Heparan Sulfate Proteoglycans from Mouse Mammary Epithelial Cells CELL SURFACE PROTEOGLYCAN AS A RECEPTOR FOR INTERSTITIAL COLLAGENS*

A heparan sulfate-rich proteoglycan is on the surface of NMuMG mouse mammary epithelial cells apparently intercalated into their plasma membranes. Mild treat- ment of the cells with trypsin releases the GAG-bearing region (ectodomain) of this molecule as a discrete pro- teoglycan which is readily purified. At physiological pH and ionic strength, the ectodomain binds collagen types I, 111, and V but not types 11, IV, or denatured type I. The proteoglycan binds to a single class of high affinity saturable sites on type I collagen fibrils, sites which are selective for heparin-like glycosaminogly- cans. The binding of NMuMG cells to type I collagen duplicates that of their cell surface proteoglycan; cells bind to native but not denatured collagen, and binding is inhibited by heparin but not by other glycosaminoglycans. These binding properties suggest that cell sur- face heparan sulfate proteoglycans could act as receptors for interstitial collagens and mediate changes in cell behavior induced by collagenous matrices. ~ mammary I resem-bling hormonal

Culture of mammary epithelial cells on or in gels of type I collagen fibrils markedly alters their behavior. For example, when cultured within a gel, the cells proliferate, form a basal lamina and apical microvilli, organize into structures resembling branching mammary ducts, and will show a lactogenic response to hormonal stimuli (Yang et al., 1979;Bennett, 1980;Haeuptle et al., 1983). The nature of the interaction which mediates the effect of the collagen on the cells is unknown. By analogy with other exogenous effectors, the interaction likely involves molecules acting as cell surface receptors for the collagen. Although receptors for interstitial collagens have been described for platelets (Chiang and Kang, 1982) and chondrocytes (Mollenhauer and von der Mark, 1983) and have been proposed for fibroblasts (Goldberg, 1979), there is little analogous information for epithelial cells.
Mouse mammary epithelial cells have a heparan sulfaterich proteoglycan (PG') on their cell surfaces. Because a soluble extracellular heparan sulfate PG from these cells binds type I collagen with high affinity (Koda and Bernfield, 1984), we hypothesized that the cell surface PG would have similar properties. This cell surface PG appears to be an integral membrane protein (Rapraeger and Bernfield, 1985), but its * This work was supported by National Institutes of Health Grant CA 06763, New Investigator Research Award HD17146, and Fellowship DE 05273, and by a fellowship from the Cystic Fibrosis Foundation. 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.
The abbreviations used are: PG, proteoglycan; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PVC, polyvinyl chloride; PBS, phosphate-buffered saline. ectodomain' can be released as a discrete soluble PG. Therefore, we examined the collagen-binding properties of the ectodomain and compared these with the collagen-binding ability of intact cells. At physiological salt concentrations, the ectodomain binds collagen types I, 111, and V, but not types I1 or IV and hinds to a single class of high affinity saturable sites on type I collagen fibrils. The ectodomain and intact cells bind to native but not denatured collagens, and this binding is inhibited by heparin-like but not other glycosaminoglycans. These characteristics suggest that the cell surface PG may bind collagen fibrils and act as a receptor to mediate the effect of the collagenous matrices on the epithelial cells.

Binding of the Ectodomain to Type I Collagen3
Affinity of the Ectodomain for Other Collagen Types-Binding of 35S-PG to collagen immobilized on polyvinyl chloride wells was used to compare binding to different collagen types as well as to native and heat-denatured type I collagen. Native collagen types I, 11, 111, IV, and V (including two different preparations each of types IV and V), the 7 S domain of type IV collagen, and heat-denatured type I were immobilized on separate microtiter wells. After washing, the amount of each collagen type immobilized was assayed (Bailey, 1962) and was found to range from 1 to 10 pglwell. To test for binding, 35S-PG in PBS containing 1% BSA was added to collagen-treated wells, and the reactions were incubated for varying periods at room temperature. Binding to types I and 111 collagen was initially rapid and then slowed after 30 min of incubation while binding to type V progressed linearly during the 90-min incubation (Fig. 1). In contrast to these interstitial collagens, no appreciable binding was observed with types I1 and IV collagen or with heat-denatured type I collagen under these conditions (Fig. 1). These differences were not due to variation in the amount of collagen on the well. Therefore, differences Ectodomain is the GAG-bearing region of the cell surface proteoglycan released by mild treatment with trypsin (Rapraeger and Bernfield, 1985).
Portions of this paper (including "Materials and Methods," part of "Results," and in the ability of the PG to bind must be due to differences in the structure of the collagens. The quaternary structure of the collagens immobilized on the well is not known. However, it is clear that a native, possibly fibrillar, structure is required since the PG binds to native but not to denatured type I collagen. Binding may not occur to type IV collagen because only the 7 S domain of this molecule resembles a fibril, and this structure, which is maintained by covalent cross-links, is probably destroyed when the type IV collagen is extracted under reducing conditions, as was done here. T o determine if the native cross-linked 7 S region of type IV collagen interacts with the proteoglycan, the isolated unreduced domain was immobilized on wells. However, no binding is detected (Fig. l). Therefore, under these conditions, the PC binds to interstitial collagen types I, 111, and V, but not to type I1 collagen, type IV collagen, or to the 7 S aggregate derived from type IV.
Binding of Cells to Collugen-The affinity of the ectodomain for interstitial collagens suggests that the cell surface P C could function as a collagen receptor. Therefore, we examined the ability of intact cells to duplicate the selectivity of the PG binding to type I collagen. Microtiter wells coated with BSA, native, or denatured type I collagen were incubated with NMuMG cells suspended by scraping after EDTA treatment. The binding of the cells exhibited the same specificity as the binding of the ectodomain. The cells bound to native but not to denatured collagen or BSA, and the binding was unaltered by including 100 pg/ml chondroitin 6-sulfate, chondroitin 4sulfate, dermatan sulfate, or hyaluronic acid in the incubation mixture (Fig. 2). In contrast, binding was prevented by including 5-10 pg/ml heparin or 100 pg/ml dextran sulfate (M, 500,000), polyanions which interfere with the binding of cell surface PG to collagen (Fig. 2). The inhibition of binding by heparin is due to blocking of binding sites on the collagen because identical results are obtained when the wells are pretreated with heparin and the assay is run in the absence of heparin (not shown).

