Laminin E8 alveolarization site: heparin sensitivity, cell surface

Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading

LANLIN CHEN,’ VLADISLAV SHICK,l MICHELLE L. MATTER,l SUSAN M. LAURIE,l ROY C. OGLE,‘y2 AND GORDON W. LAURIE1 IDepartment of Cell Biology and 2Department of Neurological Surgery, University of Virginia, Charlottesville, Virginia 22908

Chen, Lanlin, Vladislav Shick, Michelle L. Matter, Susan M. Laurie, Roy C. Ogle, and Gordon W. Laurie. Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L494-L503, 1997.-Cell adhesion to amino acids 2179-2198 (SN-peptide) of the laminin-1 al-chain is required for lung alveolar formation in vitro (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The nature of the SN-peptide receptor(s) was probed with neutralizing anti-integrin monoclonal antibodies (MAb), cells lacking integrin subunits, soluble heparin, and SN-peptide columns. Cell adhesion and spreading studies confirmed the specificity of SN-peptide and revealed adhesion to be unaffected by inclusion of anti-pi-, anti-cxZe6- or anti-a&-integrin MAb. Cells lacking PI- or cx6-integrin subunits were fully adherent. Adhesion was heparin, but not chondroitin sulfate or heparinase, sensitive, much as is cx-dystroglycan-laminin-1 binding. Heparin eluted - 155 and 180-kDa cell-surface proteins from SN-peptide columns. An additional -9l-kDa protein was eluted by EDTA. All were unrecognized by anti-Pi-integrin MAb. SN-peptide therefore interacts with three cell-surface proteins for which the iden- tity remains to be determined.

lung; alveolar; cell adhesion; integrin; dystroglycan

CELLULAR ADHESION to basement membranes is a funda- mental function of many living cells for which a number of diverse ligands and receptors have been identified (12, 20). Prominent not only in embryogenesis, such interactions play a key role in the numerous homeo- static events that mark the daily life cycle of adult epithelia, endothelia, muscle, nerve, hemopoietic, and lymphoblastic cells. The basement membrane glycoprotein laminin-1 (2) has proven to be a particularly potent source of cell adhesion activity, a function coincident with its intimate association with the basal cell surface where it is found polymerized with collagen IV, perle- can, growth factors, and possibly other components.

Elucidation of how cells interact with laminin-1 has been initiated by proteolytic fragmentation (8). As a result, it is known that the 250-kDa elastase fragment E8, which is the most adhesive domain of laminin-1, is the specific ligand of cx& (24, 25>-, (x& (16)-, and c& (15)-integrins, as well as cell surface pi 4-galactosyltransferase, which interacts with E8 oligbsaccharides during cell motility and spreading (23). Subfragmenta- tion of E8 (8) and use of an anti-recombinant G domain antibody (32) has identified an important as-dependent cell adhesion site somewhere within the first three loops of the G domain, which is conformationally dependent and/or requires a second site in the rod domain for full activity (8). Receiving increasing attention is the

- 156-kDa cell-surface protein a-dystroglycan (5), for which interaction with laminin-1 or laminin-2 is heparin and EDTAinhibitable and thought to occur through oligosaccharides (5) on the last two loops of the G domain, known as the E3 region, although interaction with E8 has also been demonstrated (11).

In a screen for basement membrane molecules required for lung alveolar formation, we discovered a novel 20-amino acid cell adhesion site (SN-peptide; @l-chain amino acids 2179-2198; see Ref. 19) in the first loop of the laminin-1 G domain, which, when presented to type II alveolar cells as soluble inhibitor, blocked alveolar morphogenesis in vitro. The same site was later identified by Nomizu et al. (designated AG- 10; Ref. 22) in a 113 overlapping synthetic peptide screen for G domain cell adhesion activity. Here we report on studies that reveal this site to play an important role in laminin-l-dependent cell spreading and that identify the cell surface receptor(s) as a triad of - 155-, 180-, and 91-kDa molecules for which interaction with SN-peptide is heparin and EDTA inhibitable.

METHODS

Preparation of substrates. Mouse laminin-1 was prepared according to the method of Kleinman et al. (14) or was purchased from Upstate Biotechnology (Lake Placid, NY). Collagen I was purchased from Collaborative Biomedical Products (Bedford, MA). Heparin (sodium salt; purified grade) was purchased from Fisher Scientific (Fair Lawn, NJ). Bovine serum albumin (BSA), chondroitin sulfate A (sodium salt), and heparinase I, II, and III were purchased from Sigma Chemical (St. Louis, MO). Fragments E8 and Pl, isolated from mouse laminin-1, were generously supplied by Drs. Peter Yurchenco (Robert Wood Johnson Medical School, Pis- cataway, NJ) and Rupert Timpl (Max Planck Institut fur Biochemie, Martinsried, Germany). Fragment E 1’ was generously supplied by Dr. Peter Yurchenco. Fragment T8 was generously supplied by Dr. Reiner Deutzman (Universitat Regensburg, Regensburg, Germany). SN-peptide (SINNNR- WHSIYITRFGNMGS; amino acids 2179-2198 from mouse laminin al-chain; see Fig. 1 and Ref. 19) was synthesized by the Biomolecular Research Facility (University of Virginia, Charlottesville, VA). Also synthesized was SN-peptide with a COOH-terminal cysteine for column coupling. All synthetic peptides were purified by reverse phase-high-performance liquid chromatography and were verified by NHz-terminal sequencing and mass spectrometry. An isoelectric plot of SN-peptide was performed using the isoelectric program of the Genetics Computer Group (Madison, WI).

