JOURNAL OF CELLULAR PHYSIOLOGY 14451 1-522 (19901

Collagen-Binding Proteins of Mammary Epithelial Cells Are Related to Ca2+- and Phospholipid-Binding Annexins GERHARD WIRL* AND REINHARD SCHWARTZ-ALBIEZ lnstitute of Molecular Biology of the Amtrian Academy of Sciences, A-5ULU Salzburg, Austria (C.W.) and Cerman Cancer Research Center, lnstitute of lmmunology and Genetics, D-6900 Heidelberg, Federal Republic of Germany (RS-A.) Three major proteins of 34, 36, and 38 kDa were isolated from mebrane preparations of chemically induced mammary tumors of the rat by collagen type I affinity chromatography and therefore were termed collagen-binding proteins (CBP). Three proteins in the same molecular weight range isolated from cell extracts by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on hydroxylapatite were demonstrated to be immunologically related to CBP. As shown by immunoblot analysis, an antiserum directed against the cluster of the 34-38 kDa proteins reacted strongly with porcine intestinal protein I, weakly with porcine lipocortin I, and very weakly with porcine intestinal protein II. Antiserum against the 34 kDa protein reacted weakly with protein I but strongly with protein I I . All three CBP reacted with protein kalpactin I-specific antiserum of immunoblots and in immunoprecipitation experiments. However, antisera directed against CBP failed to show crossreaction with collagen-binding protein anchorin II from chicken chondrocytes. Conversely, antisera against anchorin II did not react with CBP. Antiserum AS/87 immunoprecipitated CBP of 38 kDA that was labeled in a Iactoperoxydase-catalyzed iodination, suggesting that this polypeptide is associated with the cell surface. Further, all three CBP were found to be phosphorylated by incubating mammary cells with 3zP-orthophosphate. CBP bound to epithelial cell membranes in a CaZf dependent manner ( = Triton x 100 insoluble form). Fractionated extraction and immunofluorescence microscopy also show that another form of CBP ( = Triton x 100 soluble form) exists in these cells and is associated with a granular fraction. We therefore conclude that mammary collagen-binding proteins represent members of a family of Ca2'-binding membrane proteins. The 38 kDa CBP seems closely related to the pp60"' kinase substrate protein I/ calpactin I monomer, the 34 kDa CBP seems to be related or equivalent to protein II, while the relationship of the 36 kDa CBP to other defined proteins is still unclear.

Collagen, as a major component of the extracellular matrix, is an important regulator of cell proliferation (Hay, 1982; Kleinman et al., 1981), differentiation (Wakimoto and Oka, 19831, and specific gene expression (Li et al., 1987; Lee et al., 1985). Direct interaction of cells with this substrate is apparently required to exert these effects. We have isolated collagen-binding proteins (CBP) from mammary epithelial cells (Wirl and Pfaffle, 1988). The interaction of mammary epithelium with collagen gels has been well characterized for a number of species (Emerman and Pitelka, 1977; Hall et al., 1982; Haeuptle et al., 1983; Yang et al., 1979). CBP from mammary epithelial cells differ from the more commonly studied collagen receptors of the integrin family in two important respects: 1) the most prominent proteins of CBP are of approx. 34, 36, 38, and 68/70 kDa, respectively, and thus can be distinguished by their molecular size from integrins (Hynes, 0 1990 WILEY-LISS, INC.

1987; Buck and Horwitz, 1987); 2) CBP do not contain glucosamine moieties and do not bind to Concanavalin A or wheat germ agglutinin, while integrins can readily be labeled metabolically with 'H-glucosamine and bind t o different lectins (Carter and Wayner, 1988; Tomaselli et al., 1988).Indeed, CBP are more similar to proteins binding various types of collagen, which have been described for a variety of cell types. Anchorins mediate the binding of chicken chondrocytes and sheep skin fibroblasts to interstitial collagens (Mollenhauer and von der Mark, 1983; Mauch et al., 1988). In epithelial cells, functional evidence for the existence of collagen receptors has been provided for hepatocytes (Gullberg et al., 1988) and corneal epithelium (Sugrue, 1987). Received June 21, 1989; accepted May 24, 1990, *To whom reprint requests/correspondence should be addressed.

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CBP bind to a collagen type I Sepharose matrix and can be eluted a t 100-150 mM NaCl. Three major proteins with apparent molecular masses of 34,36, and 38 kDa were isolated; pulse-chase studies and comparative peptide maps indicate that the 34 kDa polypeptide is not related to the two other forms. Small amounts of these proteins are released into the culture medium. Polyclonal antibodies were raised against CBP (AS/85) that show that a t least one of these proteins is located a t the cell surface, These results prompted us to investigate CBP in greater detail. CBP appeared similar to a group of 3036 kDa molecular weight proteins in normal murine mammary epithelial cells that bind to phenylthiazine in a calcium-dependent manner (Braslau et al., 1984) and that have been proposed to be homologous with calcium-binding “calcimedins” (Moore and Dedman, 1982). Furthermore, Fernandez and others (Fernandez et al., 1988) reported that the collagen-binding protein anchorin I1 of chicken chrondrocytes is related to calpactins or lipocortins. These proteins together with calcimedins (Moore and Dedman, 1982), calelectrins (Geisow et al., 1986; Sudhof et al., 19841, proteins I and I1 (Gerke and Weber, 1984; Weber et al., 19871, p35 and p36 (Shadle et al., 19851, and chromobindins (Creutz et al., 1987) are members of the Ca’+-dependent phospholipid-binding “annexins” (Geisow, 1986). Members of this multigene family share structural features such as the “endonexin fold” (Geisow et al., 1986; Crompton et al., 1988>,are associated with the cortical cytoskeleton (Gerke and Weber, 1984; Glenney et al., 19871, and are proposed to participate in membrane fusion and exocytosis (Siidhof et al., 1984; Drust and Creutz, 19881, regulation of phospholipase A, activity (Davidson et al., 1987; Hirata et al., 1984), and regulation of blood coagulation (Tait et al., 1988; Grundmann et al., 1988). In this report we show that CBP are immunologically very similar to protein I (calpactin I, lipocortin 11) and protein 11, but are distinct from anchorin 11. CBP are also similar if not identical to proteins extracted fi-om the same cells with EGTA and purified by hydroxylapatite chromatography. In addition, CBP bind to cell membranes in a calcium dependent manner. These properties may indicate considerable functional and structural similarities with other members of the annexin family.

