Conditional ablation of beta1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.

The major epidermal integrins are alpha3beta1 and hemidesmosome-specific alpha6beta4; both share laminin 5 as ligand. Keratinocyte culture studies implicate both integrins in adhesion, proliferation, and stem cell maintenance and suggest unique roles for alphabeta1 integrins in migration and terminal differentiation. In mice, however, whereas ablation of alpha6 or beta4 results in loss of hemidesmosomes, epidermal polarity, and basement membrane (BM) attachment, ablation of alpha3 only generates microblistering due to localized internal shearing of BM. Using conditional knockout technology to ablate beta1 in skin epithelium, we have uncovered biological roles for alphabeta1 integrins not predicted from either the alpha3 knockout or from in vitro studies. In contrast to alpha3 null mice, beta1 mutant mice exhibit severe skin blistering and hair defects, accompanied by massive failure of BM assembly/organization, hemidesmosome instability, and a failure of hair follicle keratinocytes to remodel BM and invaginate into the dermis. Although epidermal proliferation is impaired, a spatial and temporal program of terminal differentiation is executed. These results indicate that beta1's minor partners in skin are important, and together, alphabeta1 integrins are required not only for extracellular matrix assembly but also for BM formation. This, in turn, is required for hemidesmosome stability, epidermal proliferation, and hair follicle morphogenesis. However, beta1 downregulation does not provide the trigger to terminally differentiate.

Animals↗
PubMed2000

Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains.

Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.

Amino Acid Substitution↗
PubMed2005

Cholesterol distribution in plasma membranes of beta1 integrin-expressing and beta1 integrin-deficient fibroblasts.

The effect of integrin receptors on the level and transmembrane localization of cholesterol molecules was investigated in beta1 integrin-expressing (beta1) and beta1 integrin-deficient (beta1 null) cells. We found that the content of specific raft components-cholesterol, sphingomyelin, and caveolin-was increased in integrin-expressing cells. Integrin presence affected as well the transmembrane distribution of cholesterol-a higher percent was found in the plasma membrane outer monolayer of beta1 compared to beta1 null cells. Sphingomyelin depletion reduced the presence of cholesterol in the outer membrane monolayer of both cell lines, but the differences in cholesterol asymmetry, observed between beta1 and beta1 null cells before sphingomyelinase treatment were preserved. These findings implied that integrin receptors affected the non-random transmembrane distribution of cholesterol. Finally, a higher percent of detergent-resistant membranes was obtained from beta1 integrin-expressing cells, suggesting that the presence of these receptors in the membranes influenced the formation and/or stabilization of lipid raft domains.

Cell Line↗
PubMed2002

Aberrant N-glycosylation of beta1 integrin causes reduced alpha5beta1 integrin clustering and stimulates cell migration.

Altered expression of cell surface N-linked oligosaccharides is associatedwith the oncogenic transformation of many types of animal cells. One of the most common forms of glycosylation in transformed cells and human tumors is the highly elevated beta1,6 branching of N-linked oligosaccharides caused by increased transcription of N-acetylglucosaminyltransferase V (GnT-V). To characterize the effects of increased beta1,6 branching on cell-matrix adhesion-mediated phenotypes, human fibrosarcoma HT1080 cells were transfected with retroviral systems encoding GnT-V that used both noninducible and tetracycline-inducible promoters. Increased GnT-V expression resulted in a >25% inhibition of cell attachment to and a >50% inhibition of cell spreading on fibronectin. Both cell adhesion and spreading were suppressed by function-blocking antibodies specific for the alpha(5) and beta(1) integrin subunits of the fibronectin receptor. Cell migration toward fibronectin and invasion through Matrigel were both substantially stimulated in cells with induced expression of GnT-V. Induction of GnT-V had no effect on the level of cell surface expression of alpha(5) and beta(1) integrin subunits but did result in a more diffuse staining of the alpha(5) and beta(1) integrin subunits on the cell surface, suggesting that inhibition of integrin clustering may be causing these cells to be less adhesive and more motile. Surprisingly, there was no detectable expression of N-linked beta1,6 branching on the alpha(5) subunit purified from HT1080 cells before and after induction of GnT-V; by contrast, however, the beta(1) subunit showed a basal level of beta1,6 branching that was greatly increased after induction of GnT-V. These results suggest that changes in N-linked beta1,6 branching that occur during oncogenesis alter cell-matrix adhesion and migration by modulating integrin clustering and subsequent signal transduction pathways. These effects most likely result from altered N-linked carbohydrate expression on the beta(1) integrin subunit.