DISCUSSION
A heparan sulfate-rich proteoglycan is on the surface of NMuMG mouse mammary epithelial cells and behaves as an integral membrane protein (Rapraeger andBernfield, 1983, 1985). The ectodomain of this PC, the region containing the glycosaminoglycan chains, is cleaved from its putative membrane-associated domain by mild trypsin treatment, producing a PG which is no longer lipophilic and can be readily handled in the absence of detergent. This ectodomain binds specifically to interstitial collagen types I, 111, and V at physiological pH and ionic strength and saturates a single class of high affinity sites on type I collagen fibrils (see Miniprint Section). The binding of the ectodomain is inhibited by heparin and dextran sulfates but not by other glycosaminoglycans at the concentrations tested (see Miniprint Section). NMuMG cells also bind to type I collagen and mimic, in several respects, the binding of their cell surface PG; the cells bind to native but not denatured type I collagen, and this binding is inhibited by heparin and dextran sulfate, but not by other glycosaminoglycans. Because the behavior of mammary epithelial cells is markedly altered by culture on and within type I collagen gels, they apparently have a mechanism for recognizing collagen fibrils. The cell surface P C could function as a receptor for the interstitial collagens, potentially mediating the collagen-induced changes in cell behavior.
Binding of the Ectodomin Is Similar to the Binding of a Basal Extracellular Proteoglycun-The ectodomain of the cell surface PG bears primarily heparan sulfate chains (M, of 36,000) but also 15-24% chondroitin sulfate in chains (M, of 17,000) attached to the same core protein (Rapraeger et ul