Preparation of Collagen- a n d Calcium-Binding Proteins The isolation of CBP from mammary epithelial cell membranes has been described (Wirl and Pfaffle, 1988). This procedure, in brief, entails sucrose gradient centrifugation of membranes, extraction of membrane proteins with Triton x -100, and affinity chromatography on a collagen type I Sepharose column. CBP were then eluted with 150 mM NaC1, rechromatographed, concentrated, and frozen at -20°C. For preparation of calcium-binding proteins, mammary cell cultures were lysed in 10 mM HEPES (pH 6.91, 100 mM KC1, 1mM CaCl,, 2 mM MgCl,, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 0.5% Triton x-100 on ice for 2 min. The remaining cellular fraction was reextractd with 10 mM HEPES, 100 mM KCl, 2 mM MgC12, 1mM PMSF, and 5 mM ethylendiaminetetraacetic acid (EDTA) for 2 min at room temperature and centrifuged a t 12,0009 for 10 min a t 4°C. The supernatant was dialyzed overnight against 10 mM HEPES (pH 7.41, 0.1% Triton X-100. Calcium was then added t o the dialysate to a final concentration of 2 mM and the mixture was incubated for 30 min on ice and centrifuged for 20 min at 15,OOOg. The pellet was reextracted with 10 mM HEPES (pH 7.41, 1 mM PMSF, and 5 mM ethyleneglycol-bis-2-aminoethylether-tetraaceticacid (EGTA)for 15 min at room temperature. The extract was then centrifuged again a t 12,OOOg and aliquots of the supernatant were applied to 15% SDS-polyacrylamide gels (Laemmli, 1970). For partial purification of calcium-binding proteins, the crude EGTA extract was dialyzed against 20 mM Tris, 100 mM NaC1, 0.1% Triton x-100 (pH 8.0) and applied to a 6 ml bed-volume column (0.7 x 10 cm) of hydroxylapatite (Biogel HTP, Biorad). After a 50 ml wash with the same buffer bound proteins were eluted with a gradient (total volume 200 ml) of 0-200 mM Na,HPO, (pH 8.0). Fractions from the column were concentrated on an Amicon ultrafiltration system and analyzed by SDS-PAGE on 15% gels. Protein content was measured by the method of Lowry and others (Lowry et al., 1951) using bovine serum albumin (BSA) as a standard.

Polyclonal Antibodies Polyclonal antiserum AS/85 against collagenbinding proteins was prepared as previously described (Wirl and Pfaffle, 1988). Polyclonal antiserum AS87 against the most prominent band of approx. 34 kDa MATERIALS AND METHODS was generated in a manner similar t o that described for ASl85. Essentially, rabbits were immunized with proCells teins that had been electroeluted from 15%CoomassieRat mammary epithelial cells were obtained from stained SDS-polyacrylamide gels and dialyzed against chemically(7,12-dimethylbenz(a) anthracene) induced phosphate-buffered saline (PBS), Rabbit polyclonal antiserum to protein I from portumors and cultured in medium 199 containing 15 mM N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid cine intestinal cells was obtained from K. Weber (Max (Hepes), 5% fetal calf serum (FCS), 1 pgiml insulin, Planck Institute for Biphysical Chemistry, Gottingen, and 0.1 pg hydrocortisone (Wirl et al., 1984). Like the FRG). Rabbit antiserum A126 directed against the colnormal mammary gland, mammary tumor derived ep- lagen-binding protein from chicken chondrocyte memithelial cells (51 pm organoids) consist of two principal branes (anchorin 11) was a generous gift from K. von cell types, the cuboidal (ductal) and myoepithelial cells der Mark (Max Planck Institute for Connective Tissue that form two-layered colonies in culture. When grown ResearchiRheumatology, Erlangen, FRG). Purified on floating collagen gels or inside of collagen gels, cells protein I1 of porcine intestinal cells and anchorin I1 of organize themselves into glandular structures (Emer- chicken chondrocytes were kindly provided by K. Weber and K. von der Mark. man and Pitelka, 1977; Yang et al., 1979).

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acetone (2 min) on ice. Cells were then treated with 1% Immunoprecipitation and Western Blotting BSA in PBS for 3 x 5 min and subsequently incubated Cultures of mammary epithelial cells (51 pm orga- with the antisera (1:20 dilution in 1%BSAIPBS) for 30 noids) were preincubated in 2 ml of methionine-free min at room temperature. After extensive washing medium (using MEM and 1% dialyzed FCS) for 30 min with PBS, the samples were finally incubated for 30 and then 200 pCiiml of 35S-methionine (specific activity 1,300 CiirnM, Amersham) was added for 1 h. Cells min with fluorescence-labeled second antibody. After were scraped off the culture flasks and lysed in 10 mM washing thoroughly, the preparations were mounted in Tris-HC1 (pH 7.4), 2 mM CaCl,, 10 mM MgCl,, 1 pg/ml Gelvatol and evaluated with a Zeiss Photomicroscope aprotinin, and 1 mM PMSF, followed by ultrasonica- 111. Controls, omitting the first antibody, were always tion ( 3 x 30 sec on ice) and centrifugation at 12,OOOg incubated in parallel. In some experiments, cells were for 2 min at room temperature. The supernatant was extracted for 4 min on ice with a cytoskeleton stabilizsubsequently centrifuged for 1h a t 100,OOOg in a SW ing buffer (10 mM HEPES, 2 mM MgCl,, 100 mM KC1, 50.1 rotor a t 4°C. The pellet was then resuspended in 1 mM CaCl,, 0,5% Triton X-100, and 1mM PMSF, pH 10 mM Tris-HC1 (pH 7.4), containing 150 mM NaC1, 6.9) before fixation. 1% NP40, 5 mM EGTA, and protease inhibitors (lysis buffer), sonicated 3~ 30 sec on ice, centrifuged at RESULTS 12,OOOg for 5 min, and further used for immunoprecipitation. The supernatant was cleared by two sequential preadsorptions (each for 60 rnin 4°C) using protein A Preparation of CBP and Sepharose 4B (Pharmacia) to reduce nonspecific bindCalcium-BindingProteins ing. Specific immunoprecipitation was performed by Mammary epithelial cells were enzymatically isoincubating the supernatant (300 p1) together with 10 lated from chemically induced mammary tumors and p1 of the antisera and 20 ~1 of protein A-Sepharose (50%suspension in PBS) overnight at 4°C under gentle cultivated as described in Materials and Methods. Cells rotation. The immune complex was washed three times were homogenized and plasma membranes were colwith PBS plus 0.25% NP-40, solubilized in Laemmli's lected from a discontinuous sucrose gradient: Triton sample buffer with dithiothreitol, and analyzed on 10% X-100 extracts were applied to a n equilibrated collaSDS-polyacrylamide gels. Gels were fixed overnight in gen type I-Sepharose column, washed extensively, and a n isopropanollacetic acidlwater mixture (2.5/1/6.5,~1 eluted with a 0-1.0 M NaCl gradient. The peak fracv/v) and then treated with a dimethylsulfoxide solution tion was concentrated and resolved on 15% polyacrylcontaining 2,5-diphenyloxazol according to Bonner and amide gels, and the separated proteins were visualized by silver staining. The eluted fraction contained three Laskey (1974). Immunoblots were prepared from gels transferred to principal protein bands of 34-38 kDa with a small nitrocellulose filters (Towbin et al., 19791, followed by amount of protein in the range of 68/70 kDa (Fig. 1, blocking of the filters with 0.1% gelatine in PBS, reac- lane 2). To compare CBP with calcium-binding proteins of tion with the antiserum (1:400 dilution), and horseradthe calpactinilipocortin family, we took advantage of ish peroxidase conjugated goat-anti-rabbit IgG (Biotheir calcium-sensitive association with membrane-cyrad) and detection with 4-chloro-1-naphthol. toskeleton complexes. Cell cultures of mammary epiSurface Iodination thelial cells were therefore treated with 1mM CaCl, in For surface iodination, harvesting of cell monolayers HEPES and 0.5% Triton X-100. After this step cells was achieved by pipetting in PBS containing 0.258 were extracted with 5 mM EDTA to release calciumEDTA. Viability of the cells was controlled by trypan binding proteins. The addition of 2 mM CaC1, to the blue staining. Comparable numbers of cells (approx. 5 dialyzed lysate resulted in the precipitation of calciumbinding proteins. These proteins were then dissolved X 106cells, viability over 95%) were labeled with 0.5 mCi of sodium lZ5J (Amersham) using the lactoperox- with 5 mM EGTA and analyzed by SDS-PAGE. Silveridase procedure (Goding, 1980). Cells were then stained calcium-binding proteins are presented in Fig. washed three times and added to the lysis buffer (see 1,lane 3. These proteins comigrate with the triplet of above). Equal amounts of supernatant counts (approx. CBP. In contrast to the single protein of 68/70 kDa 3 x lo7 cpm lz5J) were then used for each immunopre- obtained after collagen affinity chromatography, a protein doublet is usually observed in the case of Ca2+ cipitation as described above. extraction. Lane 1 in Fig. 1 depicts purified protein I1 Detection of Phosphorylation isolated from porcine intestinal epithelium with a Prior to labeling, cultures of 51 pm organoids were slightly different migration behavior (35 kDa). Since studies from other laboratories have shown preincubated in phosphate-free medium (aMEM containing 5% dialyzed FCS) for 1h and then labeled over- that calpactin I and I1 can be further fractionated on a night with "P-ortho-phosphate (1 mCii20 ml culture hydroxylapatite column (Glenney et al., 19871, we medium, carrier-free, Amersham), containing 5% dia- tested this technique to separate calcium-binding prolyzed FCS in phosphate-free MEM. Protein extraction teins from mammary cell extracts. As seen on the siland immunoprecipitation iapprox. 2 x lo5 cpm 32Pper ver-stained gel, the two leading proteins of 34 and 36 kDa could be quantitatively separated from the 38 kDa specific immunoprecipitation) was done a s described. polypeptide. The 38 kDa protein eluted later in the Immunofluorescence Microscopy gradient, suggesting that this protein interacts with Cells cultured on glass coverslips were briefly rinsed higher affinity with Ca2' than the 34 and 36 kDa proin Hanks' solution and fixed with methanol (5 min) and teins (Fig. 2).

