©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Human Integrin 81 Functions as a Receptor for Tenascin, Fibronectin, and Vitronectin (*)

(Received for publication, February 27, 1995; and in revised form, July 18, 1995)

Lynn M. Schnapp (1)(§) Nan Hatch (1) Daniel M. Ramos (1) (2) Irina V. Klimanskaya (1) Dean Sheppard (1) Robert Pytela (1) (3)

From the  (1)Lung Biology Center, Cardiovascular Research Institute, Department of Medicine and Departments of (2)Stomatology and (3)Pharmacology, University of California, San Francisco, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The integrin family of adhesion receptors consists of at least 21 heterodimeric transmembrane proteins that differ in their tissue distribution and ligand specificity. The recently identified alpha8 integrin subunit associates with beta1 and is predominantly expressed in smooth muscle and other contractile cells in adult tissues, and in mesenchymal and neural cells during development. We now show that alpha8beta1 specifically localizes to focal contacts in cells plated on the extracellular matrix proteins fibronectin or vitronectin. In addition we show that human embryonic kidney cells (293), transfected with alpha8 cDNA, express alpha8beta1 on their surface and use this receptor for adhesion to fibronectin and vitronectin. Furthermore, alpha8beta1 binds to both fibronectin- and vitronectin-Sepharose and can be specifically eluted from either matrix protein by the arginine-glycine-aspartic acid (RGD)-containing peptide, GRGDSP. Because fibronectin and vitronectin adhesion appeared to be mediated by RGD, we examined additional RGD-containing proteins, including tenascin, fibrinogen, thrombospondin, osteopontin, and denatured collagen type I. We found that only tenascin was able to mediate adhesion of alpha8-transfected 293 cells. By using recombinant fragments of tenascin in adhesion assays, we were able to localize the alpha8beta1 binding domain of tenascin to the RGD-containing, third fibronectin type III repeat. These data strongly suggest that tenascin, fibronectin, and vitronectin are ligands for alpha8beta1 and that this integrin binds to the RGD site in each of these ligands through mechanisms that are distinct and separate from alpha5-and alphav-containing integrins.


INTRODUCTION

Integrins are a class of cell adhesion glycoproteins composed of two noncovalently associated subunits, alpha and beta. Each subunit contains a large extracellular domain, a transmembrane domain, and a short cytoplasmic domain. Integrins are known to bind to a wide variety of extracellular matrix proteins, including fibronectin, vitronectin, collagens, and laminins. The specificity of protein binding is determined by particular combinations of alpha and beta subunit pairing. The ligand binding site is formed by the extracellular domain of both subunits and requires the presence of divalent cations. Many integrins interact with ligands through the tripeptide arginine-glycine-aspartic acid (RGD).

The alpha8 integrin subunit was originally identified by Bossy et al.(1) in the chick embryo nervous system and was shown to be a partner for beta1. We have identified human alpha8, cloned and sequenced the cDNA, raised antibodies to the predicted cytoplasmic domain sequence, and determined its distribution in adult mammalian tissues (2) . We found that alpha8 is predominantly expressed in a variety of visceral and vascular smooth muscle cells, kidney mesangial cells, and lung myofibroblasts(2) .

To gain insight into potential functions of alpha8beta1 in vivo, we sought to determine potential ligands. We tested various ligands for their ability to direct alpha8beta1 to focal contacts, to bind to alpha8beta1 by affinity chromatography, and to support adhesion of alpha8-transfected cells. All of the results provide strong evidence that alpha8beta1 can function as an RGD-dependent receptor for tenascin, fibronectin, and vitronectin.


EXPERIMENTAL PROCEDURES

Materials

Fibronectin was prepared from human plasma as described by Engvall and Ruoslahti(3) , and vitronectin was prepared from human plasma according to Yatohgo and co-workers(4) . Type I collagen from rat tail, type IV collagen from human placenta, and fibrinogen were purchased from Sigma. Type I collagen was heat-denatured by incubating at 100 °C for 15 min(5) . Tenascin was purchased from Life Technologies, Inc. Recombinant tenascin fragments were a gift from Dr. Kathryn Crossin, Scripps Research Institute, La Jolla, CA(6) . Osteopontin was a gift from Dr. Cecilia Giachelli, University of Washington, Seattle, WA. Laminin was a gift from Dr. Randy Kramer, University of California, San Francisco.

