the elastin receptor shows structural and functional ... · 1987) and several tumor cell lines...

THE J O U R N A L OF BIOLOGICAL CHEMISTRY Vol. 264, No. 28, Issue of October 5, pp. 16652-16657,1989 Printed in V.S.A.

The Elastin Receptor Shows Structural and Functional Similarities to the 67-kDa Tumor Cell Laminin Receptor*

(Received for publication, May 15, 1989)

Robert P. MechamSfi11, Aleksander Hinek$, Gail L. Griffin$, Robert M. Senior$, and Lance A. Liottall From the $Respiratory and Critical Care Diuision, Department of Medicine, Jewish Hospital and §Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, Missouri 63110 and the IILaboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

Laminin- and elastin-binding proteins were isolated by ligand affinity chromatography from plasma membranes of fetal bovine auricular chondroblasts and human A2068 melanoma cells. From both cell types, a 67-kDa protein was identified which bound to either elastin or laminin affinity resins. Structural and functional similarities between the elastin and laminin- binding proteins were suggested by 1) cross-reactivity between antibodies directed against the two proteins; 2) elution of the laminin receptor from laminin columns with soluble elastin peptides; and 3) modulation of substrate binding by galactoside sugars. In addition, extraction properties indicate that both receptors are peripheral membrane proteins whose association with the cell surface is mediated by their lectin properties. Mapping of the binding site on laminin suggests that the 67-kDa chondroblast receptor interacts with a hydrophobic elastin-like sequence in domain V of the B1 chain, and chemotaxis studies indicate that cell migration to elastin peptides and laminin involves the same receptor.

Many cells have surface receptors that interact with extra- cellular matrix macromolecules. Several classes of extracel- lular matrix receptors have been identified, with the integrin superfamily being the best characterized (Ruoslahti and Pierschbacher, 1987; Hynes, 1987; Buck and Horwitz, 1987). Integrin receptors share a common structural motif consisting typically of noncovalently linked a- and @-subunits that bind to Arg-Gly-Asp (RGD)-related sequences present in numer- ous proteins.

Considerable progress has also been made in defining receptors for laminin (Liotta et al., 1986; von der Mark and Kuhl, 1985; Martin and Timpl, 1987). By using laminin affinity chromatography, laminin-binding proteins have been isolated from normal cells (Lesot et al., 1983; Timpl et al., 1983; Hall et al., 1988; Huard et al., 1986; Bryant et al., 1987; Yoon et al., 1987) and several tumor cell lines (Malinoff and Wicha, 1983; Rao et al., 1983; Terranova et al., 1983). In addition, micro- organisms such as streptococcus bacteria bind laminin (Lopes et al., 1985; Switalski et al., 1984). Laminin-binding proteins seem to be of two types: proteins of 62-75 kDa which do not

* This work was supported by National Institutes of Health Grants HL-26499 and HL-29594. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll To whom correspondence and reprint requests should be ad- dressed Respiratory/Critical Care Division, The Jewish Hospital of St. Louis, 216 S. Kingshighway Blvd., St. Louis, MO 63110. Tel.: 314- 454-7522.

belong to the integrin superfamily and do not bind fibronectin (Liotta et al., 1986; von der Mark and Kuhl, 1985); and a laminin-binding protein related to the fibronectin receptor (Gehlsen et al., 1988; Douville et al., 1988; Tomaselli et al., 1988; Sonnenberg et al., 1988). These two types of receptors bind at different domains on the laminin molecule, with the 62-75-kDa receptor interacting with a site on the B1 chain and the integrin receptor binding to the globular end of the long arm.

We have recently characterized a 67-kDa cell surface elastin-binding protein that interacts with a hydrophobic hexapeptide sequence in elastin, Val-Gly-Val-Ala-Pro-Gly (VGV- APG), shown to be a cell recognition domain (Senior et al., 1984; Wrenn et al., 1986, 1988; Hinek et al., 1988; Mecham et al., 1989). This 67-kDa peripheral membrane protein also demonstrates lectin properties with specificity for galactoside sugars. The bifunctional nature of the receptor may have important physiological consequences since binding of carbohydrate lowers the affinity for elastin and releases the receptor from the cell surface. These sugar-binding properties have been useful for extracting the 67-kDa elastin-binding protein from tissues and cells and for releasing it from elastin affinity columns (Hinek et al., 1988; Barondes, 1988; Mecham et al., 1989).

During the course of our studies, several observations suggested that the 67-kDa elastin-binding protein resembled the 67-kDa laminin receptor. In this report, we show that the two proteins are functionally and immunologically similar and that both interact with a hydrophobic elastin-like sequence in domain V of the B1 chain of laminin.