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Fig. 1. Comparison o f membrane bound collagen-binding proteins of mammary cells, calcium-binding proteins from cell extracts, and porcine intestinal protein 11. Collagen- and calcium-binding proteins were isolated as described under Materials and Methods. Eluates from the collagen type I-sepharose column (lane 2) and EGTA-eluates of Ca2'-precipitates (lane 3) were subjected to 156 polyacrylamide gels and visualized by silver staining. Lane 1: porcine intestinal protein 11. Arrows at the right denote proteins of 34, 36, and 38 kDa.

CBP and Calcium-BindingProteins of Mammary Cells are Related to Annexins Partially purified mammary CBP and calciumbinding proteins were resolved on 12% polyacrylamide gels, transferred to nitrocellulose, and incubated with four different antisera. As expected, AS185 reacted strongly with CBP of 34, 36, and 38 kDa, the proteins used for immunization (Fig. 3, lane 1). The same proteins also gave a reaction with AS187, which was raised against the 34 kDa band (Fig. 3, lane 2j, and with anti-protein I (Fig. 3, lane 3). These results suggest that: 1) there is a n extensive immunological cross-reactivity within this family of proteins; and 2) these proteins are also similar to protein I. Interestingly, the recently prepared anti-anchorin I1 antiserum (A126) gave no reaction with CBP (Fig. 3, lane 4).The antisera AS185 and AS187 also reacted with calcium-binding proteins of 34 and 38 kDa, which were precipitated from cell lysates with Ca", resolubilized with EGTA, and chromatographed either on hydroxylapatite (Fig. 3, lanes 5 and 6 ) or on a newly prepared collagen type I-Sepharose column (data not shown). Anti-protein I reacted with the protein of 38 kDa but not with the 34 kDa polypeptide (Fig. 3, lane 7). Both antisera reacted with a protein species of about 42 kDa whose identity is unknown. In the case of the collagen affinity bound proteins only antiserum AS187 reacted with this protein (Fig. 3, lane 2). Again, the antiserum

Fig. 2. Fractionation of mammary cell calcium-binding proteins by hydroxylapatite chromatography. EGTA-soluble proteins were loaded onto a hydroxylapatite column and eluted with a linear 0-200 mM Na,HPO, gradient. Fractions of 20 ml (lanes 1-91 were collected and concentrated using an Amicon ultrafiltration system. Samples of these fractions were subjected to 15% polyacrylamide gels and silver stained. Arrows at the right denote proteins of 34, 36, and 38 kDa.

against anchorin I1 gave no signal with mammary calcium-binding proteins (Fig. 3, lane 8). Incubation of three Ca2 -regulated proteins with antisera AS185 and AS187, raised against CBP, was performed to verify whether mammary CBP share structural properties with protein I (calpactin I, lipocortin II), calpactin I1 (lipocortin I), and protein 11. Antiserum AS187 gave a weak reaction with protein I but showed a principal reaction with lipocortin I and protein I1 (Fig. 4, lane A). In contrast, antiserum AS185 strongly reacted with protein I from porcine intestinal epithelium but very weakly with purified lipocortin I from porcine lung and with protein I1 (Fig. 4, lane B). It is clear therefore from these data that the mammary 34 kDa CBP is immunologically related to protein I1 while the 38 kDa polypeptide is related to protein I monomer. We have additionally probed both antisera with purified anchorin I1 but did not observe a reaction by either AS185 or AS187 (data not shown). +

Characterization of Calcium-BindingProteins by Immunoprecipitation To evaluate the ability of polyclonal antisera AS185 and AS187 to bind to mammary calcium-binding proteins, the EGTA-eluting fraction of mammary cell lysates was examined using different radioactive mark-

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A

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1 2 3 1 2 3 1

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Fig. 3. Immunological relationship of mammary collagen- and calcium-binding proteins from cell extracts. Reaction was performed with various antisera. Collagen-binding proteins (lanes 1-3) and calcium-binding proteins (lanes 5-7) were loaded onto 12% polyacrylamide gels and transferred to nitrocellulose paper for immunoblot detection using antisera (1:400dilution) ASl85 (lanes 1,5), ASi87 (lanes 2,6), anti-protein I antiserum (lanes 3,7)and anti-anchorin I1 antiserum (lanes 4,s). Arrows at left denote proteins.