CeZZs. Human fibrosarcoma (HT-1080), rat adrenal pheochromocytoma (PC-12), osteogenic sarcoma (Saos-2), choriocarcinoma (JAR), ovarian adenocarcinoma (OVCAR-3), and chronic myelogenous leukemia (K-562) cells were obtained from American Type Culture Collection (Rockville, MD).

L494 1040-0605/97 $5.00 Copyright o 1997 the American Physiological Society

SN-PEPTIDE RECEPTOR L495

Mouse NII-I/3T3 cells were also obtained from American Type Culture Collection. Human colon carcinoma (Clone A), rectal carcinoma (RKO), and mouse monocyte-macrophage [P388D1 (IL-l)] cells were kindly provided by Dr. A. Mercurio (New England Deaconess Hospital, Boston, MA). Mouse M2 and Clone 10 are high and low metastatic clonal variants, respectively, of K-1735 melanoma cells (1). Mouse F9 teratocarcinoma and TKO cells (the latter is an F9 cell in which all three copies of the PI-gene were knocked out by homologous recombination; see Ref. 27) were kindly supplied by Dr. C. Damsky (University of California San Francisco, CA). Mouse GD25 cells, derived from ES cells via knocking out the P1-integrin gene by homologous recombination (lo), were kindly provided by Dr. Reinhard Faessler (Max Planck Institut fur Bioche- mie). Media used included Eagle’s minimum essential medium containing 10% fetal bovine serum (FBS; HT-1080); McCoy’s 5a with 15% FBS (Saos-2); RPM1 1640 with 10% FBS (JAR; K-562, PC-12), 15% FBS (P388D1), or 20% FBS and 10 pg/ml insulin (OVCAR-3); and Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, and 2 mM glutamine (F9, TKO). All media and sera were from GIBCO- BRL (Gaithersburg, MD).

Antibodies. Antibodies ab[SN] and ab[IK], prepared against SN-peptide and laminin al-chain amino acids 2097-2108, respectively (designated ab-A[SN] and ab-A[IK] in Ref. 19), were kindly provided by Dr. Y. Yamada (National Institute of Dental Research, Bethesda, MD). Ab[SN] and ab[IKJ were purified from antisera with protein A Sepharose (Pharmacia, Piscataway, NJ) before use. Neutralizing mouse anti-human monoclonal antibodies (MAb) specific for the (x2 (PlE6)-, (x3 (PlB5)-, (~4 (P4C2)-, cx5 (PlD6)-, and PI (P4ClO; Refs. 6,30, and 31)-integrin subunits were purchased from GIBCO-BRL. For immunoprecipitation, a rabbit antibody prepared against the conserved cytoplasmic domain of chicken P1-integrin subunit (18) was kindly provided by Dr. D. DeSimone (University of Virginia). Neutraliz- ing rat anti-mouse cxG-integrin MAb GoH3 (26) was purchased from Amac (Westbrook, ME) in purified form. The partially blocking mouse anti-human P4-integrin subunit MAb UM-A9 was kindly provided by Dr. T. Carey (University of Michigan, Ann Arbor, MI; Ref. 29). Also used was the partially blocking mouse anti-human P4-integrin subunit MAb 3El (13) purchased from GIBCO-BRL.

CeZZ adhesion assay. Cell adhesion studies were carried out using the method ofAumailley and Timpl(4). Briefly, 96-well culture plates (Costar, Cambridge, MA) were coated overnight (4°C) in triplicate with increasing micromolar amounts of BSA, fragment E8, T8, El’ or Pl, laminin-1, or SN-peptide diluted in water. Wells were blocked with 1% BSA (Sigma) for 4 h and incubated with cells (3 x lo5 cells/ml) in serum-free medium for 60 min (37°C). Wells were washed twice with medium, fixed with glutaraldehyde, and stained with crystal violet. Stained adherent cells were subsequently solubilized with Triton X-100 and analyzed in an enzyme-linked immuno- sorbent assay (ELISA) plate reader (Molecular Devices, Menlo Park, CA) at 595 nm. For adhesion inhibition studies with ab[SN] or ab[IK], substrate-coated wells (0.35 and 35 uM coating solutions of E8/Pl and SN-peptide, respectively) were blocked, treated with antibody (50 ug/well) for 60 min (37”C), washed two times, and then incubated with cells. For adhesion inhibition experiments with anti-integrin antibodies, plates were coated as for spreading studies. An equal volume of cells (6 X lo5 cells/ml) and media-diluted anti-integrin antibodies (10 and 100 pg/ml for anti-a- and anti-P-subunit antibodies) were coincubated for 45 min at room temperature with gentle agitation every 10 min and then added to plates. Use of a lo-fold higher concentration of anti-o16- or anti-PI- antibody was found to nonspecifically inhibit adhesion to

SN-peptide, as revealed by coincubation of cells lacking o6 or PI with each antibody. Substrate coating efficiency was determined by supplementation of substrate with a minor amount of 1251-labeled fragment E8 or SN-peptide according to the method ofAumailley et al. (3). Coated wells were washed with DMEM and were treated with 2 M NaOH to release radioac- tivity for analysis in a gamma counter.