EXPERIMENTAL PROCEDURES

Materials-Antibody to 14-kDa rat lung lectin was a gift from Dr. Samuel Barondes, University of California San Francisco; and platelet-derived growth factor (PDGF)’ was provided by Dr. T. F. Deuel, Jewish Hospital, St. Louis, MO. Affi-Gel 10 was purchased from Bio- Rad. Colloidal gold and silver enhancement kits were purchased from Janssen Life Science Products (Piscataway, NJ), and species- and type-specific second antibodies conjugated with peroxidase were from Boehringer Mannheim. Antibodies to bovine serum albumin were from Miles Scientific, Naperville, IL. Human neutrophil elastase, antibody to neutrophil elastase, and bovine elastin were purchased from Elastin Products Company (Pacific, MO). Peptides composed of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), Leu-Gly-Thr-Ile-Pro-Gly (LGTIPG), Val-Ser-Leu-Ser-Pro-Gly (VSLSPG), Cys-Asp-Pro-Gly- Tyr-Ile-Gly-Ser-Arg (CDPGYIGSR), Tyr-Ile-Gly-Ser-Arg (YIGSR), and Tyr-Ile-Gly-Ser-Gln (YIGSQ) were synthesized using a Biosearch model 9600 peptide synthesizer and purified using reverse phase

The abbreviations used are: PDGF, platelet-derived growth factor;

droxyethy1)-1-piperazineethanesulfonic acid; OBG, octyl p-glucoside; HPLC, high performance liquid chromatography; Hepes, 4-(2-hy-

EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; SDS, SO- dium dodecyl sulfate; FCL, fetal calf ligament fibroblasts.

16652

67-kDa Elastin and Laminin Receptors 16653

HPLC. The composition of each peptide was confirmed by amino acid analysis. All other chemicals were from Sigma.

Preparation of Laminin and Elastin Peptides-Laminin was purified from Engelbreth Holm-Swarm tumor as described by Kleinman et al. (1982). Elastin peptides were prepared using the following modification of the procedure of Senior et al. (1980). Human leukocyte elastase was added to a suspension of bovine ligamentum nuchae- insoluble elastin (1 part elastase to 100 parts elastin, w/w) in phosphate-buffered saline, pH 8.0, and allowed to incubate at 37 "C overnight. The reaction mixture was passed over a Trasylol column to remove active enzyme and then absorbed with immobilized antibody to human neutrophil elastase. The elastin peptide mixture was free of elastase as assessed by the tritium release assay (Banda et al., 1981) and by immunoblots developed using anti-elastase immuno- globulin.

Cell Isolation and Culture-Fibroblasts from ligamentum nuchae were grown from explants of fetal bovine tissue (Mecham et al., 1981). Chondroblasts from ear cartilage of 120-180-day bovine fetuses were isolated following collagenase digestion of the cleaned tissue (Me- cham, 1987). All experiments were performed on first or second passage cells. A2058 melanoma cells as well as the chondroblasts and fibroblasts were maintained in Dulbecco's modified Eagle's medium containing high glucose and high bicarbonate and supplemented with antibiotics, nonessential amino acids, and 10% (v/v) bovine calf serum.

Chemotaxis to Elastin Peptides-Ligamentum nuchae fibroblasts (first passage) and A2058 cells were assayed for directed migration to elastin-derived and synthetic peptides using a modified Boyden chamber chemotaxis assay (Senior et al., 1980, 1982, 1984). The effect of lactose on cell movement was determined by incubating cells overnight in normal growth medium supplemented with 20 mM lactose and by including lactose in both top and bottom compartments of the chamber during the chemotaxis assay (Mecham et al., 1989). Chemo- taxis to PDGF served as the positive control.

Affinity Chromatography and Elastin Receptor Isolation-Elastin peptides and laminin (20 and 1 mg/ml of resin, respectively) were dissolved in 0.1 M sodium bicarbonate buffer, pH 8, and reacted overnight in a polypropylene tube with Affi-Gel 10. Active ester sites on the resin were blocked by a 1-h incubation with 0.1 M ethanolamine (pH 8). Affi-Gel incubated with ethanolamine served as the control resin to evaluate nonspecific binding.