Fig. 4. Reactivity of polyclonal antisera ASi85 and AS/87 on Western blots of three Ca”-regulated proteins. Purified protein I1 of porcine intestinal epithelium (A,B, lane l), lipocortin I of porcine lung (A,B, lane 2), and protein I of porcine intestinal epithelium (A$, lane 3) were run on polyacrylamide gels, blotted onto nitrocellulose, and reacted with ASi87 (A) and AS85 (B). Arrows at the right denote the three proteins.

34-38 kDa CBP were detected by antiserum AS185 with the exception of a faint band at 36 kDa, whereas the 38 kDa polypeptide strongly and the 36 and 34 kDa polypeptides weakly reacted with AS/87 (Fig. 5B, lanes 1,2). Therefore?the 38 kDa band seems to represent the major surface-expressed CBP. Since calpactinsllipocortins have been shown to be phosphorylated on tyrosine residues (Gerke and Weber, 1984) we examined the possible phosphorylation of these proteins by labeling mammary cell cultures overnight with 32Pi. Cell lysates were prepared as described. Cell lysates were prepared a t a Ca2+ concentration of 1mM followed by EGTA extraction. 32Piwas incorporated into CBP though to a small extent as shown after immunoprecipitation with antisera AS185 and AS87 (Fig. 5C). In a third sample, the antiserum to protein I imrnunoprecipitated in a somewhat stronger fashion CBP of 34,36, and 38 kDa (Fig. 5C, lane 4).

ers in immunoprecipitation assays. Mammary epithelial cells were labeled with 35S-methioninefor 60 min and lysed in HEPES buffer containing 2 mM CaC1,. The final pellet was extracted in EGTA-containing buffer and immunoprecipitated with antisera AS185 and ASl87. The reaction complex was resolved in polyacrylamide gels and proteins were visualized on autoradiograms. Imrnunoprecipitation with AS185 results in a faint band a t 38 kDa while with AS187 a n additional strong band a t approx. 34 kDa was seen (Fig. 5A, lanes 1,2). The inability of ASi85 to immunoprecipitate 35S-methionine-labeled polypeptides (see also below) indicates that the relevant epitopes are masked unless p36 is denatured. Since AS187 was Distribution of Calcium-Binding Proteins raised against the 34 kDa CBP it is very likely that the corresponding Ca2+-binding34 kDa protein is identiCalpactins/lipocortins are believed to be membranecal to this CBP. Normal rabbit serum failed t o react associated proteins that interact with hospholipids with any of the mammary CBP (Fig. 5A, lane 3). and some cytoskeletal elements in a Cay+-dependent Because earlier experiments with CBP demonstrated manner (Gerke and Weber, 1984; Glenney e t al., 1987). t h a t about 5% of these proteins were found in the cul- To further compare the behavior of CBP with these ture medium and external protein was detected by irn- calcium-binding proteins?mammary cell cultures were munofluorescence microscopy (Wirl and Pfaffle, 1988), initially ruptured for 2 min with the cytoskeleton stasuggesting that some of these proteins must be secreted bilizing buffer and the supernatant was recovered. The or are attached to the cell surface, mammary cells were remaining cell fraction was reextracted with the same surface-labeled with lz5J.The labeled cells were pel- procedure as above except that Triton X-100 was omitleted and lysed directly in lysis buffer. None of the ted and 5 mM EGTA was added instead of CaC1,. In a

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100-

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Fig. 5. Immunoprecipitation of mammary calcium-binding proteins. Cell lysates (in lysis buffer containing 5 mM EGTA) were prepared and immunoprecipitated as described in Materials and Methods. Immunoprecipitates in sample buffer were analyzed on 12% polyacrylamide gels. Arrows a t the left indicate 34,36, and 38 kDa proteins. A: Mammary cell cultures were labeled with 200 pCi of 35S-methionine for 1h. Cell lysates were immunoprecipiated with AS185 (lane 1) and AS/87 (lane 2);lane 3: normal rabbit serum. B: Mammary cells were detached from culture dishes with EDTA and surface-labeled with 0.5

mCi Na'26J. Proteins were immunoprecipitated with A S 8 5 (lane l), AS187 (lane 21, and normal rabbit serum (lane 3);C : Mammary cells were incubated overnight with 50 pCi 32Pi/ml.Cell lysates were immunoprecipitated with AS185 (lane l),AS187 (lane Z), normal rabbit serum (lane 3),and anti-protein I antiserum (lane 4). Protein bands of approx. 45 kDa (A,B) and 50 kDa (B) represent nonspecific precipitation of presumably actin regularly observed in immunoprecipitates. Molecular weight markers are in kilodaltons.

parallel experiment cells were first extracted with 0.5% Triton X-100 and 5 mM EDTA followed by a second extraction step using the same buffer. In all cases supernatants were electroblotted and incubated with ASl87. The data show that the 34 and 38 kDa proteins could be detected in three of the four extracts (Fig. 6, lanes 1-3). In the presence of calcium the level of both proteins is low in the Triton-soluble fraction (lane 1) whereas EGTA released the bulk of these proteins (lane 2). When EDTA was included in the Triton-containing buffer, the release of these proteins was most extensive with very little release of the 38 kDa protein in the second buffer (Fig. 6, lanes 3,4) indicating that both proteins require calcium in mM concentrations for maximal binding to the Triton-insoluble fraction.

87. We found that all cuboidal cells examined showed essentially a similar distribution pattern of CBP. When fixed with methanoliacetone an intense reticular network was observed (Fig. 7A). It should be noted that this staining pattern was not seen with the myoepithelial-like cell type, in which the fluorescence was either absent or only weak (Fig. 7B). Therefore, staining with AS187 discriminates cuboidal and myoepithelial-like cells, which is the natural cell combination in cultures of 51 pm organoids. Some of the mammary cells, however, showed an unusual pattern of CBP distribution, in which staining seemed to exist partly in association with not yet identified large vesicles (Fig. 7D and E). The pattern of distribution of CBP in cuboidal mammary epithelial cell cultures was completely different using AS/85. Fluorescence staining showed a rather evenly distributed granular pattern (Fig. 7C). These granula not only accumulated perinuclearly, but staining was also extended further out to the edge of the

Immunofluorescence Microscopy We first studied the distribution of CBP in cultures of 51 K r n organoids using the polyclonal antiserum AS/