CeZZ spreading assay. Plates were coated in triplicate with subsaturating micromolar concentrations of each substrate individually determined from dose-response studies to sup- port adhesion at a level -50% of maximum per substrate. BSA-blocked wells were incubated with cells (3 X lo5 cells/ ml) in serum-free medium for 60 min (37°C). Photographs of the same 4-o’clock region of each well were analyzed by tracing cell perimeters from enlarged negatives using a digitizing tablet (GTCO, Columbia, MD), thereby determin- ing cellular area. For spreading inhibition studies, SN- peptide was diluted in medium and added to cells 30 min (37°C) before plating using a final peptide concentration of 10 uM.

Heparin binding assay. Wells of 96-well ELISA plates (Dynatech, Chantilly, VA) were coated (10 uM coating solution) with BSA, laminin-1, or SN-peptide, blocked with 1% BSA-0.05% Tween 20, and incubated for 1 h (at 37°C) with heparin-gold (Sigma) diluted 1: 1,000 in phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 0.05% BSA. Wells were subsequently washed with PBS-0.05% Tween 20 using a Titertek Plus (Flow Labs, Irvine, Scotland) plate washer and were incubated with silver enhancement solution (Amersham, Arlington Heights, IL). Kinetic monitoring of color development was performed over 24 min at 650 nm using a UV,,, microplate reader (Molecular Devices); values were expressed as the maximal velocity (V,&

Afinity chromatography of receptor: SN-peptide was coupled at 1 mg/ml gel to SulfoLink (Pierce, Rockford, IL) through an added COOH-terminal cysteine according to the manufactur- er’s suggestions. As a control, BSA was reduced with 2-mercap- toethylamine, separated on a Sephadex G-25 column, and coupled with SulfoLink at 4.5 mg/ml gel. Coupling efficiencies were 90-95%. Cells were biotinylated according to the method of Stephens et al. (27). Briefly, confluent cells (six 150-mm plates) were washed two times with PBS, treated with 50 ug/ml NHS-LC biotin (Pierce) in PBS for 90 min on ice, detached, and washed two times in PBS containing 50 mM glycine. Pellets were resuspended in lysis buffer containing 200 mM octyl-P-D-glucopyranoside (Sigma), 50 mM tris(hy- droxymethyl)aminomethane (Tris), 100 mM NaCl, 5 mM MnC12, 2 mM phenylmethylsulfonyl fluoride (PMSF), 21 uM leupeptin, 6 uM pepstatin A, and 2 uM aprotinin (pH 7.4). Lysates were centrifuged, and supernatants were passed over a blank SulfoLink precolumn before incubation for 6 h to overnight (4°C) with paired columns of immobilized SN- peptide or BSA. Unbound material was removed by washing with 20 column volumes of running buffer (50 mM Tris, 50 mM octyl-P-D-glucopyranoside, loo-150 mM NaCl, 5 mM MnC12, and 2 mM PMSF, pH 7.4). Bound material was eluted with running buffer containing 1 ug/ml of heparin, 10 mM EDTA in place of MnC12, or both 1 ug/ml of heparin and 10 mM EDTA. Finally, a wash with 1 M NaCl in running buffer was performed. Control columns were run in parallel using the same reagents. Equal aliquots of column fractions were acetone precipitated, separated by 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose, and detected with streptavidin- peroxidase (Amersham, Arlington Heights, IL) using the enhanced chemiluminescence method. Column fractions were precleared for 30 min with protein G agarose (Pierce), incu-

L496 SN-PEPTIDE RECEPTOR

bated with a rabbit anti-chicken PI-integrin antibody overnight, and precipitated with protein G agarose using a goat anti-rat bridge all at 4°C. Washing was performed as de- scribed by Stephens et al. (27). After boiling, samples were separated by 8% SDS-PAGE in the absence of dithiothreitol, transferred to nitrocellulose, and detected with streptavidin- peroxidase and enhanced chemiluminescence.

Statistical analysis. All values are expressed as means t SE.

RESULTS

Adhesion to SN-peptide is dose dependent. The SN- peptide site (amino acids 2179-2198 of laminin-1 al- chain; arrow on Fig. 1) was originally identified in a screen for a basement membrane activity(s) required for in vitro lung alveolar formation. Functional dissec- tion revealed the activity to be mainly attributable to laminin-1 in a fragment E8 and subsequently anti-SN- peptide or SN-peptide inhibitable fashion. When presented as a 35 uM coated substrate SN-peptide supported type II alveolar and HT-1080 fibrosarcoma cell adhesion, an interaction that was EDTA inhibitable (19). As a fir s s t t ep in characterizing the relative role of SN-peptide in laminin- l-dependent cell adhesion and the nature of the cell receptor, comparative adhesion experiments were carried out with a number of different cell lines (Fig. 2). HT-1080 fibrosarcoma (Fig. 2, A and B), Clone 10 melanoma, and NIH/3T3 embryonic cells (Fig. 2B) all adhered particularly well in a strong dose-dependent manner (Fig. 2A, shown for HT-1080). Adhesion by M2 melanoma, RKO rectal carcinoma, Clone A colon carcinoma, OVCAR-3 ovarian carcinoma, and JAR choriocarcinoma was moderate (Fig. 2, B and C). Less adhesive were F9 teratocarcinoma (Fig. 2B) and PC-12 pheochromocytoma (Fig. 2, A and B) cells, with Saos-2 osteogenic sarcoma cells showing little or no affinity (Fig. 2C). In all cases, plateau adhesion was similar to that obtainable with fragment E8, but significantly higher micromolar amounts of SN-peptide were

