©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Recombinant Domain III of Perlecan Promotes Cell Attachment through Its RGDS Sequence (*)

(Received for publication, October 14, 1994; and in revised form, November 11, 1994)

Shukti Chakravarti (1) Teresa Horchar (2) Bahiyyah Jefferson (2) Gordon W. Laurie (3) John R. Hassell (2)(§)

From the  (1)Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4955, (2)Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, and (3)Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Perlecan has been previously been shown to support attachment of a wide variety of cells through interactions of its core protein with the cell surface. The core protein domains involved in cell adhesion are, however, unknown. The laminin-like domain III of murine perlecan contains an RGDS sequence and is a likely candidate for supporting integrin-mediated cell attachment. We made a cDNA construct corresponding to domain III and containing an in frame signal peptide at the 5` end as well as in frame a stop codon at the 3` end by using cDNA clones to perlecan. The construct was inserted into the pRC/CMV vector and transfected into HT1080 cells, and the secreted recombinant domain III, a 130-kDa protein, was purified from the medium. The size of proteolytic fragments produced by digestion with V8 protease as well as analysis of the rotary shadowed image of the recombinant protein indicated it was produced in a native conformation. Recombinant domain III coated on tissue culture dishes, supports adhesion of an epithelial-like mouse mammary tumor cell line MMT 060562 in a dose-dependent manner. This interaction was inhibited specifically by the RGDS synthetic peptide and intact perlecan, but not laminin. This domain III RGD-dependent cell attachment activity indicates a role for perlecan in integrin-mediated signaling.


INTRODUCTION

Perlecan, a heparan sulfate proteoglycan with a large (M(r) = 400,000) core protein, is a major component of all basement membranes. The primary structure deduced from sequencing murine and human cDNA clones indicates that the core protein has five distinct domains (Noonan et al., 1988, 1991; Murdoch et al., 1992; Kallunki and Tryggvason, 1992). The N-terminal end of the core protein, domain I, consists of amino acid sequences unique to perlecan and has three potential heparan sulfate attachment sites. Domain II is homologous to low density lipoprotein binding region of the low density lipoprotein receptor. Domain III consists of three cysteine-free laminin-like globules that alternate with cysteine-rich EGF(^1)-like units and is most homologous to the N-terminal third of laminin A chain. Domain IV contains variably spliced immunoglobulin type repeats similar to those found in neural cell adhesion molecule. Domain V, at the C terminus of perlecan, consists of three cysteine-free neurexin-like globules (Ushkaryov et al., 1992) that alternate with EGF-like units and is most homologous to the C-terminal third of agrin (Patthy and Nikolics, 1994).

Perlecan is implicated in multiple and diverse functions (Timpl, 1993; Hassell et al., 1993). Determining the functions of the specific structural domains is critical to understand the functional diversity of this complex molecule. The heparan sulfate chains affect filtration of macromolecules (Farquhar, 1981), while the core protein interacts with itself (Yurchenco et al., 1987) and other basement membrane components, such as fibronectin (Hermans et al., 1990) and entactin/nidogen (Battaglia et al., 1992). A majority of the core protein interactions with other matrix components contribute to the assembly (Laurie et al., 1986) or maintenance of the matrix ultrastructure (Laurie, 1985). Both the core protein and the heparan sulfate chains can also bind growth factors and affect hemostasis (Timpl, 1993). Several studies have shown that cells can attach to the core protein; however, the signaling pathways involved are not yet understood. Hayashi et al. (1992) demonstrated attachment of endothelial cells to the intact perlecan core protein, and this attachment could be partially inhibited by the RGD synthetic peptide as well as an anti-beta1 integrin antibody. Murine perlecan has a single RGD sequence located in the second globular subdomain of domain III (Noonan et al., 1991). In the current study, we prepared a cDNA construct for domain III of perlecan from existing cDNA clones and used this construct in a mammalian expression vector system to express a recombinant domain III of perlecan. We show that the recombinant protein is secreted, folded properly, and is biologically active in supporting cell adhesion through cellular interactions of its RGDS sequence.