COLLAGEN-BINDING PROTEINS BELONG TO ANNEXINS

Fig. 6. Calcium dependence of the association of mammary calciumbinding proteins with cell membranes. Mammary cell cultures were subsequently extracted with a cytoskeleton stabilizing buffer containing 0.5% Tritonil mM CaCI, (lane 1) and EGTA (lane 2). In a parallel experiment cells were treated two times with 0.5% Tritoni5 mM EGTA (step 1: lane 3;step 2: lane 4). Extracted proteins (10 +g/lane as estimated by trichloracetic precipitation and Lowrie's reaction) were subjected to 15%polyacrylamide gels, transferred to nitrocellulose, and reacted with ASi87. Markers on the left indicate the 34,36, and 38 kDa molecular weight proteins.

cells. Such a staining pattern was generally seen also with the myoepithelial-like cell type (not shown). When cells were extracted with a cytoskeleton stabilizing buffer before fixation in methanollacetone and then treated with ASl87, apart from a fine terminal web, staining was always concentrated over the nuclei and at the intercellular boundaries as sharp lines (Fig. 8C). After treatment with this buffer a similar staining pattern was visualized using antiserum ASi85 (not shown) and the polyclonal antibody against protein I (Fig. 8B). Staining with the antiserum against protein I was generally weaker compared to that with antiserum ASi87. Common to these staining patterns was the absence of fluorescence in certain areas of the cell previously identified as actin-containing stress fibers (not shown). Reduction of Ca2+ i n the cytoskeleton stabilizing buffer from 1 mM CaC1, t o 0.1 mM CaC1, resulted in a total absence of fluorescence at the boundaries, indicating again that these proteins are associated with Triton-soluble cytoskeletal elements in a Ca2' -dependent manner (Fig. 8D).

DISCUSSION Our study shows that at least two major collagenbinding proteins of mammary epithelial cell cultures

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are immunologically related t o calcium-binding proteins proteins I and I1 of porcine intestinal epithelium. Antiserum raised against the 34-38 kDa cluster of CBP reacted strongly with protein I and weakly with lipocortin I from porcine lung and protein 11. Antiserum to the 34 kDa CBP band bound primarily to protein I1 and lipocortin I and weakly to protein I. On the other hand, antiserum to protein I reacted with CBP in immunoblot and immunoprecipitation assays. We have also shown that the antisera AS185 and ASi87 used in this study reacted with the same polypeptides on Western blots but are specific for their respective proteins in immunoprecipitation experiments, suggesting t h a t CBP have common epitopes that are partially masked in the native proteins. Collagen-binding proteins are also presumed to be identical with calcium-binding proteins of 34-38 kDa that have been isolated from mammary cell lysates using the Ca'+-precipitation technique. Calcium-binding proteins of 34, 36, and 38 kDa react with antiera against CBP as well as with anti-protein I serum. Mammary CBP of 38 kDa therefore resembles the 36 kDa subunit of protein I that was shown to be identical to the major cytoplasmic target substrates of retrovirally coded tyrosine-specific protein kinase (p36) present in transformed fibroblasts (Gerke and Weber, 1984). Previously, a 98% sequence homology was found between protein I and calpactin I of bovine lung and human placenta (Glenney e t al., 1987; Haigler et al., 1987). Furthermore, calpactin I monomer is identical to lipocortin I1 (Saris et al., 1986). Calpactin I monomer is also immunologically related to the 33 kDa polypeptide of human lymphocytes (Davies and Crumpton, 1985)and the chromaffine granule-binding chromobindin 8 (Geisow et al., 1984). Recently, a n antiserum against calpactin I monomer has been found to possess a cross-reacting activity with the 36 kDa protein of the 30-36 kDa complex of normal mouse mammary gland epithelium (Hom et al., 1988). Calpactin I (protein I) is a n abundant cellular protein that remains associated with membranes in the presence of Ca' and nonionic detergents. There, the heavy and light chains of this protein colocalize with actin and actin-binding proteins like fodrin (Gerke and Weber, 1984; Glenney e t al., 1987), suggesting t h a t calpactin I plays a role in the organization of the network underlying the plasma membrane. Binding to actin seems to be regulated by tyrosine phosphorylation, which requires high Ca2 ' concentrations and phospholipids (Glenney, 1985). In agreement with these findings, autoradiograms of SDS gels of immunoprecipitates presented here indicate that the 38 kDa CBP incorporated "Pi. Further, most of this protein remained bound to the Triton-insoluble cellular fraction and could be released by EGTA. It was shown by immunofluorescence microscopy that its colocalization with the p€asma membrane was not affected by the detergent treatment. In previous experiments we detected secretion but not accumulation of "S-methionine-labeled CBP in the culture medium (Wirl and Pfaffle, 1988). Consistent with these results, we now detected a strong 38 kDa band after surface labeling with lz5J and immunoprecipitation with AS187 which speaks in favor of a cell surface expression. However, it remains to be solved +

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Fig. 7. Immunofluorescence microscopy of cells stained with antisera against collagen-binding proteins. Polyclonal antiserum AS87 was used to localize the antigens in cuboidal (A) and mixed culture of cuboidal (CUB) and myoepithelial-like (ME) mammary epithelial cells (B). D and E: Unidentified large granules occurring occasion-

ally in mammary epithelial cells, stained with ASi87. C: Fine granular localization of collagen-binding proteins in cuboidal mammary epithelial cells stained with AS/85. Magnifications A$-E x 500; B x 300.

how the 38 kDa CBP is secreted and anchored to the cell surface. For instance, the collagen-binding anchorin I1 of chicken chondrocytes is partially released from cells and binds to the cell surface although hydrophobic domains are lacking (Pfaffle et al., 1988). Similarly, human placental anticoagulant protein (PP4) is

secreted through the cellular membrane, but cDNA for PP4 does not encode a signal peptide (Grundmann et al., 1988). Collagen-binding protein of 36 kDa is stained by AS/ 85, AS/87, and anti-protein I on immunoblots. Like the 38 kDa CBP, it is phosphorylated and can be released

COLLAGEN-BINDING PROTEINS BELONG TO ANNEXINS

519

Fig. 8. Immunofluorescen staining of collagen-binding proteins. Cells were incubated in cytoskeleton stabilizing buffer for 4 min before fixation in methanoliacetone. A Control mammary cells, stained with ASi87. B and C: Buffer-treated mammary cells, stained with

anti-protein I antiserum (B) and AS87 iC). D: Effect of reduction of Ca'+ from the cytoskeleton stabilizing buffer. Staining was performed with ASiS7. Note the disappearance of intensive staining from the cell periphery. Magnification x 500.

from cells by EGTA treatment. Peptide maps of lz5Jlabeled CBP have previously shown that the 36 kDa protein is not identical but shares many sequence strains with the 38 kDa protein. The 36 kDa CBP may

therefore be related to calpactin 11, which is the analogue of lipocortin I. In this case an explanation is needed for the fact that normal murine mammary glands have been reported to produce no calpactin I1