.').
Although prepared by proteolytic treatment, the ectodomain behaves as a single P C species on DEAE-cellulose, gel filtration, and collagen affinity chromatography (Rapraeger et aL4). Based on gradient PAGE, it shows a broad molecular weight distribution, possibly, as in other PGs, due to variation in the number of glycosaminoglycan or N-linked oligosaccharide chains (Hascall and Hascall, 1981).
The ectodomain is slightly smaller in molecular size range but identical in other respects to an extracellular heparan sulfate-rich PG deposited beneath the basal surfaces of the NMuMG cells when they are cultured on plastic (Koda and Bernfield, 1984). The ectodomain binds to type I collagen with properties that are nearly identical to those previously reported for this extracellular PG (Koda and Rernfield, 1984). Both PGs (i) bind to type I collagen at physiological pH and ionic strength, (ii) demonstrate specificity for native collagen, (iii) bind with nearly equal affinity and stoichiometry, and (iv) are displaced by only selected polyanions, suggesting that the binding is via the heparan sulfate chains. The relationship of these PGs to one another is not known.
The structural features of the inhibitory polyanions suggest a selective binding site on collagen fibrils. Heparan sulfate contains highly sulfated regions that are also present in heparin and potentially occur in dextran sulfate, polyanions which compete with PG binding to collagen. These highly sulfated regions are lacking in the polyanions which are ineffective competitors of the binding; for example, the sulfates occur uniformly along the chondroitin sulfate polymer rather than in "block" regions. Extensive sulfation alone is insufficient for inhibitory potential. Dextran sulfate, which has 3.3 sulfate residues/disaccharide (Windholz et al., 1976), on the average, is 10-fold less potent than similarly sized heparin, which bears 2 to 3 sulfate residues/disaccharide. Interestingly, increasing the sulfate concentration by using a larger-sized dextran sulfate increases its inhibitory potency. However, the binding is not simply a function of sulfate concentration because the sulfate concentrations provided by the noninhibitory glycosaminoglycans were 10-to 500-fold greater than the effective sulfate concentration required of any of the inhibitory polyanions. However, the chondroitin sulfates do bind to collagen (Lindahl and Hook, 1978), although apparently at sites different than heparan sulfate, suggesting that the heparan sulfate-rich PG binds to specific sites on the collagen which are selective for polysaccharides containing regions of extensive sulfation interspersed with regions of low or no sulfation.
T h e Ectodomain Binds Selectively to Interstitial Collagens-At physiological pH and ionic strength, the ectodomain binds to collagen types I, 111, and V, but not to collagen types I1 and IV. Thus, the ectodomain shows selectivity for the interstitial collagens with the exception of type 11. The reason for this is not known but may be because the type I1 collagen fibril formed in vitro under physiological ionic conditions differs from that of the other interstitial collagens.
Our failure to show binding to type IV collagen under physiologic conditions is also consistent with the requirement for collagen fibrils. Type IV collagen does not form fibrils but does form a multimeric complex of amino termini, the 7 S domain (Timpl et al., 1979;Risteli et al., 1980) which may resemble a fibril. However, the failure of PG binding to this isolated domain indicates that it does not satisfy this structural requirement.
The fibrils formed by collagen types I, 111, and V under physiological conditions have a similar distribution of clustered polar residues (Kuhn, 1982). Thus, these collagens may have a similar heparan sulfate PG binding site. Collagen types I, 111, and V also have a similar tissue localization, appearing together in extracellular matrices of many tissues (Kuhn, 1982). The tissue distribution of type IV collagen differs, being localized to the basal lamina (Kefalides, 1973). It is puzzling that the ectodomain does not bind to type IV collagen because mammary epithelial cells do lie on a basal lamina i n situ. Interactions of other basal laminar components may be required to facilitate complex formation of the cell surface PG with type IV collagen.
T h e Cell Surface Proteoglycan May Be a Receptor for Interstitial Collagens-In vitro, fibrillar type I collagen causes mammary epithelial cells to accumulate matrix materials, organize into distinct structures, and respond to physiological stimuli. For example, when cultured on top of collagen gels, mouse mammary epithelial cells form a basal lamina-like layer (Emerman and Pitelka, 1977;David and Bernfield, 1979). When embedded in collagen gels, branched ductlike structures form (Yang et al., 1979;Bennett, 1980) and, in such cultures, the cells respond to lactogenic hormones and secrete a-lactalbumin and casein (Haeuptle et al., 1983). These effects may involve a receptor on the mammary epithelial cell surface that recognizes the collagen.
Receptors are defined by their (i) high affinity, (ii) saturability, (iii) reversibility, (iv) ligand specificity, and (v) biological response produced by the receptor-ligand interaction (Hollenberg and Cuatrecasas, 1979). The cell surface PG satisfies certain of these criteria; the ectodomain reversibly binds type I collagen fibrils with high affinity ( K d -M), saturates a finite number of highly specific sites on the fibrils, and binds to only interstitial collagens. The cell surface PG may be a cellular receptor for collagen fibrils because its binding specificity is duplicated by the NMuMG cells. The cells bind to native but not denatured type I collagen and, based upon inhibition studies, bind at an identical site as their isolated cell surface PG.
Cells appear to have multiple mechanisms for attachment to or recognition of collagens. Indeed, the number and type of mechanisms may vary with the type of cell. Other cell types may also use heparan sulfate PGs, suggested by the finding that heparan sulfate or heparin inhibits the attachment of mouse myeloma cells to type I collagen fibrils (Stamatoglou and Keller, 1983). Other types of membrane-associated molecules interact with collagens but differ from the cell surface PG in their binding and chemical properties. Some bind to denatured collagen (gelatin) (Koehler et al., 1980) or to isolated a chains (Chiang and Kang, 1982) or show a different specificity for the collagen types (Mollenhauer and von der Mark, 1983;Kurkinen et al., 1984). Where described, these molecules are glycoproteins of molecular weights ranging from 31,000 to 95,000 and apparently do not contain glycosaminoglycan chains. The function of these cell surface collagenbinding molecules is not clear. Their presence on the cell surface may be to bind cells to collagens or to assist in the secretion and assembly of collagens at the cell surface. Alternatively, they may appear on the cell surface only transiently. For example, they may have a role in secretory vesicles and appear on the cell surface upon fusion of these vesicles with the plasma membrane and then be reinternalized.
If the ectodomain is, as proposed, a receptor which binds cells to interstitial collagens, then some mechanism is required to release cells from this association. Release of the cells could be accomplished by cleavage of the ectodomain from the cell surface. In the presence of a collagen substratum, such release would result in a stable collagen-proteoglycan complex. In the absence of a collagen substratum, the release mechanism could result in a soluble PG, perhaps the basal extracellular PG described previously.
In addition to binding interstitial collagens, apparently via its heparan sulfate chains as shown here, heparan sulfate PG or heparan sulfate or heparin chains are also thought to bind to fibronectin (Yamada et al., 1980), laminin (Ruoslahti andEngvall, 1980;Woodley et al., 1983), and a complex of fibronectin with collagen (Ruoslahti and Engvall, 1980). Thus, the cell surface proteoglycan may interact, as a receptor, with various matrix components.