MATERIALS AND METHODS

Construction of Domain III

All cloning procedures were derived from Sambrook et al.(1987). A cDNA encoding all of domain III (amino acids 490-1680) was prepared from restriction enzyme digests of cDNA clones 54, 5, and 72 (Noonan et al., 1991), in pBIISK vector (Stratagene), by using sites in the multicloning region and within inserts. The resulting construct was a 3.6-kb cDNA with a HindIII and a XbaI site at the 5` and 3` ends, respectively, and with a stop codon at the XbaI site. A 180-base pair cDNA containing perlecan's start codon and in frame signal peptide was prepared from clone 16 by creating a HindIII site 14 amino acids (residue 35) after the signal peptide that would put the amino acid sequence in frame with the sequence at the 5` end of the domain III construct. The construction of a HindIII sites on the signal peptide and the domain III constructs added five amino acids, so that a sequence of five amino acids (Asp-Pro-Ser-Leu-Ilu) were created between residues 35 and 490 of perlecan. The signal peptide/domain III construct (3.7 kb) was ligated into the NotI/XbaI sites of the pRC/CMV vector (Invitrogen).

Cell Transfections

The signal peptide/domain III construct in pRC/CMV was transfected into HT1080 cells (a cell line derived from a human fibrosarcoma) using Lipofectin (Life Technologies, Inc.). Cells were seeded in 35-mm dishes at 3 times 10^5 cells/well and allowed to recover overnight and transfected using a 12 µl of Lipofectin to 6 µg of DNA ratio, for 6 h in serum-free medium. The medium was changed to one containing serum, and the cells were cultured for an additional 48 h. The cells were then removed by trypsinization and plated on 100-mm dishes and cultured in the presence 450 µg of G418/ml medium for 2-3 weeks. Clones resistant to G418 were isolated individually with a cloning cylinder, removed by trypsinization, and grown in G418-containing medium.

Detection of Recombinant Domain III

(i) For Western blot analysis, clonal cell lines resistant to G418 were grown to confluence in 35-mm dishes, the medium was changed to serum-free medium, and the cultures were maintained for an additional 48 h. The media were collected, concentrated 10-fold by centrifugation in Microsep tubes containing membranes with 10-kDa molecular mass cut-offs (Filtron, Northborough, MA). The concentrated media were fractionated by SDS-PAGE and transferred to nitrocellulose. The presence of domain III was detected with perlecan antiserum followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Klein et al., 1988). (ii) For Northern analysis, total RNA was isolated from immunopositive clonal cell lines by the guanidine thiocyanate-phenol-chloroform extraction method (Chomcznski and Sacchi, 1987). The RNA was electrophoresed in 1% agarose, transferred to nitrocellulose, and evaluated for the presence of a domain III message by Northern blot analysis using P-labeled (random-prime oligolabeling kit, Pharmacia Biotech Inc.) clone 54 DNA as a probe (Sambrook et al., 1987). (iii) For [S]methionine radiolabeling and immunoprecipitation, immunopositive cell lines were pulse-labeled with [S]methionine for 20 min in serum-free medium followed by a 5-h chase period with unlabeled methionine in serum-containing medium. Recombinant domain III was immunoprecipitated from pulse-radiolabeled cell lysates and medium using protein A-Sepharose beads bound to anti-perlecan IgG as described previously (Ledbetter et al., 1985). The radiolabeled immunoprecipitated proteins were reduced with dithiothreitol and separated by SDS-PAGE. The gel was embedded in Flouro-hance (Research Products International), and the dried gel was exposed to X-Omat film (Kodak) at -80 °C.

Purification of Recombinant Domain III

Clonal cell lines that were judged by Western blot to produce high levels of recombinant protein were grown to confluence in 100-mm dishes in the presence of G418. The medium was then changed to serum-free medium, and after 2 days of culture the medium was collected. An initial ammonium sulfate precipitation was performed by adjusting the medium to 20% saturation with solid ammonium sulfate. The precipitate was discarded, and the medium was adjusted to 60% saturation ammonium sulfate. The 20-60% precipitate containing domain III was redissolved in 2 M urea containing 0.05 M Tris, pH 6.8, and 0.1 M NaCl and applied to Superose 6 (Pharmacia) column. The elution position of domain III was monitored by enzyme-linked immunosorbent assay using an anti-perlecan antiserum. Those fractions containing domain III were combined, dialyzed against 2 M urea containing 0.05 M Tris, pH 6.8. The material was applied to a column of 15 Q (Pharmacia) and eluted with a 0.0-1.0 M NaCl gradient. The elution position of domain III was again monitored by enzyme-linked immunosorbent assay, and fractions containing domain III were combined, concentrated by centrifugation in Microsep tubes, and dialyzed against phosphate-buffered saline. The protein concentration was determined with a protein assay kit (Bio-Rad).