Actins↗
PubMed1999

Cooperative role for activated alpha4 beta1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5.

We recently reported that the heparin (Hep) III domain of fibronectin contains the H2 cell adhesion site in repeat III5 which binds activated alpha4 integrins. We have now further characterized the heparin and cell binding activities of this domain. A recombinant fragment containing repeats III4-III5 (FN-III4-5) induced Jurkat cell adhesion upon integrin activation with Mn2+ or TS2/16 monoclonal antibody (anti-beta1). Adhesion of Mn2+-treated cells to FN-III4-5 or FN-III5 fragments was inhibited by chondroitinase ABC and ACII but not by the anti-alpha4 monoclonal antibody HP2/1. In contrast, HP2/1 completely blocked adhesion of TS2/16-treated cells while chondroitinase had a partial (FN-III4-5) or minor (FN-III5) effect. Thus, the role of each receptor depended on the stimulus used to activate alpha4 beta1. The combination of HP2/1 and chondroitinase at dilutions which did not inhibit when used individually abolished adhesion of Mn2+ or TS2/16-treated cells to both fragments, indicating a cooperative effect between alpha4beta1 and chondroitin sulfate proteoglycans (CSPG). Furthermore, we have identified a 20-amino acid sequence in III5 (HBP/III5) which binds heparin and induces cell adhesion via CSPG exclusively. Although soluble HBP/III5 was a poor inhibitor, when combined with H2, it abolished adhesion to FN-III4-5 and FN-III5 fragments. These results establish that adhesion to the Hep III domain involves the cooperation of activated alpha4 beta1 and CSPG and show that HBP/III5 is a novel heparin and CSPG-binding site contributing to cell adhesion to this domain.

Amino Acid Sequence↗
PubMed1999

Expression of beta1 integrins in glomerular tissue of Streptozotocin-induced diabetic rats.

Based upon the importance of integrins as receptors for extracellular matrix components as well as transducers of extracellular signals, and since major alterations take place in the renal extracellular matrix during diabetes, it is important to study the role played by integrins in the development of the diabetic glomerulosclerosis. Expression of the beta1 subunit by renal glomerular cells was evaluated by biochemical and morphological means in short- and long-term diabetic rats. Western blots of isolated rat renal glomeruli demonstrated that the expression of beta1 increases along with age as well as with the hyperglycaemic state. These changes were significant as early as 6 weeks of hyperglycaemia. This was further demonstrated by immunocytochemistry, which revealed the presence of the beta1 subunit at the level of the plasma membranes of endothelial, epithelial, and mesangial cells. Quantitation of the immunolabelings confirmed the increased expression of beta1 under diabetic conditions. Further to this, expression of the focal adhesion kinase (FAK) was evaluated by immunoblotting showing little increase in diabetic conditions. On the other hand, testing the tyrosine phosphorylation of FAK, revealed significant increases in diabetes. To recover the fraction of FAK associated with the beta1 subunit, immunoprecipitation of isolated glomeruli homogenates was carried out with the anti-beta1 antibody. This demonstrated that the amounts of FAK co-precipitated with beta1, as well as its tyrosine-phosphorylation, are in fact reduced in diabetic conditions. Since the changes reported were observed at time points prior to any morphological alteration of the renal extracellular matrix, it appears that modifications in integrins and in their intracellular relays constitute early events that precede the onset of the diabetic nephropathy and must then be associated with the hyperglycaemic condition.

Age Factors↗
PubMed1996

Cell adhesion via murine alpha4 human beta1 integrin chimera on transfected K562 cells to endothelial cells.