Plasma membranes, prepared from cultured cells as described previously (Wrenn et al., 1988), were extracted with membrane solu- bilization buffer (3 M guanidine HCI, 10 mM Hepes, pH 8, 0.1 M dithiothreitol, 0.5% (w/v) octyl 8-glucoside (OBG), and proteinase inhibitors).* Extraction was a t 4 "C for 5 h with constant stirring. Insoluble material was pelleted by centrifugation in a microcentrifuge for 3 min a t 10,000 X g. The supernatant was dialyzed a t 4 "C against 0.1 M sodium bicarbonate, pH 8, containing protease inhibitors and mixed with elastin or laminin-containing affinity resin for 2-4 h. The resin and supernatant were transferred to a siliconized glass column, and unbound material was removed by washing the resin with 0.1 M sodium bicarbonate buffer, pH 8, until the A m of the eluent returned to the background level. The columns were then eluted with either: 1) 3 M guanidine HC1 containing 0.5% OBG (guanidine-OBG); 2) 0.5 mg/ml synthetic peptide CDPGYIGSR, YIGSR, YIGSQ, VGVAPG, LGTIPG, and VSLSPG; 3) 100 mM lactose, fucose, or mannose; or 4) 5 mM EGTA or EDTA in 100 mM Hepes buffer, pH 8. Synthetic peptides and carbohydrates were dissolved in 0.1 M sodium bicarbonate buffer, pH 8. Affinity isolation on immobilized asialofetuin was as described previously (Hinek et al., 1988).

Electrophoresis and Immunoblot Analysis-Proteins eluted from the affinity columns were dialyzed against water a t 4 "C and concen- trated by lyophilization. The dried samples were suspended in SDS sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis on 0.45-mm thick 7.5-12% gradient gels (Wrenn et al., 1987). Protein bands were visualized by silver staining. Proteins were transferred from SDS gels to nitrocellulose, and immunoblots were developed with appropriate antibodies according to Wrenn et al. (1986). Antibodies containing immunogold were visualized by amplification using a silver enhancement kit as described by the manufacturer.

* Proteinase inhibitors included 5 mM EDTA, 5 mM benzamidine, 0.1 M 6-aminocaproic acid, and 1 mM N-ethylmaleimide.

RESULTS

To determine whether the elastin receptor binds laminin, detergent extracts of plasma membranes from cultured bovine auricular chondroblasts were applied to elastin or laminin affinity columns, and bound protein eluted with buffers containing either the synthetic peptide VGVAPG, which defines the receptor-binding region in elastin, or with lactose, a carbohydrate that decreases the affinity of the elastin receptor for elastin. Fig. 1 shows that chondroblast membranes contain laminin-binding proteins of 67, 61, and 55 kDA which are similar in size to proteins eluted from the elastin column. NO protein was released from either column with mannose or fucose (not shown), confirming the known specificity of the elastin receptor for galactose-like sugars (Hinek et al., 1988). The presence or absence of calcium did not affect binding to either column, and EGTA-containing buffers were ineffective at releasing bound protein (not shown).

In our previous studies, we noted that the 61- and 55-kDa proteins do not bind to elastin individually but bind as a complex with the 67-kDa protein (Mecham et al., 1989). We have also shown that the 67-kDa component behaves as a peripheral membrane protein that dissociates from the cell surface in the presence of lactose-containing buffers. To determine whether the laminin-binding proteins behave similarly, chondroblast membranes were extracted with bicarbonate buffer containing 100 mM lactose, and the dialyzed extract was absorbed with immobilized elastin. Retained proteins were eluted with lactose, an aliquot taken for analysis on SDS-polyacrylamide gel electrophoresis, and the remaining sample was absorbed with immobilized laminin. The presence of 67-kDa protein in eluents from both columns (Fig. 2, lanes

Elastin Laminin

A B A B

- kD

- 67 - 61 - 55

FIG. 1. Affini ty chromatography of elastin- and laminin- binding proteins. Plasma membranes from auricular chondroblasts were extracted with 3 M guanidine HCI, 0.1 M dithiothreitol, 0.5% (w/v) octyl @-glucoside, 10 mM Hepes, pH 8, and proteinase inhibitors, dialyzed a t 4 "C against 0.1 M sodium bicarbonate, pH 8, and mixed with elastin- or laminin-containing affinity resin for 2-4 h. After washing with 0.1 M sodium bicarbonate buffer to remove unbound protein, retained protein was eluted from each resin using either 100 pg/ml Val-Gly-Val-Ala-Pro-Gly (lanes A ) or 0.1 M lactose (lanes 13) in sodium bicarbonate buffer. Column eluates were electrophoresed on 7.5-12% SDS-polyacrylamide gradient gels and visualized with silver staining.