Antibodies and Reagents

Rabbit polyclonal antibody to the human alpha8 cytoplasmic domain was previously characterized (2) and shown to cross-react with rat alpha8 (see ``Results''). Anti-vinculin antibody was purchased from Sigma. Monoclonal antibody L230 (anti-alphav) (7) was prepared in our laboratory from hybridoma cells obtained from American Type Culture Collection (ATCC); P1B5 (anti-alpha3) (8, 9) was obtained from Telios Pharmaceuticals (San Diego, CA); P1H5 (anti-alpha2) (10) and P3D10 (anti-alpha5) were a gift from Dr. William Carter (Fred Hutchison Cancer Center, Seattle, WA); P1F6 (anti-alphavbeta5) (7) and P5D2 (anti-beta1) (11) were gifts from Dr. Elizabeth Wayner (University of Minnesota, Minneapolis, MN). The peptide GRGDSP was obtained from Telios Pharmaceuticals. The peptides ACRGDGWMCG (RGDGW), DSCRRETAWACRL (CRRETAWAC), and ACDCRGDCFCG (4C) were gifts from Drs. Erkki Koivunen and Erkki Ruoslahti, La Jolla Cancer Research Foundation, La Jolla, CA (12, 13) .

Cell Lines

Human intestinal smooth muscle cells (HISM), rat embryo fibroblasts (REF 52), human embryonic kidney cell line 293, and human colon carcinoma cell line SW 480 were obtained from ATCC and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin. The alpha9-transfected 293 (14) and beta3-transfected 293 cells were previously characterized(15) .

Immunofluorescence

Coverslips were coated overnight at 4 °C with 10-20 µg/ml of the following extracellular matrix proteins: fibronectin, vitronectin, collagen types I and IV, denatured collagen type I, laminin, and fibrinogen. Coverslips were blocked with 3% bovine serum albumin (BSA), (^1)phosphate-buffered saline (PBS) at 37 °C for 30 min. Cells grown to 80% confluence were removed from tissue culture plates with 2 mM EDTA, PBS, washed with PBS, resuspended in serum-free Dulbecco's modified Eagle's medium, and plated onto the coverslips. Three hours after plating, cells were fixed and permeabilized with 2% paraformaldehyde, 0.1% Triton X-100 for 10 min. Coverslips were blocked with 3% BSA and then incubated with anti-alpha8 antibody (10 µg/ml) and anti-vinculin antibody (2 µg/ml) for 1 h at room temperature. Unbound primary antibody was removed by washing with PBS. Coverslips were incubated with biotin-conjugated donkey anti-rabbit IgG (1:50) (Amersham Corp.) and Texas Red-conjugated goat anti-mouse IgG (1:100) (Caltag, South San Francisco, CA) for 1 h at room temperature, followed by PBS wash. Coverslips were then incubated with fluorescein conjugated streptavidin (1:100) (Amersham) for 15 min at room temperature, washed with PBS, and mounted with Vectashield (Vector Laboratories, Burlingame, CA).

To block endogenous production of extracellular matrix proteins, cells were incubated with the protein synthesis inhibitor cycloheximide (30 µg/ml) for 3 h in serum-free medium. Cells were then detached with 2 mM EDTA and seeded onto fibronectin- and vitronectin-coated coverslips in the presence of cycloheximide.

Preparation of cDNA Expression Constructs

A cDNA containing the entire coding region of human alpha8 was initially constructed in pBluescript (Stratagene, La Jolla, CA). The full-length alpha8 cDNA was amplified by PCR using the previously reported cDNA clone HA33A (2) as a template. This clone lacks the signal peptide and contains a deletion of nucleotides 688-732. Because the signal sequence of human alpha8 has not been determined, we joined the N terminus of mature alpha8 with a synthetic signal sequence derived from the alphaM (Mac-1) integrin subunit. We chose alphaM because the N-terminal four amino acid residues of alphaM and alpha8 are identical (FNLD). We designed two overlapping forward PCR primers that encoded for the signal peptide of the human alphaM integrin subunit (16) and the amino terminus of mature alpha8 (Mac 2F, 5`- CCTCCTCGAAAGCTTCTCCTTCCAGCCATGGCTCTCAGAGTCCTTCTCTTAACAGC-3`; Mac 1F, 5`-AGAGTCCTTCTCTTAACAGCCTTATGTCATGGGTTCAACCTGGACGTGGAAAA-3`). The Mac 2F primer contains a HindIII recognition sequence (underlined), 12 nucleotides of the alphaM 5` untranslated region, followed by the ATG initiation codon (double underlined) and the sequence encoding the first nine amino acids of the alphaM signal peptide (ALRVLLLTA). The Mac 1F primer has a 20 nucleotide overlap (underlined) with the 3` end of the Mac 2F primer and encodes the remainder of the alphaM signal peptide (ALCHG) followed by the N terminus of mature alpha8 (FNLDV). The first PCR reaction used forward primer Mac 1F and a alpha8-specific 3` reverse primer and HA33A plasmid DNA as a template. The second PCR reaction used forward primer Mac 2F and the same alpha8-specific reverse primer and the first PCR reaction cDNA as a template. In order to correct the deletion, we amplified the region from clone HA15-A that does not contain the deletion (2) and used unique restriction sites (BclI and HpaI) in the alpha8 clone in order to ligate the fragment. All polymerase chain reactions were performed with Vent DNA polymerase (New England Biolabs) which has been reported to provide significantly higher fidelity than Taq polymerase. The plasmid was then completely sequenced using Sequenase 2.0 (U. S. Biochemical Corp., Cleveland, OH) and found to be free of mutations that would alter the encoded amino acid sequence. The plasmid was cut at the unique HindIII and XbaI sites in the pBluescript polylinker, and ligated into the mammalian expression vector pCDNAIneo between the unique HindIII and XbaI sites to generate pCDNAIneoalpha8.