Fig. 1. Schematic diagram of the COOH-terminal region of laminin-1 with an arrow marking the SN-peptide cell adhesion and alveolarization site. This site, SINNNRWHSIYITRFGNMGS (amino acids 2 179- 2198), is located in the first loop of the globular (G) domain and is contributed solely by the laminin al-chain. Amino to the G domain, l31- and yl-chains combine with al-chain to form a rod. Fragment E8 consists of all of the diagramed region with the exception of the last two G domain loops, the latter of which comprise the E3 fragment. Bar, -5 nm.

0.8

HT1080

v) 6, u)

d . 0

0 10 20 30 40 50 60

SNpeptide (PM)

Fig. 2. SN-peptide is adhesive for a number of different cells but not all cells. A: dose-dependent adhesion of HT-1080 fibrosarcoma vs. PC-12 pheochromocytoma cells to SN-peptide. HT-1080 cells adhere well, and PC-12 cells adhere moderately poorly with plateau adhesion achieved at 20-30 uM. Values derive from a mean of 6 (PC-12) to 10 (HT-1080) independent measurements. Bovine serum albumin (BSA) background values (0.09 and 0.17, for HT-1080 and PC-12 cells, respectively) have been subtracted away in Figs. 2, 3, 7 and 9. Inset: isoelectric plot of SN-peptide indicating a net charge of +2.5 at pH 7.4. B: comparative SN-peptide (20-25 uM) and E8 (0.1-l FM&dependent adhesion of HT-1080, PC-12, Clone 10, M2 melanoma, F9 teratocarcinoma, 3T3 fibroblast, RKO rectal carcinoma, and Clone A colon carcinoma cells. Values derive from a mean of 5 (E8) to 10 (SN-peptide) independent measurements. C: comparative adhesion of OVCAR-3 ovarian carcinoma, Saos-2 osteogenic sarcoma, and JAR choriocarcinoma cells to SN-peptide (35 PM). Means of 9 independent measurements. OD, optical density

required (20-30 uM for SN-peptide vs. 0.1 pM for E8). To determine whether these latter differences were in part because of differential substrate adsorption, we included trace amounts of iodinated SN-peptide or fragment E8, revealing adsorption to be approximately threefold less for SN-peptide (Table 1). Correcting half-maximal coating values for substrate actually adsorbed suggested an 80-fold difference in adhesion activity between SN-peptide and E8, a value pointing to a requirement for additional sites and/or E8 folding.

SN-peptide could simply represent a highly charged region of E8 that interacts in a nonselective way with a negatively charged cell surface for which the strength might vary among cell types. To examine charge and specificity, several approaches have been used. Charge was analyzed via Isoelectric (Genetics Computer Group) revealing SN-peptide to have a relatively low net charge of +2.5 at pH 7.4 (Fig. 2A, inset). Second, a


Table 1. Determination of coating eficiencies (70 adsorbed)

Substrate Coating Solution, PM

0.05 0.1 5 25

SN-peptide ND ND 0.2 (4) 0.48 2 0.2 (4) ES fragment 0.6 (3) 1.7 2 0.3 (4) ND ND

Values for SN-peptide with 25 pM substrate coating solution and for E8 fragment with 0.1 pM substrate coating solution are means -C SE; no. of measurements are in parentheses. ND, not determined.

scrambled version of the peptide site exhibited no cell adhesion activity (21). Finally, the anti-SN-peptide antibody ab[SNl was examined and was found to inhibit HT-1080 cell adhesion to coated SN-peptide and fragment E8 but not to affect adhesion to the Pl fragment originating from the center of the laminin-1 cross (Fig. 3). Control antibody ab[IKl directed to al-chain amino acids 2097-2108 in the rod domain of E8 had no effect (Fig. 3). Taken together with previous specificity studies (191, these data are in keeping with SN-peptide representing a highly selective adhesion site.

SN-peptide specificity further revealed by selective inhibition of cell spreading. We next asked whether the SN-peptide site has a role in laminin-l-dependent cell spreading. HT-1080 cells were plated on SN-peptide alone or on laminin-1 in the absence or presence of soluble SN-peptide (Fig. 4). Examination 1 h later revealed no apparent spreading on SN-peptide (Fig. 4A), although small projections could be occasionally distinguished on careful examination (Fig. 4A, inset). In contrast, cells rapidly spread on laminin-1 (Fig. 4B, inset a), a process that was suppressed by preincuba-

80

7 @

60

5 .- z 40 6 a

5 20

+ +

ab[SN] ab[lK] Fig. 3. HT-1080 cell adhesion to SN-peptide (SNpep) is inhibitable with anti-SN-peptide (ab[SN]) but not anti-IK-peptide (ab[IKl) antibodies. AblSNl also inhibits adhesion to fragment E8 but has no apparent effect on adhesion to the Pl fragment originating from the cross-region of laminin-1. Coating solutions were 35 pM (SN-peptide) and 0.35 pM (E8); antibody was used at 50 pg/well. Ab[IKl was raised against laminin al-chain sequence AASIKVAVSADR (amino acids 2097-2108) located in the rod region of E8. Each value represents the mean of 9 independent measurements with BSA background subtracted.