16654 67-kDa Elastin and

A B C D Laminin Receptors

A B C D Lactose Lactose VGVAPG Lactose L E L E L E L E

~~

kDa 67- 61 - 55 -

43 -

FIG. 2. Elastin and laminin-binding properties contained in 67-kDa protein. Plasma membranes from auricular chondroblasts were extracted for 4 h a t 4 "C with 0.1 M sodium bicarbonate, pH 8.0, containing 0.1 M lactose. Following dialysis against 0.1 M sodium bicarbonate buffer, the extract was chromatographed over an elastin affinity column, and bound protein was eluted with 0.1 M sodium bicarbonate, pH 8.0, containing 0.1 M lactose. An aliquot of the eluate was taken for analysis by SDS-polyacrylamide gel electrophoresis ( l o n e A ) , and the remaining sample was dialyzed to remove lactose and absorbed with immobilized laminin. Protein bound to the laminin ( l o n e B ) was released with lactose and analyzed by SDS-polyacrylamide gel electrophoresis. The 61- and 55-kDa components described in Fig. 1 are evident only if detergent is included in the membrane extraction buffer ( l a n e C). Lane D shows A2058 tumor cell-derived elastin-binding proteins that were released from the elastin column with lactose. All samples were separated on 7.5-1276 SDS-polyacrylamide gradient gels and visualized with silver stain.

A and B) confirms that the elastin-binding protein also binds laminin. The 61- and 55-kDa components described in Fig. 1 were evident only if detergent was included in the membrane extraction buffer (Fig. 2, lane C), which is consistent with their identification as integral membrane proteins (Mecham et al., 1989).

In view of the size similarity between the elastin/laminin- binding protein and laminin receptors described previously, we next determined whether the chondroblast elastin receptor was related immunologically to laminin receptors from tumor cells, and whether the tumor cell laminin receptor could bind elastin. Membrane extracts of A2058 melanoma cells, a cell line shown to be rich in laminin receptor (Wewer et al., 1987) were absorbed with immobilized elastin and bound protein eluted with guanidine-OBG buffer. As with the chondroblast membranes, a 67-kDa protein was the major elastin-binding protein in the membrane of the tumor cell (Fig. 2, lane D).

Immunological similarity between the chondroblast and tumor cell protein was investigated using two antibodies: 1) a monospecific polyclonal antibody (3801) raised against a 20- mer synthetic peptide deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (Wewer et al., 1986) and 2) a monoclonal antibody (BCZe7) to the 67-kDa elastin-binding protein (Mecham et al., 1988). Fig. 3 shows that 3801 and BCZs7 antibodies reacted

FIG. 3. Immunological similarity between laminin- and elastin-binding proteins from chondroblasts and tumor cells. Laminin (lanes L ) - or elastin (lanes E)-binding proteins from detergent extracts of auricular chondroblast or A2058 tumor cell membranes were transferred from SDS gels to nitrocellulose. The blots were then reacted with monoclonal antibody to the 67-kDa elastin receptor (BCZC?) and antibody 3801 prepared against synthetic peptide Prom-Ala deduced from a cDNA sequence corresponding to the COOH-terminal end of the tumor cell laminin receptor.

with the 67-kDa chondroblast protein released from elastin and laminin affinity columns by lactose or VGVAPG (Fig. 3, A-C). Reactivity with BCZ6, was also observed for the 67- kDa laminin and elastin-binding proteins from detergent extracts of A2058 melanoma cells (Fig. 30) .

Elution of the tumor cell receptor from elastin and laminin columns by lactose suggests that the tumor cell laminin- binding protein, like the elastin receptor, has lectin-like properties. In an earlier study, we found that antibodies to the elastin receptor cross-react with a group of small molecular weight galactose-binding lectins and that antibodies raised against these highly purified lectins react with the elastin receptor (Hinek et al., 1988). To determine whether the same was true for the tumor cell laminin receptor, affinity-purified receptor protein was transferred from SDS-polyacrylamide gel electrophoresis to nitrocellulose, and the blots developed with antibody to a 14-kDa galactoside-binding lectin obtained from rat lung (Cerra et al., 1984). Fig. 4 demonstrates that anti-lectin antibody recognizes the 67-kDa laminin- and elastin-binding proteins.

The release of 67-kDa protein from immobilized laminin with reagents that specifically elute the receptor from elastin suggests that this protein interacts with an elastin-like sequence in the laminin molecule. As described in our previous study, the 67-kDa elastin-binding protein recognizes the hydrophobic sequence VGVAPG in elastin (Mecham et al., 1989). The A, B1, and B2 chains of laminin do not contain VGVAGP sequences (Pikkarainen et al., 1988a, 1988b; Sasaki et al., 1988), but two hexapeptide sequences in the B1 chain have hydrophobic properties similar to the VGVAPG sequence in elastin. The first, LGTIPG (amino acids 442-447), is located in domain V. The other, VSLSPG (amino acids 634-639), is in globular domain IV. To test whether either of these sequences defines a binding site for the 67-kDa elastin- binding protein, synthetic peptides were constructed and tested for their ability to compete with elastin or laminin for


67 kDa-

B C

FIG. 4. Immunoreactivity of 67-kDa laminin-binding protein with anti-lectin antibody. Plasma membrane extracts from A2058 tumor cells were passed over a laminin or elastin affinity column and laminin-binding proteins eluted with lactose (lane A ) or VGVAPG (hne B ) and elastin-binding proteins eluted with lactose (lane C ) . Eluted proteins were separated on 7.5-12% SDS-polyacrylamide gels, transferred to nitrocellulose, and the blots developed with antibody to a 14-kDa rat lung galactoside-binding lectin.