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WIRL AND SCHWARTZ-ALBIEZ

(Hom et al., 1988). It could be argued that normal mu- in the detergent-soluble fraction whereas the bulk of rine and transformed rat mammary epithelial cells ex- this proteins was retained in the detergent-insoluble press a different pattern of calpactinsllipocortins, but it cytoskeletal fraction, indicating that two functionally should also be borne in mind that the 36 kDa CBP distinct populations of CBP exist in these cells. Both protein could result from proteolytic degradation of the soluble and cytoskeletal forms of calpactin I and pro38 kDa CBP. Degradation of calpactin I to smaller tein I1 have been described for several other cell types forms has been described for several other cell types including intestinal cells (Gerke and Weber, 1984), fi(Glenney, 1985). Recently, a 33 kDa fragment has been broblasts (Zokas and Glenney, 19871, and lymphocytes obtained by limited proteolysis of 36 kDa mammary (Owens et al., 1984). The insoluble pool of these procalpactin (Hom et al., 1988). teins may represent membrane-bound forms of calpacThe collagen-binding protein of 34 kDa is about tin I and protein I1 that are stabilized by interacting 1,000 D smaller than protein I1 of porcine intestine. It with the cytoskeleton and overlying plasma memreacted with ASl85, ASl87, and anti-protein I on im- brane. The membrane location of CBP, which is munoblots. Anti-protein I also immunoprecipitated strengthened by our immunof luorescence data, raises this protein in its phosphorylated form. Polyclonal an- the possibility that the presence of these proteins is tibodies against this protein gave a strong reaction correlated with mammary cell attachment and movewith protein I1 and a comparatively weaker reaction ment. This question is currently under investigation. with lipocortin I but no reaction with protein I. With Triton-soluble forms of protein I1 may be partially other annexins this protein shares the properties of bound to intracellular organelles. ImmunocytochemiCa2 -dependent precipitation and subsequent solubili- cal studies with antisera against CBP directly demonzation by EGTA. Finally, this polypeptide is localized strated that a t least one of the three CBP is located a t cell membranes where it may be associated with almost exclusively on yet unidentified cytoplasmic cytoskeletal elements. These data make i t very likely granules of both the cuboidal and myoepithelial-like that the 34 kDa CBP is related or equivalent to protein cell type. In this regard, our findings are similar to 11. Protein 11, together with a 35 and 68 kDa polypep- observations on the localization of calelectrins in adtide, was extracted from bovine tissues. These proteins herent cells, which exhibit extensive exocytosis. Using were called “calelectrins” because they cross-react with polyclonal antiserum against the 32 kDa protein from the antiserum against Torpedo calelectrin (Sudhof et Torpedo electric organ, the existence of this protein in al., 1984). Recent primary structural information for epithelial cells of bile and pancreatic ducts (Silva et al., calelectrin (Geisow e t al., 1986; Huang et al., 1986; 1986), on synaptic vesicles of the electric organ (Fiedler Saris et al., 1986, Weber et al., 1987) suggests that and Walker, 1985), and on secretory granules of r a t these proteins have several copies of a common 17 adrenal medulla (Walker et al., 1983) and human granamino acid consensus sequence and that calelectrins of ulocytes (Sudhof et al., 1983) was strikingly confirmed. 35 kDa and 32.5 kDa correspond to proteins I and I1 Further, Creutz et al. (1987) have described several (Shadle e t al., 1985). Consistent with this notion is the chromaffin granule-binding proteins that they termed fact that antiserum to 32.5 bovine liver calelectrin “chromobindins” (Creutz e t al., 1987). They bind to gave a reaction with the leading protein of the 30-36 secretory vesicle membranes of the adrenal medulla in kDa complex of murine mammary gland calcium- a Ca2+-dependent way and represent candidates for binding proteins (Hom e t al., 1988). mediators of membrane interactions during exocytosis. Despite the 40-50% homology between collagenImmunostaining of cultures of 51 pm organoids with binding anchorin I1 of chicken chondrocytes with bo- AS187 showed different results with regard to the apvine and murine calpactin I and porcine protein 11, an- pearance of the 34 kDa CBP in cuboidal (ductal) and tiserum A126 against anchorin I1 showed no myoepithelial-like cell types. Cuboidal cells always immunological reaction with any of the rat mammary contain this protein while the other cell type was deCBP. Additionally, AS185 and AS187 did not cross-react void of staining. However, in preliminary experiments, with purified anchorin 11.These results suggest that a n monolayers of isolated cuboidal and myoepithelial-like equivalent protein is not expressed by transformed rat cell both demonstrate comparable high levels of this mammary epithelial cells. Alternatively, the absence protein in immunoprecipitation experiments and on of cross-reactivity may result from species-specific dif- immunoblots (data not shown). As yet there is no obferences in amino acid sequences that reduce binding vious explanation of the discrepancy. Possibly there is capabilities. This observation is consistent with the a n altered synthesis andlor higher turnover of the 34 fact t h a t peptide maps of radiolabeled CBP and those kDa CBP in the myoepithelial-like cell type. A mechfrom anchorin I1 were completely different (Wirl and anism may also exist through which cuboidal cells can Pfaffle, 1988).In contrast to the 98% sequence identity regulate expression of this protein in the other cell between murine and bovine calpactin I (Saris et al., type. 1986; Kristensen et al., 19861, interspecies conservaThe relationship of CBP with calelectrins, which do tion of rat 34 kDa CBP and chicken anchorin I1 is much not have hydrophobic sequences typical for transmembrane domains, argues against their function a s intelower. Since all of the mammary CBP demonstrate exten- gral membrane receptors for collagen. The ability to sive immunological cross-reactivity with AS185 and radiolabel the 38 kDa protein by lactoperoxidase-meASl87, the localization and function of these proteins in diated iodination on cells detached from the substrate mammary epithelial cells remains to be clarified. Ex- by EDTA does not necessarily imply its transmemtraction of cell culture with a cytoskeleton stabilizing brane location. Appearance of this protein on the cell buffer at moderate (1 mM) CaC1, concentration re- surface may be a transient event, possible related to vealed that little of the 38 and 34 kDa CBP was present the secretion of this protein. Surface binding of colla+

52 1

COLLAGEN-BINDING PROTEINS BELONG TO ANNEXINS

gen may have a role for signal transduction during differentiation, proliferation, and migration of mammary cells (Blum et al., 1987). Collagen binding of CBP would also occur intracellularly, e.g., during collagen synthesis and processing. Indeed, several other collagen-binding proteins like the 47 kDa “colligin” (Kurkinen et al., 1984) and the 47 kDa heat-shock protein (Saga et al., 1987) have been originally postulated to be surface collagen receptors, but recently they have been shown to be located in the endoplasmic reticulum. Despite the apparent functional significance of CBP, we cannot completely exclude that collagen binding occurs during affinity chromatography by ionic interactions with contaminants such as phospholipids. For example, the use of calcium-dependent hydrophobic chromatography on phenothiazine columns for purification of membrane-bound calcimedins (Moore and Dedman, 1982) suggests indirect binding of CBP to collagen type I. However, due to the highly reproducible and specific separation of CBP on collagen type I columns, a n interaction with contaminants seems to be very unlikely. Our data speak in favor of a physiological importance of these collagen-binding proteins that is in a yet unknown way related to their Ca2+-binding properties.