A 40

0

B 40

Area (pm2 x 10) Fig. 4. Coated SN-peptide promotes little or no HT-1080 cell spreading, but soluble SN-peptide suppresses spreading on laminin-1. A: area distribution of individual HT-1080 cells (n = 200) adherent to SN-peptide for 1 h. Inset: appearance of 1 h adherent cells. Cells remain rounded, but small projections are occasionally evident. B: area distribution of individual HT-1080 cells adherent for 1 h to laminin-1 in the absence (Ln; n = 359) or presence (Ln + SNpep; n = 320) of soluble SN-peptide (10 pM) as inhibitor. Presence of soluble SN-peptide inhibits cell spreading, thereby decreasing cellular area. Area distribution curve resulting from use of 1 uM SN-peptide as inhibitor (not shown) resides between the two shown. Inset: appearance of cells adherent to laminin-1 plated in the absence (a) or presence (b) of soluble SN-peptide (10 PM). Area measurements in this and subsequent figures were determined by tracing the perimeters of all clearly distinguishable cells in randomly selected fields. Coating solutions were 10 (SN-peptide) and 0.006 (laminin-1) uM, which give rise to approximately half-maximal adhesion, Bars, 200 Pm.

tion of cells with 10 pM SN-peptide (Fig. 4B, inset b). This inhibitory effect was manifested by a leftward shift in the area distribution of individually measured cells to a curve appropriate for a round or near-round phenotype (Fig. 4B). Decreasing the concentration of inhibitory SN-peptide lo-fold shifted the curve to an intermediate position between round and spread pheno-


types (not shown). The same experiment was performed using plates coated with collagen I, fragment El’ (similar to Pl) or fragment E8 (Figs. 5 and 6). HT-1080 spreading on collagen I was unaffected by soluble SN-peptide (10 pM; Fig. 5, compare insets a and b). SN-peptide similarly had no apparent effect on the small amount of spreading observed on El’ (Fig. 6A, compare insets a and b). However, E8-dependent spreading was suppressed (Fig. 6B, compare insets a and b).

Search for the SN-peptide receptor argues against ,BI-integrins. With both adhesion and spreading studies supporting the specificity of SN-peptide, we next turned to our main goal of identifying the SN-peptide receptor. HT-1080 cell adhesion to E8 is partially inhibitable with antibodies directed against o&i-integrin, a major laminin-1 receptor that is thought to bind E8 within the first three loops of the G domain (32). To ask whether adhesion to SN-peptide is mediated by o&i, HT-1080 cells were incubated with neutralizing anti-cxs- or anti- pi-antibodies (10 and 100 ug/ml) under conditions that inhibited adhesion to E8 or the similar T8 fragment (Fig. 7, inset). Both antibodies, however, had little or no effect on adhesion to SN-peptide (Fig. 7). Other integrins expressed by HT-1080 cells include CX&, cx&, (Y&, and possibly cq$&, but use of neutralizing anti-c+., -03, -04, -05, and -c~vl& MAb or soluble RGDS or RGES peptide at 100 pM (not shown) was also without inhibitory effect (Fig. 7). Another E8 binding integrin, CY&, appears not to be expressed by HT-1080 cells based on lack of HT-1080 adhesion to anti-p4 MAb- coated plates (not shown). Because F9 cells express a&, we tested the partially neutralizing anti-&-MAb

r

Area (pm* x 10) Fig. 5. SN-peptide has no effect on HT-1080 cell spreading on collagen I. Area distribution of individual HT-1080 cells adherent for 1 h to collagen I without ((301; n = 196) or with (Co1 + SNpep; n = 197) soluble SN-peptide (10 PM). Inset: appearance of cells adherent to collagen I in the absence (a) or presence (b) of soluble SN-peptide. Collagen I coating solution was 0.002 pM, which supports half- maximal cell adhesion. Bar, 200 pm.

E8 + SNpep Y

Fig. 6. SN-peptide partially inhibits HT-1080 cell spreading on fragment E8 but not on fragment El’ from the cross region of laminin-1. A: area distribution of HT-1080 cells on El’ (n = 197) reveals slight spreading after 1 h, which appears to be unaffected by soluble SN-peptide (El’ + SNpep; n = 1971. Inset: without (al or with (b) soluble SN-peptide. Fragment El’ is similar to the Pl fragment with some differences. B: in contrast, HT-1080 cells spread well on E8 in the absence (E8; n = 199) but not presence (E8 + SNpep; n = 171) of soluble SN-peptide. Inset: without (a) or with (b) soluble SN- peptide. Coating solutions for half-maximal adhesion were 0.1 (El’) and 0.01 (E81 pM. Soluble SN-peptide was 10 uM. Bars, 200 pm.

3El and UM-AS, but neither inhibited adhesion to SN-peptide (not shown).