A B C

116-

68 -

45 -

FIG. 5. Elastin-like sequences in laminin elute 67-kDa protein from laminin and elastin columns. Membrane extracts of auricular chondroblasts were chromatographed over laminin affinity columns and eluted with 100 pg/ml HPLC-purified synthetic peptides LGTIPG (lane A ) , VGVAPG (lane B ) , or VSLSPG (lane C ) in 0.1 M NaHC03 buffer. Column eluates were electrophoresed on 7.5-12% SDS-polyacrylamide gradient gels and visualized with silver stain.

receptor binding. Fig. 5 shows that LGTIPG, but not VSLSPG, released 67-kDa protein from elastin and laminin columns. No protein was eluted from either affinity resin with VGVAPG following elution with LGTIPG, indicating that LGTIPG is as effective as VGVAPG in releasing the 67-kDa

0 1 .01 .1 1 10 100 1000

Peptide Concentralion (nM)

0 2 4 6 8 1 0 1 2 Elastin Peptides (pglml)

LGTIPC (nM)

0 2 4 6 8 1 0 1 2 Elastin Peptides (pg/rnl)

LGTIPC (nM)

FIG. 6. Chemotaxis of FCL fibroblasts and A2058 tumor cells to elastin and laminin sequences. Panel A shows the chemotactic response of FCL fibroblasts to VGVAPG, LGTIPG, and VSLSPG peptides. Preincubation of FCL cells with lactose (Lac) blocked the chemotactic response to LGTIPG and to elastin peptides (panel E ) . The effects of lactose on cell movement were determined by incubating cells overnight in normal growth medium supplemented with 20 mM lactose and by including lactose in both top and bottom compartments of the chamber during the chemotaxis assay. Lactose had no effect upon chemotactic reactivity to PDGF. Fibroblast migration to PDGF was 68 cells/high power field (H.P.F.) for cells not exposed to lactose and 60 cells/high power field for cells exposed to lactose. Panel C compares chemotaxis of A2058 tumor cells and FCL fibroblasts with elastin peptides. As with FCL cells, chemotaxis of A2058 tumor cells to elastin peptides was blocked by incubation with lactose. All experiments were done at least twice in triplicate with five readings a t each test concentration. Shown are the mean and S.E., n = 15.

protein? In contrast, when synthetic CDPGYIGSR or YIGSR peptides were evaluated for their ability to displace chondroblast membrane receptor protein from elastin and laminin affinity resins, no 67-kDa protein was eluted from elastin with either peptide, whereas a small but variable amount (usually less than 20%) of the total bound protein was eluted from laminin (not shown). Protein that remained on the column following elution with the YIGSR peptide could be eluted with VGVAPG, elastin peptides, or guanidine-OBG buffers. No protein was eluted with the control peptide YIGSQ.

In chemotaxis assays, LGTIPG but not VSLSPG induced a chemotactic response in ligament fibroblast (Fig. 6A) with peak activity occurring at approximately 10 nM, equal to that observed for VGVAPG. The peak response for the LGTIPG peptide (Le. the maximal number of cells that migrated) was similar to the maximal response observed using intact laminin ~ ~ ~~

67-kDa protein from A2058 tumor cells, FCL fibroblasts, and lung extract was also eluted from the elastin resin with LGTIPG peptide (not shown).

16656 67-kDa Elastin and Laminin Receptors

(not shown). Of particular interest, fibroblast chemotaxis to LGTIPG was inhibited when ceIls were preincubated with lactose, although there was a full response to PDGF (Fig. 6B). A similar effect of lactose has been observed in studies of chemotaxis to elastin peptides and to VGVAPG (Mecham et al., 1989). LGTIPG and elastin peptides were also chemo- attractants for A2058 cells (Fig. 6C) and movement of the tumor cell to these peptides, like the ligament fibroblast, was blocked by lactose.

DISCUSSION

In this study, we identified and compared laminin- and elastin-binding proteins associated with the plasma membane of bovine ear cartilage chondroblasts and A2058 melanoma cells. These cell types provide an interesting comparison in that cultured auricular chondroblasts produce elastin but not laminin and express a 67-kDa elastin receptor (Mecham et al., 1989), whereas A2058 tumor cells synthesize laminin but not elastin and express a 67-kDa laminin receptor (Wewer et al., 1987). When the binding specificity of each receptor was compared, the elastin receptor was found to bind to laminin, and the laminin receptor could bind elastin. Evidence for structural similarity between the two receptors was obtained with antibodies directed against both proteins. On immunoblots, antibody to the elastin receptor reacted with the laminin-binding protein, and antibodies to the laminin receptor recognized the elastin-binding protein.