Transfection of Mammalian Cells

The human embryonic kidney cell line 293 and human colon carcinoma cell line SW 480 were transfected using the Lipofectin reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Stably transfected cell lines were selected in medium containing the neomycin analog G418 (0.4 mg/ml). Cells were transfected with either pCDNAIneoalpha8 (alpha8-transfected cells) or pCDNAIneo alone (mock-transfected cells).

Flow Cytometry

Cells were detached with 2 mM EDTA, washed with PBS, resuspended in normal goat serum and incubated at 4 °C for 10 min. After centrifugation, cells were resuspended in PBS containing the appropriate antibody: P1H5 (anti-alpha2), P1B5 (anti-alpha3), P3D10 (anti-alpha5), L230 (anti-alphav), P5D2 (anti-beta1) and incubated for 20 min on ice. After subsequent washes with PBS, cells were incubated with phycoerythrin-conjugated goat anti-mouse antibody (Boehringer Mannheim) for 20 min on ice. After washing, the cells were processed for flow cytometric analysis using a FACScan (Becton Dickinson, Rutherford, NJ).

Cell Adhesion Assays

Non-tissue culture-treated plates were coated with increasing concentrations (0.3, 1, 3, 10, 20 µg/ml) of fibronectin, vitronectin, thrombospondin, osteopontin, denatured collagen type I, fibrinogen, and intact tenascin. Plates were also coated with increasing concentrations of the recombinant tenascin fragments containing the third fibronectin type III repeat (TNfn3), the third fibronectin type III repeats in which the RGD site had been mutated to RAA (TNfn3RAA) or the fourth to sixth fibronectin type III repeats (TNfn4-6). As a negative control, wells were coated with 1% BSA, PBS. Wells coated at 37 °C for 1 h were washed with PBS and blocked with 1% BSA for 30 min at 37 °C. Cells were detached with 2 mM EDTA, washed with PBS, and resuspended in serum-free Dulbecco's modified Eagle's medium (containing 200 µg/ml CaCl(2), 200 µg/ml MgCl(2)). 50,000 cells were added to each well, centrifuged at 10 times g for 3 min to ensure uniform settling of cells, and incubated for 1 h at 37 °C. Nonadherent cells were then removed by centrifugation (top-side down) at 10 times g for 5 min. The attached cells were fixed and stained with 1% formaldehyde, 0.5% crystal violet. After washing with PBS, adherence was determined by absorption at 595 nm in a Microplate Reader (Bio-Rad). The data were reported as the mean absorbance of triplicate wells ± S.E., minus the mean absorbance of BSA-coated wells.

For antibody-blocking experiments, cells were incubated with no antibodies, with P5D2 (anti-beta1) (1:10), with P3D10 (anti-alpha5) (1:5), with L230 (anti-alphav) (1:10), with P5D2 and P3D10 or with P5D2 and L230, in a plate precoated with 1% BSA. After 15 min of incubation at 4 °C, cells were then transferred to protein coated wells (3 µg/ml of fibronectin, vitronectin, or TNfn3) and processed as above. All antibodies were used at saturating concentrations. The data were expressed as the mean absorbance of triplicate wells ± S.E., minus the mean absorbance of BSA-coated wells.

Peptide blocking experiments were carried out in an analogous manner. Peptides were used at the following final concentrations: 125 µM RGDGW, 125 µM CRRETAWAC, 1 µM 4C, and 100 µg/ml GRGDSP.