To test these data in a different way, adhesion studies (Fig. 8) were carried out with cells lacking 06- or pl-integrin subunits. TKO and GD25 cells, in which the pi-integrin gene had been knocked out by homologous recombination (10, 27>, attached to SN-peptide (Fig. 8A) in a dose-dependent manner (Fig. 8A, inset) at plateau levels within the range previously observed for HT-1080 cells. Both cells displayed low (TKO) or good (GD25) adhesion to laminin-1, and no specific adhesion to E8 (Fig. SA). P388Di and K-562 cells, which express


Antibody Fig. 7. Neutralizing anti-integrin antibodies do not significantly inhibit HT-1080 cell adhesion to coated SN-peptide. Cells were preincubated with antibodies at a final concentration of 10 (all anti-a) or 100 (anti-pi) ug/ml. At a lo-fold higher concentration of anti-cw6-, -P1-, or +-antibodies, complete (anti-q) or partial (anti-p) inhibition of cells to SN-peptide was observed (7). This is now suspected to be nonspecific because cells lacking the o6- or Pi-subunits are also inhibited from adhesion to SN-peptide at 100 (anti-q) or 1,000 (anti-pi) ug/ml, respectively. Inset: positive control inhibition of adhesion to fragments E8 or T8 by anti-o6 (10 ug/ml) or -pi (100 ug/ml), respectively. T8 is identical to E8 with the exception of shortened amino ends of the (Y-, p-, and y-chains. Coating solution was 2.5 uM (SN-peptide) and 0.1 uM (E8/T8). Values represent a mean of 10 (SN-peptide) or 3 (E8/T8) independent measurements.

little or no q-subunit on their surface (24), also adhered well to SN-peptide (Fig. 8, B and C) with a similar dose-dependent relationship (Fig. 8A, inset) and showed no apparent avidity for laminin-1 (P388DJ or E8 (K-562). These results underlined the conclusion that (x&i is not the primary receptor for SN-peptide and that other q- or P1-receptors are also not likely to be directly involved. Apparently contradictory, however, was an avidity for SN-peptide in the absence of adhesion to E8, an observation made more curious by the capacity of SN-peptide to inhibit E8-dependent cell spreading.

Interaction with receptor is inhibited by heparin but not chondroitin sulfate or heparinase. Another laminin-1 receptor is a-dystroglycan, for which the interaction with laminin-1 is heparin and EDTA inhibitable but which is thought to be mediated mainly through the E3 domain (11). Cell adhesion to SN-peptide was EDTA inhibitable (19). Is it also heparin inhibitable? HT-1080 cells were plated on SN-peptide in the presence of increasing amounts of soluble heparin revealing a dose-dependent inhibition of adhesion that was maximal at l-10 ug/ml (Fig. 9A). Soluble chondroitin sulfate at similar levels had no effect (Fig. 9A). Cells plated in parallel on E8 displayed no heparin inhibition of adhesion (Fig. 9A). Parallel experiments with Clone 10 melanoma cells (Fig. 9B) revealed a similar pattern of heparin (1 ug/ml) but not chondroitin sulfate inhibition of adhesion to SN-peptide and little or no effect on adhesion to E8 (not shown). Differing somewhat were

M2 melanoma cells, for which the adhesion to SN- peptide was less heparin sensitive and somewhat chondroitin sulfate sensitive (Fig. 9B). To ask whether this response was attributable to a cell-surface heparan sulfate proteoglycan, for which a direct interaction of its glycosaminoglycan side chains with SN-peptide might be expected, we examined heparin-SN-peptide binding and whether heparinase pretreatment altered adhesion. Little or no heparin bound SN-peptide (Fig. lo), in contrast to laminin-1 that has a number of heparin binding sites (32). Pretreatment of HT-1080 cells with heparinase I, II, and III, at a level lo-fold higher than that which eliminated syndecan-l-dependent cell adhesion to the heparin binding domain of thrombospondin (17), however, had no effect on cell adhesion to SN-peptide (mean optical density 595 = 0.93 IT 0.05 with 1 U/ml heparinase I-III, n = 27).

1.2

0.8

B -L - 1.2

0.8 - 0.8

0.4

0.0

P388Dl K562 Fig. 8. Cells lacking pi- or +ntegrin subunits adhere well to SN-peptide. A: Pi-gene knockout TKO and GD25 cells display moderately good levels of attachment to SN-peptide. Slight (TKO) or robust (GD25) adhesion to laminin-1 (Ln-1), but not E8 fragment, is also apparent. Inset: dose-dependent adhesion of TKO (TK), GD25 (GD), P388Di (P3), and K562 (K6) cells to SN-peptide. P388Di (B) and K-562 (C) cells, both of which express little or no a@UbUnit, adhere well to SN-peptide but show no adhesion to laminin-1 or E8. Coating solutions were BSA(40 pg/ml), laminin-1(0.05-0.1 PM), E8 (0.1 PM), and SN-peptide (25-50 PM). Values represent a mean of 6 (inset) or 7 (histograms) independent measurements.

L500

Fig. 9. Cell adhesion to SN-peptide is heparin inhibitable. A: soluble heparin, but not chondroitin sulfate, inhibits HT-1080 cell adhesion to SN-peptide in a dose-dependent manner. Adhesion to E8 is not affected. B: adhesion of Clone 10 melanoma cells to SN-peptide is similarly inhibited by soluble heparin (hep) but not chondroitin sulfate (CS; each at 1 ug/ml), unlike M2 melanoma cells for which the adhesion to SN-peptide is less inhibited by heparin and displays partial inhibition by chondroitin sulfate (each at 1 ug/ml). Values in A represent a mean of 3 (E8) to 6 (SN-peptide) independent measurements; values in B each represent a mean of 3 (chondroitin sulfate) to 21 (heparin) independent measurements. BSA background has been subtracted.