The 67-kDa protein from chondroblasts can be eluted from laminill a d elastin columns with lactose, suggesting that it has lectin-like properties. This finding is in agreement with our previous observation that the elastin receptor is a galactoside-binding protein that has immunological similarity with a family of small molecular weight galactoside lectins (Hinek et al., 1988; Cerra et al., 1984; Barondes, 1988). Results pre- sented in this current study extend this similarity to the 67- kDa laminin-binding protein since the 67-kDa component from both chondroblasts and A2058 tumor cells reacts with antibody to a 14-kDa rat lung lectin. Similarly, the 14-kDa lectin is recognized by antibodies to the 67-kDa tumor cell protein (data not shown). The nature of this cross-reactivity is not yet understood, but it is likely that a conserved galactose-binding domain provides a common epitope on the lectin and 67-kDa protein.

Earlier studies have demonstrated that the lectin properties of the 67-kDa protein greatly influence its binding character- istics (Hinek et al., 1988). Other receptor proteins have been shown to contain binding sites for both protein and carbohydrate substrates. Binding of mannose 6-phosphate to the insulin-like growth factor II/mannose 6-phosphate receptor, for example, has been shown to act synergistically to increase receptor affinity for insulin-like growth factor I1 (MacDonald et al., 1988). In addition, cloning studies of the endothelial leukocyte adhesion molecule 1 (Bevilacqua et al., 1989) and the mouse lymph node homing receptor (Siegelman et al., 1989) have identified a mosaic architecture that includes a lectin-like domain and domains that are homologous to epi- dermal growth factor and complement regulatory proteins. Several other examples of proteins with a known carbohydrate-binding site and with an established or inferred function mediated by another domain can be found in Barondes (1988).

The 67-kDa elastin/laminin-binding protein identified in our study has properties of a peripheral and not an integral membrane protein. It can be extracted from the cell surface with lactose-containing buffers in the absence of detergent and, as such, is the only laminin- or elastin-binding protein released from the cell membrane under these conditions.

When detergent is used to extract the plasma membrane, two other proteins of 61 and 55 kDa are detected by laminin affinity chromatography. These proteins have molecular weights similar to elastin-binding proteins in extracts of the same membranes. We have shown previously that only the 67-kDa protein binds to elastin and that the 61- and 55-kDa components aggregate with the 67-kDa protein as a complex. Our findings in the present study are similar for laminin: the 61- and 55-kDa proteins did not bind laminin in the absence of the 67-kDa component. Although the nature of the 61- and 55-kDa proteins is unclear, our findings to date (Mecham et al., 1989) suggest that they are integral membrane proteins and not degradation products of the 67-kDa protein. It should be noted that the 61-kDa protein has not been detected in all cell types.

It is noteworthy that LGTIPG and VGVAPG both induced a peak chemotactic response at 10 nM. The activity of these peptides is surprisingly high for synthetic ligands lacking the extensive structural features of an intact protein. The peptides are extremely hydrophobic, however, and it is likely that the number of spatial conformations the peptide can assume in aqueous solvents is restricted to the extent that the pre- ferred conformation in solution is similar to that adopted in the intact protein. Interestingly, we have shown that VGVAPG is a major antigenic epitope on mature elastin, suggesting that despite its hydrophobic nature, it is located on the surface of the elastin polymer where interaction with the aqueous environment is likely (Wrenn et al., 1986). Al- though the secondary structure of laminin around the LGTIPG sequence has not been determined experimentally, formation of interchain disulfide bonds between cysteine residues in this region of the molecule (Sasaki et al., 1988) could form a loop structure with LGTIPG exposed at the end of the loop. Elastin has no cysteine residues outside of a small region near the carboxyl terminus, but the VGVAPG cell-binding domain is flanked on each side by lysine residues that form covalent cross-links after oxidation by lysyl oxidase. One might speculate that these cross-links position and restrict the VGVAPG sequence in a fashion similar to disulfide bonds in laminin.

Recognition of both LGTIPG and VGVAPG sequences by the elastin- and laminin-binding protein raises the question of epitopic specificity. Does this protein bind all hydrophobic sequences equally well, or does recognition involve a confor- mational determinant that tolerates several different amino acids as long as the overall hydrophobic nature of the peptide is maintained and limited spatial requirements are met? In support of specificity was our finding that VSLSPG, a VGVAPG-like sequence from domain IV of laminin, did not elute 67-kDa protein from elastin or laminin affinity columns and had no biological activity in chemotaxis assays. Also, Long et al. (1988) have described two nonapeptide sequences in elastin (AGVPGFGVG and GFGVGAGVP) which compete with VGVAPG in chemotaxis assays, suggesting that all three peptides bind to the same receptor. Importantly, several other hydrophobic peptides (AGVP, VPGVG, VPGFG, GVPVP) were not active.