ACKNOWLEDGMENTS We would like to thank Dr. N. Johnson (MPI for Biophysical Chemistry, Gottingen, FRG) for immunoblot analysis of AS185 and AS187 and for providing protein I, protein 11, and lipocortin I. We also thank Dr. Jacqueline Trotter (Neurobiology Department, University of Heidelberg) and Dr. Sonka (German Cancer Research Center, Heidelberg), for critically reading the manuscript. The excellent technical assistance of Mrs. A. Merling and of Mrs. A. Danielopol is gratefully acknowledged. LITERATURE CITED Blum, J.L,, Zeigler, M.E., and Wicha, M.S. (1987) Extracellular matrix: structure, biosynthesis, and role in mammary differentiation. In: Cellular and Molecular Biology of Mammary Cancer. D. Medina, W. Kidwell, G. Heppner, and E. Anderson, eds. Plenum Press, New York, pp.105-128. Bonner, W.M., and Laskey, R.A. (1974) A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem., 46233-88. Braslau, D.L., Ringo, D.L., and Rocha, V. (1984) Synthesis of novel calcium-dependent proteins associated with mammary epithelial cell migration and differentiation. Exp. Cell Res., 155:213-221. Buck, C.A., and Horwitz, A.F. (1987) Integrin, a transmembrane glycoprotein complex mediating cell-substratum adhesion. J . Cell. Sci., Supp1.8:231-250. Carter, W.G., and Wayner, E.A. (1988) Characerization of the class I11 collagen receptor, a phosphorylated, transmembrane glycoprotein expressed in nucleated human cells. J. Biol. Chem., 263: 4193-4201. Creutz, C.E., Zaks, W.J., Hamman, H.C., Crane, S., Martin, W.H., Gould, K.L., oddie, K.M., and Parsons, S.J. (1987) Identification of chromaffin granule-binding proteins. J. Biol. Chem., 262: 1860-1868. Crompton, M.R., Owens, R.J., Totty, N.F., Moss, S.E., Waterfield, M.D., and Crumpton (1988) Primary structure of the human, membrane-associated Ca2+-bindingprotein p68: a novel member of‘ a protein family. EMBO J . 7:21-27. Davidson, F.F., Dennis, E.A., Povell, M., and Glenney, J.R. Jr. (1987) Inhibition of phopholipase A, by “lipocortins” and calpactins. K. Biol. Chem., 262:1698-1705. Davies, A.A., and Crumpton, M.J. (1985) Identification of calciumbinding proteins associated with the lymphocyte plasma membrane. Biochem. Biophys. Res. Commun., 128571-577, De, B.K., Misono, K.S., Lukas, T.J., Mroczkowski, B,, and Cohen, S.

(1987) A calrium-dependent 35-kilodalton substrate for epidermal growth factor receptorikinase isolated from normal tissue. J. Biol. Chem., 261 r13784-13792. Drust, D.S., and Creutz, C.E. (1988) Aggregation of c h r o m a f h granules by calpactin at micromolar levels of calcium. Nature, 331t8891. Emerman, J.T., and Pitelka, D.R. (1977) Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro, 13t316-328. Fiedler, W.F., and Walker, J.H. (1985) Electron microscopic localization of calelectrin, a Mr 36000 calcium-rcgulated protein, at the cholinergic electromotor synapse of Torpedo. Eur. J . Cell Biol., 38: 34-41. Fernandez, M.P., Selmin, O., Martin, G.R., Yamada, Y., Pfaffle, M., Deutzmann, R., Mollenhauer, J., and von der Mark, K. (1988) The structure of anchorin 11, a collagen binding protein isolated from chondrocyte membrane. J. Biol. Chem., 363:5921-5925. Geisow, M.J. (1986) Common domain structure of CaZ+ and lipidbinding proteins. FEBS Lett., 302t99-103. Geisow, M., Childs, J., Dash, B., Harris, A,, Panayotou, G. Sudhof, T.C., and Walker, J. (1984) Cellular distribution of three mammalian Ca’ binding proteins related to Torpedo calelectrin. EMBO J. 3:3969-3974. Geisow, M.J., Fritsche, U., Hexham, J.M., Dash, B., and Johnson, T. (1986) A consensus amino-acid sequence repeat in Torpedo and mammalian Ca+ +-dependent membrane binding proteins. Nature, 320r636-638. Gerke, V., and Weber, K. (1984) Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to non-erythroid spectrin and F-actin. EMBO J., 3t227-233. Glenney, J.R. Jr. (1985) Phosphorylation of p36 in vitro with pp60””: regulation by Ca2+ and phospholipid. FEBS Lett., 192:79-82. Glenney, J.R., Tack, B., and Powell, M.A. (1987) Calpactins: two distinct Ca’ regulated phospholipid- and actin-binding proteins isolated from lung and placenta. J. Cell Biol., 104503-511. Coding, J.,W. (19801Structural studies of murine lymphocyte surface IgD. J . Immunol., 124:2082-2088. Grundmann, U., Abel, K.J., Bohn, H., Lobermann, H., Lottspeich, F., and Kupper, H. (1988) Characterization of cDNA encoding human placental anticoagulant protein (PP4): homology with the lipocortin family. Proc. Natl. Acad. Sci. USA, 85:3708-3712. Gullberg, D., Terracio, L., and Rubin, K. (1988) Membrane glycoproteins involved in hepatocyte adhesion to collagen type I. Exp. Cell Res., 175r388-395. Haeuptle, M.T., Suard, Y.L., Bogenmann, E., Reggio, H., Racine, L., and Kraehenbuhl, J.P. (1983) Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture. J. Cell Riol., 96:1425-1434. Haigler, H.T., Schlaepfer, D.D., and Burgess, W.H. (1987) Characterisation of lipocortin I and a n immunologically unrelated 33-kDa protein as epidermal growth factor receptodkinase substrates and phospholipase A, inhibitors. J. Biol. Chem., 262t6921-6930. Hall, G.H., Farson, D.A., and Bissell, M.J. (1982) Luman formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture. Proc. Natl. Acad. Sci. USA, 79:4672-4676. Hay, E.D. (1982) Interaction of embryonic cell surface and cytoskelton with extracellular matrix. Am. J. Anat., 165:l-12. Hirata, F., K., Matsuda, Y., Notsu, T., Hattori, T., and del Carmine, R. (1984) Phosphorylation at a tyrosine residue of lipomodulin in mitogen-stimulated murine thymocytes. Proc. Natl. Acad. Sci. USA, 81r4717-4721. Hom, Y.R., Siidhof, T.C., Lozano, J.J., Haindl, A.H., and Rocha, V. (1988) mammary gland Ca -binding(-dependent) proteins: identification as calelectrins and calpactin Iip36. J . Cell. Physiol., 135: 435-442. Huang, K.S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Two human 35 kD inhibitors of phospholipase A, are related t o substrates of pp6OV-“‘and of the epidermal growth factor receptorikinase. Cell, 46:191-199. Hynes, R.O. (1987) Integrins: a family of cell surface receptors. Cell, 48549-554. Kleinmann, H.K., Klebe, R.J., and Martin, G.R. (1981) Role of collagenous matrices in the adhesion and growth of cells. J. Cell Biol., 88:473-485. Kristensen, T., Saris, C.J.M., Hunter, T., Hicks, L.J., Noonan, D.J., Glenney, J.R., and Tack, B.F. (1986) Primary sequence of bovine calpactin I heavy chain (~361,a major cellular substrate for retro+