20 HT1080

L

ol- * **- = *.-’ a =mn’ua m “d .OOl .oi .I 1 IO 100 4!

Heparin inhibitor (pg/ml) 2 0 0

Identification of potential receptor(s) by SN-peptide afinity chromatography. We coupled SN-peptide to SulfoLink beads through an added COOH-terminal cysteine and asked what cell-surface proteins bind specifically to SN-peptide in a heparin and/or EDTA elutable fashion (Fig. 11). For this purpose, SN-peptide columns were incubated with biotinylated HT-1080 cell surface extracts in the presence of Mn2+, octylglucoside, proteolytic inhibitors, and loo-150 mM NaCl, and then washed extensively. Subsequent addition of 1 ug/ml heparin in octylglucoside column buffer eluted a prominent -155kDa band and a lighter -180-kDa band (Fig. 11B). A parallel BSA column was blank (Fig.

50 c T -I .

‘x’ a 40

c

E

L 7 30 -

s m 20- 19 21 P

23 L

g IO-

0-w a 2 Coat

Heparin-Gold Free Gold Fig. 10. SN-peptide shows little or no affinity for heparin in contrast to laminin-1 (Ln). Heparin-gold was incubated with BSA-, laminin- l-, or SN-peptide-coated wells, and binding was assessed from the maximal velocity ( Vmax > of color development after addition of silver enhancement solution. In controls, gold in the absence of heparin was added followed by silver enhancement. Inset: example mOD vs. time plot. Steepest slope through 5-7 data points was used to calculate V max. Each value in main figure represents the mean of 9 independent measurements.

- 80 .

- 60 .

- 40

- 20

-0

1lA). A similar pattern plus an -91-kDa band was apparent when SN-peptide columns were eluted with heparin (1 ug/ml) in the presence of 10 mM EDTA (Fig. 1lC) or with EDTA alone (Fig. llD>. Immunoprecipita- tion of the heparin eluate with anti-pi-antibody (Fig. 11E) yielded little or no receptor, in contrast to the abundant amount of P1-integrin in the unbound fraction (Fig. 11E).

DISCUSSION

The goal of the present study was to identify the receptor for amino acids 2179-2198 (SN-peptide) of the laminin al-chain, an evolutionarily conserved site required for lung alveolar formation in vitro (19). Cell adhesion studies pointed to a heparin-sensitive and PI-, p4-, or avP5-integrin-independent interaction involved in laminin- l-dependent cell spreading, which appears to be mediated mainly by cell-surface proteins of -155, -180, and -91 kDa.

Various reagents were used to assess the specificity of SN-peptide-cell interactions. Antibodies against SN- peptide inhibited cell adhesion to SN-peptide and to the 250-kDa E8 fragment that contains the SN-peptide site but not to the PI fragment derived from the cross- region of laminin-1. Soluble SN-peptide suppressed spreading by cells adherent to laminin-1 and E8 but did not affect cell spreading on the Pl-like fragment El’ or on collagen I. Some, but not all, cell lines tested adhered to SN-peptide. Finally, SN-peptide columns in physiological salt selectively purified three cell-surface proteins among hundreds in the starting sample.

These results are in keeping with known interactions between cells and E8 subfragments (8,28,32) that had previously suggested the presence of a cell adhesion site(s) within the first three G domain loops (8). Thus E8 subfragment T8’-A, consisting of the first three loops, has cell adhesion activity, and antibodies against it (anti-rG70) inhibit HT-1080 adhesion to fragment E8 (32), a result more precisely defined by ab[SN] in the present study. A 113 overlapping synthetic peptide screen for G domain cell adhesion activity recently

kDa

205- 121- 88-

205-

121- 88-

200- 118- 88-

205- 121-

50- 33-

Fig. 11. Heparin , and EDTA elution of the putative SN-peptide cell

surrace receptor(s) from SN-peptide columns. HT-1080 cells were surface labeled with biotin, solubilized in octylglucoside, and then incubated with BSA (A) or SN-peptide (B-D) columns in octylglucoside buffer containing 100 (A-C) or 150 (D) mM NaCl. Unbound material is represented by the flow through (FT). Last 2 column volumes of a 20-column volume wash is shown in Wsh. Elution (Elu) was with column buffer containing 1 pg/ml heparin (A and B), 20 mM EDTA (D), or heparin (1 pg/ml) plus 10 mM EDTA (Cl. NaCl is a 1 M NaCl column wash. A: little or no material is henarin elutable from

FT Wsh ElU Nat3

. (

123


F

confirmed the SN-peptide site (designated AG-10) and revealed it to be the only linear adhesive site in the first loop (22). Two and one other sites were reported in the second and third loops, respectively (22). Differing was the proposed minimum active sequence of SIYITRF rather than SINNNR, a discrepancy possibly reflecting difficulties in adsorption by synthetic peptides of this short size, for which application of longer peptides, such as SN-peptide, is recommended. Taken together, it is apparent that SN-peptide is a specific and highly reproducible tool that offers a simple but powerful new approach to analysis of the laminin-cell interface.