The data reported represent the results from one clone of mock-transfected and alpha8-transfected 293 cells. The adhesion assay results were confirmed with four independent clones of alpha8-transfected 293 cells and with wild type (untransfected) 293 cells (data not shown).

Affinity Chromatography

Cells grown to 80% confluence were detached with 2 mM EDTA in PBS. Cells were centrifuged and washed three times in labeling buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM MnCl(2), 20 mM glucose). Cells were surface labeled with I-sodium iodide using the lactoperoxidase method(17) .

Cell lysates were prepared by incubation with 200 mM octylglucoside, 100 mM Tris-HCl, 1 mM divalent cation (MnCl(2), CaCl(2) or MgCl(2)) for 1 h at 4 °C, followed by centrifugation at 14,000 rpm for 20 min.

Affinity columns were prepared by coupling the ligand (fibronectin or vitronectin) to cyanogen-bromide activated Sepharose as described previously(17) . I-Labeled cell lysates were applied to a 1-ml affinity column. The column was washed with 10 volumes column buffer (50 mM octylglucoside, 50 mM Tris-HCl, 1 mM divalent cation (MnCl(2), CaCl(2), or MgCl(2)). Elutions were carried out using 4 volumes of GRGDSP peptide (1 mg/ml), followed by 2 volumes of column buffer, and 4 volumes of 10 mM EDTA in column buffer without divalent cations. Finally the columns were washed with 4 volumes of 1 M NaCl in column buffer. Fractions (1 ml) were collected and either analyzed directly by SDS-PAGE or subjected to immunoprecipitation and then analyzed by SDS-PAGE as described previously(17) .

Immunoprecipitation

Cell lysates and eluted fractions were immunoprecipitated with anti-alpha8, -alphav (L230), -alpha5 (P3D10), or -beta1 (P5D2) antibodies at 4 °C for 2 h. Immune complexes were captured on protein A-Sepharose and washed five times with immunoprecipitation buffer (100 mM Tris-HCl, pH 7.5, 0.1% Nonidet P-40, 150 mM NaCl, 1 mM CaCl(2), 1 mM MgCl(2)). Samples solubilized in Laemmli sample buffer were analyzed by SDS-PAGE.


RESULTS AND DISCUSSION

Focal Contact Formation

Many cells in culture, including smooth muscle cells and fibroblasts, attach to extracellular matrix proteins at discrete sites called focal contacts or adhesion plaques. Focal contact localization of integrins is ligand-dependent; i.e. a particular integrin will accumulate at focal contacts only when its ligand is present in the substrate(18, 19) . We therefore examined focal contact localization of alpha8beta1 on cells plated on various extracellular matrix proteins as a first step toward identifying ligands for alpha8beta1. In our experiments we used a human smooth muscle cell line (HISM) and a rat embryo fibroblast (REF) cell line, which we found to express alpha8beta1 by immunoprecipitation (Fig. 1, lanes 1 and 2).


Figure 1: Immunoprecipitation of alpha8-containing integrins from HISM cells (lane 1), REF cells (lane 2), 293 cells (lane 3), and alpha8-transfected 293 cells (lane 4). Aliquots of I-surface-labeled lysates were immunoprecipitated with anti-alpha8 antibody. Proteins were analyzed by SDS-PAGE under nonreducing conditions. Positions of molecular size markers in kilodaltons are shown to the right.



HISM cells plated on extracellular matrix proteins were analyzed by double-labeling immunofluorescence microscopy using antibodies to vinculin (to detect focal contacts) and alpha8 (Fig. 2). Three hours after plating on fibronectin, vitronectin, or collagen, HISM cells were well spread and formed vinculin-containing focal contacts. In cells plated on fibronectin or vitronectin, alpha8 co-localized to vinculin-containing focal contacts (Fig. 2, A-D). In contrast, in cells plated on collagen type I or denatured collagen type I, alpha8 did not localize to focal contacts, despite the abundance of focal contacts identified by vinculin staining (Fig. 2, E and F). Cells plated on tenascin adhered, but did not form vinculin-containing focal contacts (data not shown). Cells plated on fibrinogen or laminin attached poorly and did not spread or form focal contacts. No localized alpha8 staining was detected in these cells (data not shown). Identical results were obtained using rat embryo fibroblast cells (data not shown). It is unlikely that alpha8 localization was due to matrix protein secretion by the cells, because control experiments using cells pretreated with the protein synthesis inhibitor, cycloheximide, yielded similar results (Fig. 3). We were not able to demonstrate focal contact formation by alpha8-transfected or untransfected 293 cells. However, alpha8-transfected SW 480 colon carcinoma cells showed new localization of alpha8 to focal contacts when plated on fibronectin and vitronectin (data not shown). Thus, vitronectin and fibronectin, but not collagen, specifically promote localization of alpha8 to focal contacts. These data suggest vitronectin and fibronectin are ligands for alpha8beta1.