To evaluate the property of binding activity of alpha4beta1 integrin in cell-cell interaction, we newly established a cell line, alpha4mK562, by transfecting cDNA of murine integrin alpha4 subunit into human erythroleukemic K562 cells. alpha4mK562 transfectant expressed both murine alpha4 and human beta1 subunit, which generated a functional heterodimer. alpha4mK562 cells more efficiently bound to murine endothelial cell lines and recombinant human TNFalpha (rhTNF)-treated human umbilical vein endothelial cells (HUVEC) than the parental K562 cells. These adhesion resulted from the interaction between alpha4beta1 and VCAM-1. Interestingly, treatment with mAb against human beta1 (4B4 clone), which has been known as inhibitory mAb, enhanced binding of alpha4mK562 cells to rhTNF-treated HUVEC but not to murine endothelial cells. This increase in binding induced by 4B4 mAb was completely inhibited by another mAb against human beta1 (mAb13), but only partially by anti-alpha4 mAb (PSsolidus2). The increase in binding induced by 4B4 mAb was also abolished by metabolic inhibitors, indicating that the increased binding is energy dependent. These observations suggest that the binding of 4B4 mAb to the chimeric alpha4beta1 induces an unique outside-in signaling and enhances the specific binding of alpha4mK562 cells to rhTNF-treated HUVEC.

Animals↗
PubMedDate not supplied

Adhesion strength of individual human bone marrow cells to fibronectin. Integrin beta1-mediated adhesion.

The purpose of this work was to study the adhesion strength of individual bone marrow cells, using a micropipette aspiration technique. The adhesion strength of the primary human bone marrow cells to fibronectin-coated substrate, by blocking the beta1 integrin with and without antibodies, was also determined. Human bone marrow stromal cells of the second passage were seeded at a density of 500 cells/cm2 on two different substrates: plastic culture dish (PCD) and PCD coated with fibronectin. In short adhesion times (15-180 min) the cells attached without spreading and remained almost spherical. A negative pressure of about 3500 Pa was applied, through the micropipette, on individual bone marrow cells and the detach process was recorded. The tip of the micropipette was bent at an 130 degrees angle to the corpus of the pipette and it was manipulated to be on the upper side of the cell and vertically to the bottom of the plate. It was observed from the experiments that the cells exhibited smaller adhesion strength at early adhesion times (30-85 min). After 85 min the adhesion strength increased abruptly and remained relatively constant for the adhesion period from 85 to 180 min for all substrates. Monoclonal antibodies against integrin subunit beta1 were used for integrin blocking experiments. The data suggested that the attachment of osteoblasts to a plastic culture dish without fibronectin coating occurred earlier than to the one coated with fibronectin PCD. In longer adhesion time the coating with fibronectin increased the adhesion strength at 107%. Blocking of integrin beta1 with monoclonal antibody resulted in decrease of the adhesion strength at 49%.

Journal Article↗
PubMed1998

The cytoplasmic domains of a beta1 integrin mediate polarization in Madin-Darby canine kidney cells by selective basolateral stabilization.

In Madin-Darby canine kidney cells, newly synthesized apical and basolateral membrane proteins are generally transported directly to their respective cell surface domain due to targeting determinants that mediate sorting in the Golgi complex. In several basolateral membrane proteins, these targeting determinants reside in the cytoplasmic domains. We show here that basolateral expression of the human alpha5beta1 integrin in stably transfected Madin-Darby canine kidney cells is also mediated by the cytoplasmic domains. Distinct regions in both cytoplasmic domains were found to be sufficient to mediate basolateral expression independently from one another. Unexpectedly, newly synthesized wild-type alpha5beta1 and basolaterally expressed chimeras containing the cytoplasmic domain of either alpha5 or beta1 were integrated into both cell surface domains, preferentially apically, during biosynthesis. The apical pools of wild-type integrin and chimeric subunits were found to become quickly degraded, whereas the basolateral pools were stabilized. Thus, the cytoplasmic domains of the alpha5beta1 integrin are independently sufficient to mediate sorting by selective basolateral stabilization.

Animals↗