The capacity of EDTA to inhibit HT-1080 adhesion to SN-peptide (19) and evidence pointing to o&i as the main laminin-1 integrin of HT-1080 cells (32) raised the possibility that c&i may serve as receptor for SN- peptide or AG-10 (21, 22). Preliminary studies (7) supported this hypothesis. Commercial GoH3 anti-as- antibody displayed a dramatic inhibitory response over a very narrow concentration range of 70-100 pg/ml, similar to the GoH3 concentration previously employed to inhibit kidney morphogenesis in vitro (9) but greater than the 0.5 pg/ml required to inhibit platelet adhesion to laminin-1 (26). The unreliability of this atypical dose-inhibition curve was later emphasized by observation of an identical inhibitory result with P388Di cells, for which the lack of cell surface (Yg had been verified by Fats analysis (24). Similar results at lo-fold higher antibody concentrations were obtained for the anti-pi MAb P4ClO. We subsequently chose the highest anti-o6 (10 pg/ml)- and anti-pi (100 pg/ml)-antibody concentrations that had no negative effect on P388Di or TKO (pi minus) cell adhesion to SN-peptide, respectively. Now anti-o6 and -pi inhibited HT-1080 adhesion to fragments E8 or T8 but not to SN-peptide. Finally, the putative receptor(s) eluted from SN-peptide columns with heparin or EDTA was not (Y&i, based on immunoprecipitation and Western blot analysis.

the control BSA column. Nonspecific binding, presumably to column beads, is apparent from the 1 M NaCl wash (also seen in SN-peptide column runs carried out in 100 mM but usually not 150 mM NaCl). B: in contrast, heparin rclcases a doublet consisting of strong -155kDa and weak -180-kDa bands from the SN-peptide column. C: in the nresence of both henarin and EDTA. a third variable -9l-kDa band is apparent. D: latt’er pattern was apparent using EDTA alone. E: anti-pi immunoprecipitation (IP) from B. Lalze 1, no primary antibody control immunoprecipitation of FT. Lane 2, anti-pi immunoprecipitation of FT. Lane 3, anti-pi immunoprecipitation of heparin eluant. Little or no pi-integrin is present in the eluant, in contrast to the flow through (unbound) fraction.

Although linear SN-peptide does not appear to bind (Y&i, could SN-peptide presented in the native confor- mation achieved by intercalation of laminin crl-, pl-, and yl-COOH-termini contribute to the o&i-dependent adhesion complex? Partially suggestive of this possibility is the capacity of SN-peptide to suppress HT-1080 spreading on laminin-1 or E8, the latter of which would be expected to be an o&i-dependent process. Also suggestive is the inhibition of E8 adhesion by anti-SN-peptide antibody. On the other hand, steric hindrance because of proximity of SN-peptide and o&-binding sites is a plausible explanation, in which binding of the SN-peptide site.to a nonintegrin receptor could be viewed as helping to stabilize the moderately weak 06P1-E8 complex. The answer to this question m, 1~ be best determined by analysis of recombinant E8 following deletion or alteration of the SN-peptide site.

Combined heparin and EDTA sensitivity suggested that linear SN-peptide-dependent adhesion may involve a cell surface heparan sulfate proteoglycan or an o-dystroglycan-like molecule. The former possibility was tentatively ruled out by the inability of heparinase I-III predigestion to affect cell adhesion and by SN-


peptide’s apparent lack of affinity for heparin. cx-Dystro- glycan has a molecular mass (156 kDa) similar to the - 155kDa cell-surface protein eluted from SN-peptide columns with heparin or EDTA. A sharp band, however, is uncharacteristic of ar-dystroglycan, which is noted for its heavy level of glycosylation (5). Moreover, interaction of a-dystroglycan with laminin- 1 is thought to involve the E3 domain, comprising the last two G domain loops, although some interaction appears to be possible (11).

In summary, SN-peptide supports the adhesion of a number of different cell lines and suppresses HT-1080 cell spreading on laminin-1 in a specific manner. Adhe- sion to SN-peptide is heparin and EDTA sensitive and is mediated by cell-surface proteins of -155, 180, and 91 kDa that bind to SN-peptide under conditions of physiological salt. Whether folding of the SN-peptide site, on intercalation of laminin-1 chains, alters the nature of its binding specificity remains to be determined.

We gratefully acknowledge Jeff Romonow and Drs. Tom Carey, Carolyn Damsky, Reiner Deutzman, Reinhard Faessler, Jay Fox, Arthur Mercurio, Leslie Shaw, Arnoud Sonnenberg, Rupert Timpl, and Peter Yurchenco for antibodies, cells, peptides, and fragments. Helpful suggestions from Drs. Arthur Mercurio, and Carol Otey are greatly appreciated.

Present addresses: L. Chen, Biomolecular Research Facility, Uni- versity of Virginia, Charlottesville, VA 22908; and M. L. Matter, The Burnham Institute Health Sciences Center, La Jolla Cancer Re- search Foundation, La Jolla, CA 92037.

Address for reprint requests: G. W. Laurie, Dept. of Cell Biology, University of Virginia, Box 439, Health Sciences Center, Charlottes- ville, VA 22908.

Received 19August 1996; accepted in final form 15 October 1996.

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Laminin E8 alveolarization site: heparin sensitivity, cell surface