Enzymatic Digestion of Recombinant Domain III

The presence or lack of heparan sulfate side chains on domain III was determined by heparatinase digestion of both purified domain III and serum-free medium containing secreted domain III followed by SDS-PAGE and Western blotting (Klein et al., 1988). Purified recombinant domain III was also digested with V8 protease and analyzed for the presence of the V8-resistant 44- and 46-kDa peptides by SDS-PAGE with Coomassie Blue staining (Ledbetter et al., 1987).

Rotary Shadowing

Recombinant domain III was prepared for examination in the electron microscope using the Heuser(1989) method. Briefly, 1.5 µg of domain III in 100 µl of 50 mM sodium phosphate, 22 mM sodium chloride, pH 7.0, was absorbed to mica flakes, cryopressed, and then freeze-fractured, etched, and rotary shadowed. Replicas were examined in a JOEL 100 cx electron microscope. The lengths of domain III molecules were determined using a digitized tablet on line with a Macintosh SE computer.

Cell Adhesion Assay

MMT 060562 mouse mammary cells (American Type Culture Collection) were grown and maintained in high glucose Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cell adhesion assays were performed as described previously (Chakravarti et al., 1990). In brief, cell monolayers were radiolabeled with [^3H]thymidine (2.5 mCi/ml) for 12 h and then briefly trypsinized. The single cells were resuspended in serum-free Dulbecco's modified Eagle's medium containing 0.05% bovine serum albumin for use in attachment assays. 24-well tissue culture plates were used for attachment assays, coated with the recombinant or native proteins and blocked with bovine serum albumin. The radiolabeled cells were added to the wells at 5 times 10^4 cells/well and allowed to attach for 1 h at 37 °C. Unattached cells were removed by washing with phosphate-buffered saline, and the attached cells were solubilized in 2% SDS and counted in a liquid scintillator. Perlecan and laminin were purified from the EHS tumor as described previously for use in cell attachment assays (Ledbetter et al., 1987; Graf et al., 1987).

Attachment Inhibition Assays

The synthetic peptides RGDS and the control peptide GRADSP (Calbiochem) were dissolved in Dulbecco's modified Eagle's medium and adjusted to pH 7 with sterile sodium bicarbonate. The peptides or proteins were added at the time of plating of the cells, and cell attachment was determined after 1 h as described above.


RESULTS

Isolation of Transfected Cells

The first cDNA clone isolated to perlecan was clone 5, and it was obtained by screening an expression vector library with rabbit antisera to intact native perlecan (Noonan et al., 1988). Since clone 5 encodes for part of domain III, we were able to use existing rabbit antisera to native perlecan to screen the medium of transfected clonal cell lines for the production of recombinant domain III. Transfection of four 35-mm dishes with the domain III construct in the pRC/CMV vector and subsequent culture in G418 resulted in 18 clonal cell lines. Screening the media and dilutions of the media from each cell line with antisera to perlecan by SDS-PAGE-Western blot showed 15 of the clonal cell lines to be immunopositive and eight of these to be strongly immunopositive for recombinant domain III (not shown). Four of these eight (cell lines 6, 8, 9, and 12) were indistinguishable from one another by SDS-PAGE-Western blot and these were used in subsequent analysis.

Synthesis and Export of Recombinant Domain III

Northern analysis was performed on total RNA from the four independent G418-resistant, immunopositive, transfected HT1080 cell lines, using perlecan cDNA clone 54 as a probe (Fig. 1). Total RNA from all of the transfected cell lines, but not wild type HT1080 cells, contained a 4-kb message that hybridized to the probe. The size of this message is consistent with the 3.7-kb size of the construct for domain III. Native perlecan mRNA is 12-13 kb, and it was not detected.


Figure 1: Northern analysis of four clonal HT1080 cell lines permanently transfected with domain III mRNA. The recombinant produce is a 3.6-kb mRNA. Native perlecan mRNA (approx12-13 kb) is not detected.



