The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.     

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.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Characterization of zinc-alpha(2)-glycoprotein as a cell adhesion molecule that inhibits the proliferation of an oral tumor cell line.

Zn-alpha(2)-glycoprotein (Znalpha(2)gp) is a soluble protein widely distributed in body fluids and glandular epithelia. We have found it to be expressed in stratified epithelia as well. Znalpha(2)gp is clinically correlated with differentiation in various epithelial tumors, including oral and epidermal tumors. We have cloned epidermal Znalpha(2)gp and report the preparation of the recombinant protein in a Baculovirus expression system. Like the native molecule, recombinant Znalpha(2)gp has RNase activity. Znalpha(2)gp functions as a matrix protein for the Tu-138 oral squamous cell carcinoma cell line. Cell attachment to Znalpha(2)gp is comparable to that for fibronectin and is inhibited by the synthetic RGD peptides RGD, RGDV, and RGDS. Attachment is also inhibited by the antibody to integrin alpha(5)beta(1) (the fibronectin receptor), but not by antibodies to integrins alpha(v)beta(3), alpha(3)beta(1), and alpha(2)beta(1). We find that the proliferation of Tu-138 cells is inhibited on a Znalpha(2)gp matrix, as compared with other matrix proteins (fibronectin, vitronectin, laminin, and collagens I and IV) on which growth resembles that on the BSA control. We believe that the role of Znalpha(2)gp in differentiation and its RNase activity are two likely suspects as agents of the inhibition of proliferation.     

Antibodies to the vitronectin receptor (integrin alpha V beta 3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells.

The amino acid sequence Arg-Gly-Asp (RGD) is highly conserved on the VP1 proteins of different serotypes and subtypes of foot-and-mouth disease virus (FMDV) and is essential for cell attachment. This sequence is also found in certain extracellular matrix proteins that bind to a family of cell surface receptors called integrins. Within the Picornaviridae family, enterovirus coxsackievirus A9 also has an RGD motif on its VP1 capsid protein and has recently been shown to utilize the vitronectin receptor integrin alpha V beta 3 as a receptor on monkey kidney cells. Competition binding experiments between type A12 FMDV and coxsackievirus A9 using BHK-21 and LLC-MK2 cells revealed shared receptor specificity between these two viruses. Polyclonal anti-serum to the vitronectin receptor and a monoclonal antibody to the alpha V subunit inhibited both FMDV binding and plaque formation, while a monoclonal antibody to the beta 3 subunit inhibited virus binding. In contrast, antibodies to the fibronectin receptor (alpha 5 beta 1) or to the integrin (alpha V beta 5) had no effect on either binding or plaque formation. These data demonstrate that the alpha V beta 3 vitronectin receptor can function as a receptor for FMDV.     

Integrin-associated protein immunoglobulin domain is necessary for efficient vitronectin bead binding

Integrin-associated protein (IAP/CD47) is physically associated with the alpha v beta 3 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg- Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-IAP antibodies also inhibit binding of Vn- coated particles to many cells expressing alpha v beta 3. However, prior studies with anti-IAP did not directly address IAP function because they could not distinguish between IAP blockade and antibody- induced signaling effects on cells. To better determine the function of IAP, we have characterized and used an IAP-deficient human cell line. Despite expressing alpha v integrins, these cells do not bind Vn-coated particles unless transfected with IAP expression constructs. Increasing the level of alpha v beta 3 expression or increasing Vn density on the particle does not overcome the requirement for IAP. All known splice variants of IAP restore Vn particle binding equivalently. Indeed, the membrane-anchored IAP Ig variable domain suffices to mediate Vn particle binding in this system, while the multiply membrane-spanning and cytoplasmic domains are dispensable. In all cases, adhesion to a Vn- coated surface and fibronectin particle binding through alpha 5 beta 1 fibronectin receptors are independent of IAP expression. These data demonstrate that some alpha v integrin ligand-binding functions are IAP independent, whereas others require IAP, presumably through direct physical interaction between its Ig domain and the integrin.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

Motility of fibronectin receptor-deficient cells on fibronectin and vitronectin: collaborative interactions among integrins.