+

+

+

522

WIRL AND SCHWARTZ-ALBIEZ

viral protein-tyrosine kinases: homology with the human phospholipase A, inhibitor, lipocortin. Biochemistry, 2.5r4497-4503. Kurkinen, M., Taylor, A,, Garrels, J.I., and Hogan, B.L.M. (1984) Cell surface associated proteins which bind native type IV collagen or gelatin. J. Biol. Chem., 259:5915-5922. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685. Lee, E.Y.H.P., Lee, W.H., Kaetzel, C.S., Parry, G., and Bissel, M. (1985) Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion. Proc. Natl. Acad. Sci. USA, 821419-1423. Li, M.L., Aggeler, J., Farson, D.A., Hatier, C., Hassell, and Bissell, M.J. (1987) Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc. Natl. Acad. Sci. USA, 84:136-140. Lowry, O.H., Rosebrough N.J., Farr, A.L., and Randall R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 19.3:265-275. Mauch. C., von der Mark. K., Helle, 0..Mollenhauer. J., Haffle. M.. and Krieg, T. (1988) A.defective cell surface collagen-binding ‘pro: tein in dermatosparactic sheep fibroblasts. J. Cell. Biol., 106:205211. Mollenhauer, J., and von der Mark, K. (1983) Isolation and characterization of a collagen-binding glycoprotein from chondrocyte membranes. EMGO J.,2r45-50. Moore, P.B., and Dedman, J.R. (1982) Calcium-dependent protein binding to phenothiazine columns. J . Biol. Chem., 257:9663-9667. Owens, R.J., Gallagher, C.J., and Crumpton, M.J. (1984) Cellular distribution of p68, a new calcium-binding protein from lymphocyes. EMBO J, 3:945-952. Pfaffle, M., Ruggiero, F., Hofmann, H., Fernandez, M.P., Selmin, O., Yamada, Y., Garrone, R., and von der Mark, K. (1988) Biosynthesis, secretion and extracellular localization of anchorin 11, a collagenbinding protein of the calpactin family. EMBO J, 7t2335-2342. Saga, S., Nagata, K., Chen, W.T., and Yamada, K.M. (19871 pH-dependent function, purification, and intercellular location of a major collagen-binding glycoprotein. J. Cell Biol., 105:517-527. Saris, C.J.M., Tack, B.F., Kristensen, T., Glenney, J.R., and Hunter T. (1986) The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin heavy chain) reveals a multidomain protein with internal repeats. Cell, 46t210-212. Shadle, P.J., Gerke, V., and Weber, K. (1985) Three Ca2+-binding proteins from porcine liver and intestine differ immunologically and physicochemnically and are distinct in CaZ+ . J. Biol. Chem., 260:16345-16360. Silva, F.G., Sherill. K., Spurgeon, S., Sudhof, T.C., and Ston, D.K. (1986) High-level expression of the 32.5-kilodalton calelectrin in ductal epithelial as revealed by immunocytochemistry. Differentiation, 33:175-183.

Sudhof, T.C., Zimmerman, C.W., and Walker, J.H. (1983) Calelctrin in human blood cells. Eur. J. Cell Biol., 30:214-218. Siidhof, T.C., Ebbecke, G., Walker, J.H., Fritsche, U., Boustead, C. (1984) Isolation of mammalian ealelectrins: a new family of ubiquitous Ca2+-regulatedproteins. Biochemistry, 23:1103-1109. Sugrue, S.P. (1987) Isolation of collagen-binding proteins from embryonic chicken corneal epithelial cells. J. Biol. Chem., 262: 3338-3343. Tait, J.F., Sakata, M., McMullen, B., Miao, C.H., funakoshi, T., Hendrickson, L.E., and Fujikawa, K. (1988) Placental and anticoagulant proteins: isolation and comparative characterization of four members of the lipocortin family. Biochemistry, 27,6268-6276, Towbin, H., Staehelin, T., and Gordon, J. (1979)Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Tomaselli, J.K., Damsky, C.H., and Reichardt, L.F. (1988) F‘urification and characterization of mammalian integrins exprssed by a rat neuronal cell line (PC12): evidence that they function as a@ heterodimeric receptors for laminin and type IV collagen. J. Cell Biol., 107r1241-1252. Wakimoto, H., and Oka, T. (1983) Involvement of collagen formation in the hormonally induced functional differentiation of mouse mammary gland in organ culture. J. Biol. Chem., 258:3775-3779. Walker, J.H., Obrocki, J., and Sudhof, T.C. (1983) Calelectrin, a “calcium dependent membrane binding protein” associated with secretory granules in Torpedo cholinergic motor nerve endings and rat adrenal medulla. J. Neurochem., 41r139-145. Wirl, G., and Pfaffle, M. (1988) Collagen-binding proteins of rat mammary tumor epithelial cells: a biochemical and immunological study. Exp. Cell. Res., 176t20-37. Wirl, G., Kronberger, A,, and Langanger, G. (1984) Epithelial organoids and mononuclear phagocytes from OMBA-induced mammary tumors of the rat secrete collagenase in vitro. Exp. Cell Res., 151: 502-518. Weber, K., Johnsson, N., Plessmann, U., Van, P.N., Soling, H.D., Ampe, C., and Vandekerckhove, J. (1987) The aminoacid sequence of protein I1 and its hos hor lation site for protein kinase C; the domain structure Ca 8+-modulated lipid binding proteins. EMBO J , 6t1599-1604. Zokas, L., and Glenney, J.R. Jr. (1987) The calpactin light chain is tightly linked to the cytoskeletal form of calpactin I: studies using monoclonal antibodies to calpactin subunits. J . Cell Bio., 105r21112121. Yang, J., Richards, J., Guzman, R., Imagawa, W., and Nandi, S. (1979) Sustained growth and three-dimensional organization of primary tumor epithelial cells embedded in collagen gels. Proc. Natl. Acad. Sci. USA, 77:2088-2092.