Figure 2: Focal contact formation by HISM cells on various extracellular matrix proteins. HISM cells were allowed to spread on 10 µg/ml fibronectin (A and B), vitronectin (C and D), or collagen type I (E and F) for 3 h then fixed, permeabilized, and double-labeled with anti-alpha8 antibody (A, C, and E) and anti-vinculin antibody (B, D, and F). Sample focal contacts are identified by arrows.




Figure 3: Focal contact formation by HISM cells in the presence of cycloheximide. Cells were preincubated with cycloheximide (30 µg/ml) for 3 h and then allowed to spread on 10 µg/ml fibronectin (A and B) and vitronectin (C and D) for 3 h in the presence of cycloheximide. Cells were fixed, permeabilized, and doublelabeled with anti-alpha8 antibody (A and C) and anti-vinculin antibody (B and D).



Heterologous Expression of alpha8 cDNA

The human embryonic kidney cell line 293 is highly permissive for transfection of heterologous cDNAs and does not express alpha8 as determined by immunoprecipitation (Fig. 1, lane 3). After transfection of 293 cells with alpha8 cDNA we obtained four independent clones that expressed alpha8 as detected by Western blot analysis (data not shown). Immunoprecipitation of surface-labeled cells demonstrated that alpha8beta1 was expressed on the cell surface (Fig. 1, lane 4).

To determine whether the alpha8 transfectants have altered levels of other beta1-associated alpha subunits, we performed fluorescein-activated cell sorting analysis using a panel of anti-integrin antibodies (Fig. 4). Mock-transfected and alpha8-transfected 293 cells contained similar amounts of cell surface alpha2-, alpha3-, alpha5-, and alphav-containing integrins. Thus, changes in adhesive properties of alpha8-transfected cells are not due to changes in the surface expression of other integrins.


Figure 4: Flow cytometry of alpha8-transfected 293 cells (black bars) and mock-transfected 293 cells (white bars). The results represent an average of four different clones of alpha8-transfected 293 cells and mock-transfected 293 cells. Cells were incubated with the following antibodies: P1H5 (anti-alpha2), P1B5 (anti-alpha3), P3D10 (anti-alpha5), L230 (anti-alphav), and P5D2 (anti-beta1) and were analyzed using a FACScan. The y axis represents fluorescence intensity (in arbitrary units).



Adhesion of alpha8-transfected 293 Cells

To determine whether alpha8beta1 can mediate cell adhesion to fibronectin and vitronectin, we compared the adhesive properties of mock-transfected and alpha8-transfected 293 cells. Fibronectin and vitronectin adhesion were not significantly affected by alpha8 expression (Fig. 5), although a slight increase in adhesion to 3 µg/ml vitronectin was noted. When endogenous receptors for fibronectin and vitronectin were blocked with monoclonal antibodies, the contribution of alpha8beta1 was more apparent ( Fig. 6and Fig. 7). The adhesion of wild type or mock-transfected cells to fibronectin was almost completely inhibited (>90%) by either anti-beta1 antibody (P5D2) or anti-alpha5 antibody (P3D10) (Fig. 6A). In contrast, alpha8-transfected cell adhesion was only partially blocked by anti-alpha5 antibody (33%), but was completely blocked by anti-beta1 antibody. These data suggest that the alpha8-transfected cells are using alpha8beta1 in addition to alpha5beta1 to adhere to fibronectin.


Figure 5: Adhesion of mock-transfected (black squares) and alpha8-transfected (open squares) 293 cells to increasing concentrations (0.3, 1, 3, 10, and 20 µg/ml) of (A) fibronectin and (B) vitronectin. The y axis represents the absorbance at 595 nm after staining attached cells with crystal violet.




Figure 6: Adhesion of alpha8-transfected (black squares) and mock-transfected (open squares) 293 cells to 3 µg/ml fibronectin. A, cells plated on fibronectin were incubated with no antibody, anti-beta1 antibody (P5D2), anti-alpha5 antibody (P3D10), or anti-beta1 and anti-alpha5 antibody in combination. B, cells plated on fibronectin were incubated alone (control), or with the peptides CRRETAWAC and RGDGW.