Cells are capable of adhering to and migrating on protein components of the extracellular matrix. These cell-matrix interactions are thought to be mediated largely through a family of cell surface receptors termed integrins. However, the manner in which individual integrins are involved in cell adhesion and motility has not been fully determined. To explore this issue, we previously selected a series of CHO variants that are deficient in expression of the integrin alpha 5 beta 1, the "classical" fibronectin receptor. Two sets of subclones of these variants were defined which respectively express approximately 20% or 2% of fibronectin receptor on the cell surface when compared to wild-type cells (Schreiner, C. L., J. S. Bauer, Y. N. Danilov, S. Hussein, M. M. Sczekan, and R. L. Juliano. 1989. J. Cell Biol. 109:3157-3167). In the current study, the variant clones were tested for haptotactic motility on substrata coated with fibronectin or vitronectin. Data from assays using fibronectin show that cellular motility of the 20% variants was substantially decreased (30-75% of wild type), while the motility of the 2% variants was nearly abolished (2-20% of wild type). Surprisingly, a similar pattern was seen for haptotactic motility of both 2% and 20% variants when vitronectin was used (approximately 20-30% of wild type). The reduced haptotactic motility of the fibronectin receptor-deficient variant clones on vitronectin was shown not to be due to reduced vitronectin receptor (alpha v beta 3) expression nor to a failure of these variants to adhere to vitronectin substrata. Transfection of the deficient variants with a cDNA for the human alpha 5 subunit resulted in normal levels of fibronectin receptor expression (as a human alpha 5/hamster beta 1 chimera) and restored the motility of the CHO variants on fibronectin and vitronectin. This indicates that expression of the alpha 5 subunit is required for normal haptotactic motility on vitronectin substrata and suggests that the fibronectin receptor (alpha 5 beta 1) plays a cooperative role with vitronectin receptors in cell motility.     

The function of PS integrins during Drosophila embryogenesis

The Drosophila position-specific (PS) antigens are homologous to the vertebrate fibronectin receptor family, or integrins. A Drosophila gene required for embryonic morphogenesis, l(1)myospheroid, codes for a product homologous to the beta subunit of the vertebrate integrins. l(1)myospheroid mutants die during embryogenesis. We show here that they lack the beta subunit of the PS antigens. In the absence of the beta subunit in mutant embryos, the PS alpha subunits are not expressed on the cell surface. We conclude that the l(1)myospheroid phenotype represents the lack-of-function phenotype for these Drosophila integrins. In wild-type embryos, PS antigens are found at the interface between mesoderm and ectoderm, and later mainly at the attachment sites of muscles to the epidermis and gut. Together these results indicate that during embryogenesis, Drosophila integrins are used to attach mesoderm to ectoderm, and are required for the proper assembly of the extracellular matrix and for muscle attachment.     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells located between retinal photoreceptors and blood vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellular matrix structure which becomes abnormal in several disease processes, including age-related macular degeneration (AMD). Ruptures or abnormalities in Bruch's membrane are frequently accompanied by choroidal neovascularization. Disturbed interaction of RPE cells with their extracellular matrix (ECM) could play a role in this process. The present study was undertaken to examine the complex interactions between hypoxia, integrin, and ECM in the regulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alphavbeta5 and adhesion to fibronectin occurs through alpha5beta1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhesion as compared to that with collagen IV or the RGD matrices such as vitronectin (alphavalpha5) , fibronectin (alpha5beta1), or thrombospondin (alpha5beta1 + alphavbeta5). Addition of soluble vitronectin, or fibrinogen to RPE cell cultures resulted in a small to moderate increase in VEGF and FGF2 in the media, while each of these growth factors was dramatically increased after addition of thrombospondin 1 (TSP1). In contrast, soluble fibronectin resulted in differential upregulation of VEGF but not FGF2. Similarly, immobilized TSP1 resulted in differential greater upregulation in VEGF but not FGF2 release from RPE as compared to other ECMs under either normoxic or hypoxic conditions. Additionally, hypoxia resulted in a time-dependent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin, or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha5beta1, anti-alphavbeta3, and anti-alphavbeta5 antibodies indicating that it may be mediated in part by TSP1 binding to those integrins. These data suggest that alterations in oxygen levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AMD, can cause increased secretion of angiogenic growth factors that might contribute to the development of choroidal neovascularization. These data also suggest the potential modulatory role of VEGF release from RPE by ECM and alphavbeta5 and alpha5beta1 integrins.     

Activation of the alpha 4 beta 1 integrin through the beta 1 subunit induces recognition of the RGDS sequence in fibronectin

Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16- activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function.     

Cell adhesion to extracellular matrix regulates the life cycle of integrins.

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.     

Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.     

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (_Vβ₃/β₅) and fibronectin (₅β₁) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.     

Novel role of presenilins in maturation and transport of integrin beta 1

Presenilins (PSs) play important roles in modulating the trafficking and maturation of several membrane proteins. However, the target membrane proteins whose trafficking and maturation are regulated by PS are largely unknown. By characterizing PS-deficient fibroblasts, we found that integrin beta1 maturation is promoted markedly in PS1 and PS2 double-deficient fibroblasts and moderately in PS1- or PS2-deficient fibroblasts; in contrast, nicastrin maturation is completely inhibited in PS1 and PS2 double-deficient fibroblasts. Subcellular fractionation analysis demonstrated that integrin beta1 maturation is promoted in the Golgi apparatus. The mature integrin beta1 with an increased expression level was delivered to the cell surface, which resulted in an increased cell surface expression level of mature integrin beta1 in PS1 and PS2 double-deficient fibroblasts. PS1 and PS2 double-deficient fibroblasts exhibited an enhanced ability to adhere to culture dishes coated with integrin beta1 ligands, namely, fibronectin and laminin. The inhibition of gamma-secretase activity enhances neither integrin beta1 maturation nor the adhesion of wild-type cells. Moreover, PS deficiency also promoted the maturation of integrins alpha3 and alpha5 and the cell surface expression of integrin alpha3. Integrins alpha3 and alpha5 were coimmunoprecipitated with integrin beta1, suggesting the formation of the functional heterodimers integrins alpha3beta1 and alpha5beta1. Note that integrin beta1 exhibited features opposite those of nicastrin in terms of maturation and trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus in PS1 and PS2 double-deficient fibroblasts. Our results therefore suggest that PS regulates the maturation of membrane proteins in opposite directions and cell adhesion by modulating integrin maturation.     