The identification of LGTIPG as a possible receptor-binding sequence in laminin is in contrast to results of Graf et al. (1987a, 1987b), who reported that peptides containing the sequence CDPGYIGSR (located in cystein-rich sequences in domain I11 of the B1 chain) and the shorter pentapeptide, YIGSR, are active in cell attachment assays and in blocking chemotaxis to laminin. In our studies, neither peptide was particularly active at eluting 67-kDa receptor from elastin. It is not surprising that YIGSR-containing peptides are inactive


toward the elastin receptor since no corresponding sequence exists in bovine elastin. Failure of YIGSR to release the receptor completely from laminin suggests that the 67-kDa elastin/laminin-binding protein from chondrobiasts has a different binding specificity than the YIGSR laminin receptor on HTlO8O and Chinese hamster ovary cells (Graf et al., 1987a, 1987b).

In summary, our results show that the 67-kDa elastin receptor is functionally and immunologically similar to the laminin receptor of sim.ilar molecular weight. Both receptors have properties of peripheral membrane proteins, and their association with the cell surface as well as their affinity for elastin or laminin are mediated by their lectin properties. It is interesting to note that Hall et al. (1988) have isolated a 66-kDa laminin-binding protein from muscle that, like the 67-kDa protein described in this study, has properties of a peripheral membrane protein. Moreover, we have recently identified a 67-kDa type IV collagen-binding protein in neu- trophils which has an amino acid composition resembling the elastin receptor and binds to both elastin and laminin (Senior et al., 1989). Further c:haracterization of the 67-kDa protein will be necessary to determine whether this diversity in binding is explained by a family of receptors or by a single receptor that interacts with multiple ligands.

Acknowledgments-We thank Loren Whitehouse, Robert W. Henry, Lisa Mecham, Gertrude Crump, and Noreen Person for tech- nical assistance and Terese Hall for excellent secretarial help. We also thank Dr. Mark Sobel for constructive comments and helpful discussion.

REFERENCES Banda, M. J., Dovey, H. F., and Werb, Z. (1981) in Methods for

Studying Mononuclear Phagocytes (Adams, D. O., Edelson, P. J., and Koren, H., eds) pp. 603-618, Academic Press, New York

Barondes, S. H. (1988) Trends Biochem. Sci. 13, 480-482 Bevilacqua, M. P., Stengdin, S., Gimbrone, M. A., and Seed, B., Jr.

(1989) Science 2 4 3 , 1180-1165 Bryant, G., Rao, C. N., Brentani, M., Martins, W., Lopes, J. D.,

Martin, S. E., Liotta, L. A., and Schiffmann, E. (1987) J. Leukocyte Biol. 41,220-227

Buck, C. A., and Horwitz, A. F. (1987) Annu. Rev. Cell Biol. 3 , 179- 205

Cerra, R. F., Haywood-Reid, P. L., and Barondes, S. H. (1984) J . Cell Biol. 98,1580-1589

Douville, P. J., Harvey, W. J., and Carbonetto, S. (1988) J. Biol. Chem. 263,14964-14969

Gehlsen, K. R., Dillner, L., Engvall, E., and Ruoslahti, E. (1988) Science 241 , 1228-1229

Graf, J., Iwamoto, Y., Sasaki, M., Martin, G. R., Kleinman, H. K., Robey, F. A., and Yamada, Y. (1987a) Cell 48,989-996

Graf, J., Ogle, R. C., Robey, F. A., Sasaki, M., Martin, G. R., Yamada, Y., and Kleinman, H. IC. (1987b) Biochemistry 26 , 6896-6900

Hall, D. E., Frazer, K. A., Hann, B. C., and Reichardt, L. F. (1988) J. Cell Bwl. 107, 687-697

Hinek, A., Wrenn, D. S., Mecham, R. P., and Barondes, S. H. (1988) Science 239,1539-1541

Huard, T. K., Malinoff, H.. F., and Wicha, M. S. (1986) Am. J. Pathol. 123,365-370

Hynes, R. 0. (1987) Cell 48,549-554 Kleinman, H. K., McGa.rvey, M. L., Liotta, L. A., Robey, P. G.,

Tryggvason, E., and Martin, G. R. (1982) Biochemistry 21, 6188- 6193

Lesot, H., Kuhl, U., and von der Mark, K. (1983) EMBO J. 2 , 861-

Liotta, L. A., Rao, C. N., and Wewer, U. M. (1986) Annu. Reu.