Figure 7: Adhesion of alpha8-transfected (black squares) and mock-transfected (open squares) 293 cells to 3 µg/ml vitronectin. A, cells plated on vitronectin were incubated with no antibody, anti-beta1 antibody (P5D2), anti-alphav antibody (L230), or anti-beta1 and anti-alphav antibody in combination. B, cells plated on vitronectin were incubated alone (control), or with the peptides 4C and RGDGW.



Vitronectin adhesion of mock-transfected and wild type 293 cells was almost completely inhibited by the blocking anti-alphav antibody, L230 (Fig. 7A), consistent with previous reports(15, 20) . In contrast, vitronectin adhesion in alpha8-transfected cells was only partially inhibited (19%) by the anti-alphav antibody, L230, and inhibited by 41% using the anti-beta1 antibody, P5D2. Vitronectin adhesion was completely abolished using both antibodies in combination. These data suggest that alpha8-transfected cells are using alpha8beta1 in addition to alphav-containing integrins, to adhere to vitronectin.

To further elucidate the binding characteristics of alpha8beta1 to fibronectin and vitronectin, adhesion assays were performed in the presence of three different synthetic peptides (Fig. 6B and 7B). Although integrins can interact through a common RGD site in the ligand, conformationally constrained peptides can discriminate between various RGD binding integrins. The cyclic peptide, CRRETAWAC, has recently shown to be highly selective for alpha5beta1(13) . At concentrations sufficient to block adhesion of alpha5beta1 to fibronectin, CRRETAWAC does not block alphavbeta1 fibronectin adhesion or alphav-mediated vitronectin adhesion(13) . In an analogous fashion, the cyclic peptide 4C selectively inhibits alphav-mediated adhesion(21) . In contrast, the peptides GRGDSP and RGDGW are able to block both alpha5- and alphav-mediated adhesion(12) . We took advantage of these selective peptides to further define the binding characteristics of alpha8beta1. We found that adhesion of alpha8-transfected cells to fibronectin was inhibited by the peptide RGDGW, but not by the CRRETAWAC peptide (Fig. 6B). The addition of the alpha5 blocking antibody, P3D10, to CRRETAWAC, did not significantly decrease fibronectin adhesion (data not shown). In contrast, adhesion of mock-transfected 293 cells to fibronectin was inhibited by either RGDGW or CRRETAWAC (Fig. 6B). Adhesion of mock-transfected and alpha8-transfected 293 cells to fibronectin was not affected by the alphav-selective 4C peptide (data not shown). These results suggest that alpha8beta1 interacts with fibronectin by mechanisms that are similar to, but distinguishable from, those used by alpha5beta1.

Adhesion of alpha8-transfected cells to vitronectin was inhibited by the peptide RGDGW, but not by the 4C peptide (Fig. 7B). The addition of the alphav blocking antibody, L230, to 4C did not further inhibit vitronectin adhesion (data not shown). In contrast, adhesion of mock-transfected 293 cells to vitronectin was inhibited by either RGDGW or 4C (Fig. 7B). Adhesion of mock-transfected and alpha8-transfected 293 cells to vitronectin were not affected by the alpha5-selective peptide, CRRETAWAC (data not shown). Thus, the 4C peptide, at the concentration used, blocks alphav- but not alpha8beta1-mediated adhesion to vitronectin, suggesting that alpha8beta1 interacts with vitronectin through mechanisms distinct from those used by alphav integrins.

Affinity Chromatography

To determine whether alpha8beta1 binds directly to fibronectin and vitronectin, we performed affinity chromatography using octylglucoside lysates of I-labeled, alpha8-transfected 293 cells. We passed cell lysates over fibronectin-Sepharose (Fig. 8A) or vitronectin-Sepharose (Fig. 8B) columns, washed with loading buffer (lanes 2-8), and eluted with GRGDSP (lanes 9-12) and EDTA (lanes 13-16). The eluate of the fibronectin-Sepharose column contained three labeled proteins with sizes corresponding to putative fibronectin receptor subunits (Fig. 8A, arrows): 120 kDa (beta1), 150 kDa (alpha5), and 170 kDa (alpha8). To determine whether the 170-kDa/120-kDa protein is indeed identical with alpha8beta1, we performed immunoprecipitations with anti-alpha8 antibody (Fig. 9A, lanes 1-3). The putative alpha8beta1 heterodimer (170 kDa/120 kDa) was detected in the cell lysate (lane 1), was not detectable in wash fractions (lane 2), and was again present in the GRGDSP-eluate (lane 3). To confirm the identity of the 150-kDa/120-kDa protein as alpha5beta1, we performed an immunoprecipitation of the GRGDSP eluate with P3D10 (anti-alpha5) antibody (lane 4).