Integrins alpha v beta 3 and alpha v beta 5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface

We investigated the role of the integrins alpha v beta 3 and alpha v beta 5 in mediating vitronectin adhesion of three phenotypically distinct cell types. M21 human melanoma cells and H2981 lung carcinoma cells use both alpha v-containing integrins in adhering to vitronectin while UCLA-P3 lung carcinoma cells adhere exclusively with alpha v beta 5. Specifically, monoclonal antibodies directed to functional epitopes on both receptors were required to block adhesion of M21 or H2981 cells while adhesion of UCLA-P3 cells to vitronectin could be blocked with a monoclonal antibody to alpha v beta 5. Although both receptors are involved in M21 and H2981 cell adhesion to vitronectin, only alpha v beta 3 can be detected in focal contacts, colocalizing with vinculin, talin, and the ends of actin filaments, while alpha v beta 5 shows a distinct, nonfocal contact, distribution on the cell surface. These results provide the first evidence that two homologous integrins that recognize the same ligand distribute differentially on the cell surface.     

The collagen receptor alpha 2 beta 1, from MG-63 and HT1080 cells, interacts with a cyclic RGD peptide.

Several receptors for the extracellular matrix protein collagen have been described which belong to the superfamily of receptors collectively known as integrins. Although several integrins have been shown to interact with extracellular matrix molecules via a common recognition site, arginine-glycine-aspartic Acid (RGD), within the beta 1 integrin subfamily, only the fibronectin receptor (alpha 5 beta 1) has been convincingly shown to interact with RGD. In the present study, we tested whether a collagen receptor could interact with RGD. Adhesion of an osteosarcoma cell line, MG-63, to immobilized collagen I was inhibited by the cyclic RGD-containing peptide, C*GRGDSPC* (where C* indicates that Cys participates in disulfide), and not by the linear GRGDSP or the non-RGD-containing cyclic peptide, C*GKGESPC*. Similarly, using collagen-Sepharose affinity chromatography, a heterodimeric protein could be specifically eluted from the column by the cyclic RGD peptide. Immunoprecipitations of the eluted material with monoclonal antibodies showed reactivity with the collagen receptor alpha 2 beta 1 and not alpha 3 beta 1. Our data demonstrate that RGD peptides can interact with the collagen receptor, and the differences seen with the linear and cyclic peptide suggest that the cyclic C*GRGDSPC* has a higher avidity for the receptor than the more flexible linear GRGDSP. In this paper, we provide supportive evidence that one possible mode of collagen interaction with alpha 2 beta 1 is via the RGD recognition sequence.     

The glycobiology of gametes and fertilization

In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human (beta)ig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-beta. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or beta-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg(2+) or Mn(2+)), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin alpha(1) or beta(1) subunit, but not to the alpha(2), alpha(3), alpha(5) or beta(2) subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the alpha(5) subunit, but not the mAb to the alpha(1) subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin alpha(1)beta(1) that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.     

Adhesion of a chicken myeloblast cell line to fibrinogen and vitronectin through a beta 1-class integrin.

The adhesive interactions of circulating blood cells are tightly regulated, receptor-mediated events. To establish a model for studies on regulation of cell adhesion, we have examined the adhesive properties of the HD11 chick myeloblast cell line. Function-perturbing antibodies were used to show that integrins containing the beta 1 subunit mediate HD11 cell attachment to several distinct extracellular matrix proteins, specifically fibronectin, collagen, vitronectin, and fibrinogen. This is the first evidence that an integrin heterodimer in the beta 1 family functions as a receptor for fibrinogen. While the alpha v beta 1 heterodimer has been shown to function as a vitronectin receptor on some cells, this heterodimer could not be detected on HD11 cells. Instead, results suggest that the beta 1 subunit associates with different, unidentified alpha subunit(s) to form receptors for vitronectin and fibrinogen. Results using function-blocking antibodies also demonstrate that on these cells, additional receptors for vitronectin are formed by alpha v beta 3 and alpha v associated with an unidentified 100-kD beta subunit. The adhesive interactions of HD11 cells with these extracellular matrix ligands were shown to be regulated by lipopolysaccharide treatment, making the HD11 cell line attractive for studies of mechanisms regulating cell adhesion. In contrast to primary macrophage which rapidly exhibit enhanced adhesion to laminin and collagen upon activation, activated HD11 cells exhibited reduced adhesion to most extracellular matrix constituents.