Long, M. M., King, V. J., Prasad, K. U., and Urry, D. W. (1988)

Lopes, J . D., DosReis, M., and Brentani, R. R. (1985) Science 2 2 9 ,

MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988) Science 239, 1134-1137

Malinoff, H. L., and Wicha, M. S. (1983) J. Cell Biol. 96,1475-1479 Martin, G. R., and Timpl, R. (1987) Annu. Reu. Cell Biol. 3, 57-85 Mecham, R. P. (1987) Methods Enzymol. 144, 232-246 Mecham, R. P., Lange, G., Madaras, J . G., and Starcher, B. C. (1981)

J. Cell Biol. 90, 332-338 Mecham, R. P., Hinek, A,, Cleary, E. G., Kucich, U., Lee, S. J., and

Rosenbloom, J . (1988) Biochem. Biophys. Res. Commun. 151,822- 826

Mecham, R. P., Hinek, A., Entwistle, R., Wrenn, D. S., Griffin, G. L., and Senior, R. M. (1989) Biochemistry 28, 3716-3722

Pikkarainen, T., Eddy, R., Fukushima, Y., Byers, M., Shows, T., Pihlajaniemi, T., Saraste, M., and Tryggvason, K. (1988a) J. Biol.

Pikkarainen, T., Kallunki, T., and Tryggvason, K. (1988b) J. Biol.

Rao, N. C., Barsky, S. H., Terranova, V. P., and Liotta, L. A. (1983)

Ruoslahti, E., and Pierschbacher, M. D. (1987) Science 238,491-497 Sasaki, M., Kleinman, H. K., Huber, H., Deutzmann, R., and Yamada,

Senior, R. M., Griffin, G. L., and Mecham, R. P. (1980) J. Clin. Znuest. 66,859-862

Senior, R. M., Griffin, G. L., and Mecham, R. P. (1982) J. Clin. Znuest. 70,614-618

Senior, R. M., Griffin, G. L., Mecham, R. P., Wrenn, D. S., Prasad, K. U., and Urry, D. W. (1984) J. Cell Biol. 99,870-874

Senior, R. M., Hinek, A., Griffin, G. L., Pipoly, D. J., Crouch, E. C., and Mecham, R. P. (1989) Am. J. Resp. Cell Molec. Biol., in press

Siegelman, M. H., van de Rijn, M., and Weissman, I. L. (1989) Science 243,1165-1170

Sonnenberg, A., Modderman, P. W., and Hogervorst, F. (1988) Nature

Switalski, L. M., Speziale, P., Hook, M., Wadstrom, R., and Timpl, R. (1984) J. Biol. Chem. 259,3732-3738

Terranova, V. P., Rao, C. N., Margulies, I. M., and Liotta, L. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,444-448

Timpl, R., Johansson, S., van Delden, V., Oberbaumer, I., and Hook, M. (1983) J. Biol. Chem. 258,8922-8927

Tomaselli, K. J., Damsky, C. H., and Reichardt, L. F. (1988) J. Cell Biol. 107 , 1241-1252

von der Mark, K., and Kuhl, U. (1985) Biochim. Biophys. Acta 8 2 3 , 147-160

Wewer, U. M., Liotta, L. A., Jaye, M., Ricca, G. A., Drohan, W. N., Claysmith, A. P., Rao, C. N., Wirth, P., Coligan, J. E., Albrechtsen, R., Mudry, M., and Sobel, M. E. (1986) Proc. Natl. Acad. Sci. U. S.

Wewer, U. M., Taraboletti, G., Sobel, M. E., Albrechtsen, R., and Liotta, L. A. (1987) Cancer Res. 47, 5691-5698

Wrenn, D. S., Griffin, G. L., Senior, R. M., and Mecham, R. P. (1986) Biochemistry 25, 5172-5176

Wrenn, D. S., Parks, W. C., Whitehouse, L. A., Crouch, E. C., Kucich, U., Rosenbloom, J., and Mecham, R. P. (1987) J. Biol. Chem. 262,

Wrenn, D. S., Hinek, A., and Mecham, R. P. (1988) J. Biol. Chem.

Yoon, P. S., Boxer, L. A., Mayo, L. A., Yang, A. Y., and Wicha, M.

865

Biochem. 5 5 , 300-311

Biochim. Biophys. Acta 9 6 8 , 300-311

275-277

Chem. 262,10454-10462

Chem. 263,6751-6758

Biochem. Biophys. Res. Commun. 1 1 1,804-808

Y. (1988) J. Biol. Chem. 263,16536-16544

336,487-489

A. 83,7137-7141

2244-2249

263,2280-2284

s. (1987) J. Zmmunol. 138, 259-265

the elastin receptor shows structural and functional ... · 1987) and several tumor cell lines...

Documents