Figure 8: Affinity chromatography of I-surface-labeled lysates from alpha8-transfected 293 cells on (A) fibronectin-Sepharose column in the presence of 1 mM MnCl(2) and (B) vitronectin-Sepharose column in the presence of 1 mM MgCl(2). Lane 1, whole cell lysate; lanes 2-8, column buffer fractions; lanes 9-12, GRGDSP-eluted (1 mg/ml) fractions; lanes 13-16, 10 mM EDTA-eluted fractions; lane 17, 1 M NaCl wash. Arrows indicate the positions of putative receptor subunits. Positions of molecular size markers in kilodaltons are shown to the left.




Figure 9: Immunoprecipitation of alpha8- and beta1- containing integrins from (A) fibronectin- and (B) vitronectin-Sepharose column fractions. Lanes 1-3, immunoprecipitations with anti-alpha8 antibody; lanes 1, whole cell lysates; lanes 2, column buffer fractions; lanes 3, GRGDSP-eluted fractions; lane 4A, GRGDSP-eluted fractions immunoprecipitated with anti-alpha5 antibody (P3D10); lane 4B, GRGDSP-eluted fractions immunoprecipitated with anti-alphavbeta5 antibody (P1F6). Proteins were analyzed by SDS-PAGE under nonreducing conditions. Positions of molecular size markers in kilodaltons are shown to the left.



The GRGDSP eluate of the vitronectin-Sepharose column contained major bands of 150 kDa and 95 kDa (Fig. 8B, arrows). However, immunoprecipitation of the eluted fractions with anti-alpha8 antibody demonstrates the 170-kDa/120-kDa complex corresponding to alpha8beta1 was specifically eluted from the vitronectin-Sepharose column by GRGDSP (Fig. 9B, lanes 1-3). As we have shown previously(15) , the 150-kDa/95-kDa-bands correspond to the alphavbeta5 heterodimer as confirmed by immunoprecipitation of the GRGDSP eluate with an anti-alphavbeta5 antibody, P1F6 (Fig. 9B, lane 4).

Adhesion of alpha8beta1 to Additional RGD-containing Proteins

Because the above experiments suggested that alpha8beta1 was binding to the RGD sites in fibronectin and vitronectin, we examined additional RGD-containing proteins for the ability to bind to alpha8beta1. We tested the ability of mock- and alpha8-transfected 293 cells to adhere to tenascin, fibrinogen, thrombospondin, osteopontin, and denatured collagen type I (Fig. 10). We found that alpha8-transfected 293 cells adhered and spread well on tenascin, whereas mock-transfected 293 cells did not adhere (Fig. 10). Neither mock-transfected nor alpha8-transfected 293 cells adhered to fibrinogen, thrombospondin, osteopontin, or denatured collagen type I (Fig. 10). In contrast, beta3-transfected 293 cells adhered well to fibrinogen, thrombospondin, osteopontin, and denatured collagen type I (Fig. 10).


Figure 10: Adhesion of alpha8-transfected (black bars), mock-transfected (white bars) and beta3-transfected 293 cells (gray bars) to 3 µg/ml fibrinogen, denatured collagen type I, tenascin, thrombospondin, and osteopontin. The y axis represents the absorbance at 595 nm after staining attached cells with crystal violet.



Tenascin is a modular protein that contains several fibronectin type III repeats. An RGD site located in the third fibronectin type III repeat of tenascin (TNfn3) has been shown to mediate adhesion of several integrins, including alphavbeta3 and probably alphavbeta6(22) . To determine whether alpha8beta1 was mediating adhesion through this RGD site, we tested the adhesion of alpha8- and mock-transfected 293 cells to various recombinant fragments of tenascin (Fig. 11). The alpha8-transfected cells adhered to the RGD-containing fragment, TNfn3, in a concentration-dependent manner. As expected, mock-transfected 293 cells did not adhere to TNfn3. In contrast, alpha8-transfected 293 cells did not adhere to a fragment containing the fourth through sixth fibronectin type III repeats, TNfn4-6 (Fig. 11). To confirm that alpha8-transfected cells were binding through the RGD site of TNfn3, we tested adhesion of alpha8-transfected cells to a mutant TNfn3 in which the RGD site had been altered to RAA (TNfn3RAA). We found that this abolished adhesion of alpha8-transfected 293 cells (Fig. 11). In a control experiment, alpha9-transfected 293 cells, which have been shown to adhere to TNfn3 at a non-RGD site(14) , adhered well to TNfn3RAA (data not shown).


Figure 11: Adhesion of alpha8-transfected cells to increasing concentrations of TNfn3 (), TNfn3-RAA (box), TNfn4-6 (bullet), and adhesion of mock-transfected cells to increasing concentrations of TNfn3 (down triangle). The y axis represents the absorbance at 595 nm after staining attached cells with crystal violet.



Adhesion to TNfn3 by alpha8-transfected cells was abolished by the anti-beta1 antibody, P5D2 (Fig. 12). We also tested the ability of the selective, synthetic peptides to block adhesion to TNfn3. The peptides, GRGDSP and RGDGW blocked adhesion of alpha8-transfected cells to tenascin (Fig. 12). In contrast, the cyclic CRRETAWAC peptide did not inhibit adhesion and the cyclic 4C peptide only partially inhibited adhesion to TNfn3 (Fig. 12). Adhesion of beta3-transfected cells to tenascin was abolished by the 4C peptide (data not shown). Taken in concert, these results suggest that alpha8beta1 is binding to the RGD site in tenascin. However, this interaction must be somewhat distinct from the alphav-tenascin interactions because it is not completely inhibited by the peptide 4C.


Figure 12: Adhesion of alpha8-transfected cells to 3 µg/ml TNfn3 alone (control) or in the presence of an anti-beta1 antibody, P5D2, the peptides GRGDSP, RGDGW, or the cyclic peptides CRRETAWAC and 4C. The y axis represents the absorbance at 595 nm after staining attached cells with crystal violet.



In summary, we have demonstrated that alpha8beta1 can bind to tenascin, fibronectin, and vitronectin by interacting with the RGD sites on these ligands. We also show that alpha8beta1 is capable of localizing to focal contacts on fibronectin and vitronectin on fibroblasts and smooth muscle cells. alpha8beta1 is eluted from both fibronectin and vitronectin affinity columns by an RGD-containing peptide. Our fibronectin adhesion data are in agreement with the recently published observations that chicken alpha8beta1 is able to support attachment, spreading, and neurite outgrowth by transfected cells(23) . Two other beta1-containing integrins are known to interact with RGD sites: alpha5beta1 and alphavbeta1. These alpha subunits, along with alphaIIb, are the most closely related to alpha8 (42-43% amino acid identity). In addition, these subunits share several other structural features with each other including the presence of post-translational cleavage and the absence of I domains. Thus, alpha8, alpha5, alphav, and alphaIIb define a subfamily of alpha subunits that have close sequence homology, bind to RGD-containing peptides, are post-translationally cleaved, and do not contain I domains. Despite the similarities to alphav and alpha5, the binding specificity of alpha8beta1 is unique. In contrast to alpha5beta1, whose only known ligand is fibronectin, alpha8beta1 is more promiscuous and can bind to vitronectin and tenascin as well as fibronectin. In addition, binding by alpha8beta1 is not affected by the peptide CRRETAWAC, which efficiently blocks alpha5beta1. When compared to alphavbeta3, the binding repertoire of alpha8beta1 is more limited. Although both alpha8beta1 and alphavbeta3 bind to tenascin, fibronectin, and vitronectin, alpha8beta1 does not bind to several other alphavbeta3 ligands, including fibrinogen, thrombospondin and denatured collagen. Additionally, alpha8-mediated adhesion is not affected by the peptide 4C. Thus, the binding characteristics of alpha8beta1 are unique and distinguishable from both alpha5beta1 and alphavbeta3.

In adult mammalian tissues, alpha8beta1 is prominently expressed in vascular and visceral smooth muscle cells, kidney mesangial cells, and lung myofibroblasts(2) . Tenascin, fibronectin, and vitronectin are thought to play a role in the response to injury and inflammation(24) . Thus, alpha8beta1 may contribute to the functional changes that occur in smooth muscle cells during tissue repair. Since smooth muscle cells also express other fibronectin, vitronectin, and tenascin receptors, such as alpha5beta1, alphavbeta5, and alphavbeta3(25) , it will be important to determine the specific functional contribution of alpha8beta1 to smooth muscle cell behavior.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA53259 and HL191551 (to R. P.), and HL/A133259 and HL47412 (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Clinical Investigator Development Award K08HL02874 and American Heart Association, California Affiliate Grant-in-Aid 94-235. To whom all correspondence should be addressed: Lung Biology Center, UCSF Box 0854, San Francisco, CA 94143. Tel.: 415-206-5959; Fax: 415-206-4123.

(^1)
The abbreviations used are: BSA, bovine serum albumen; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Drs. K. Crossin, E. Ruoslahti, E. Koivunen, C. Giachelli, Y. Yokosaki, R. Kramer, W. Carter, and E. Wayner for generously providing reagents used in this study.


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