Synthesis and secretion of the recombinant protein into the medium was analyzed in [S]methionine-labeled clonal cell line 6 by immunoprecipitation with a polyclonal anti-perlecan antiserum and followed by SDS-PAGE under reducing conditions (Fig. 2). After 20 min of pulse-labeling with [S]methionine, a 160-kDa recombinant protein could be immunoprecipitated from the cell lysate extracts. A similarly sized recombinant protein could be immunoprecipitated from the medium after a chase period of 5 h with unlabeled methionine-containing medium. Thus, the domain III cDNA construct is transcribed, translated, processed, and secreted as a protein that migrated with an apparent molecular mass of 160 kDa under reducing conditions.


Figure 2: Synthesis and secretion of domain III in transfected HT1080 clonal cell line. Cells were metabolically radiolabeled with [S]methionine, pulsed for 20 min, and chased for 5 h with unlabeled methionine. Recombinant domain III was immunoprecipitated from cell layer extracts and media with antisera to perlecan. The samples were reduced with dithiothreitol and analyzed by SDS-PAGE. CL, extracted cell layer; M, medium.



Purification

Recombinant domain III was purified from conditioned medium of clonal cell lines 6 and 12 as described under ``Materials and Methods.'' An ammonium sulfate precipitate of the medium containing recombinant domain III was solubilized in 2 M urea and purified by molecular serve and ion exchange chromatography. Immunostaining of the purified protein with perlecan antiserum revealed a single protein, approximately 130 kDa in size (Fig. 3). Reduction with dithiothreitol prior to SDS-PAGE increased the apparent molecular mass to 160 kDa (as seen in Fig. 3). This change in gel mobility upon reduction is due to the unfolding of the cysteine-rich EGF units in domain III. Heparatinase digestion of recombinant domain III did not change its apparent molecular mass or increase the intensity of the band. This indicates that this recombinant product is not made with heparan sulfate side chains.


Figure 3: Western blot of purified domain III. Purified domain III (not reduced) was resolved in a 5% SDS-polyacrylamide slab gel and transferred by Western blotting to an Immobilon filter. Domain III was detected by a polyclonal anti-perlecan antiserum and secondary anti-rabbit goat IgG conjugated to horseradish peroxidase.



Secondary Structure

Limited protease digestion is helpful in evaluating the structure of a protein. The peptide fragments produced by digesting recombinant domain III with V8 protease were analyzed by SDS-PAGE (Fig. 4). After 15 min of digestion, a 70- and a 44-kDa fragment appear. After 1 h of digestion, a 46-kDa band just above the 44-kDa band appears, and this band increases in intensity after 4 and 16 h of digestion. After 16 h of digestion the 70-kDa fragment is degraded, and the protease-resistant 44-46-kDa doublet is the major proteolytic product. A previous study showed that treatment of intact perlecan with V8 protease also initially generates the 44-kDa fragment, and prolonged enzyme treatment generates the 44-46-kDa doublet (Ledbetter et al., 1987). Amino acid sequence obtained from the 44- and 46-kDa peptides purified from V8-digested intact perlecan aligned with the deduced sequence in the two major EGF subdomains of domain III (Noonan et al., 1991). The resistance of the 44- and 46-kDa fragments to proteolysis is most likely due to the high proportion of cysteine-cysteine bonding in the EGF units. The production of the same proteolytic fragments from recombinant domain III as that obtained from intact perlecan indicates the recombinant protein is properly folded.


Figure 4: Digestion of domain III with V8 protease (at a ratio of 100:1) for 0-16 h, as indicated. Digested samples (not reduced) were resolved by SDS-PAGE on a 5% polyacrylamide slab gel and the fragments viewed by Coomassie Blue staining.



Examination of domain III in the electron microscope (Fig. 5) revealed a rod-shaped structure with several globules. This is consistent with structural predictions from sequence data and sequence homology with laminin short arms (Noonan et al., 1991). Domain III lengths measured 20 ± 1 nm long (n = 48). This length is approximately 25% of the total core protein length (Laurie et al., 1988; Paulsson et al., 1987) and this compares favorably with molecular mass of domain III (126 kDa) relative to the total mass (396 kDa) of perlecan core protein.


Figure 5: Electron micrographs of rotary shadowed domain III. a, low power field. The bar represents 15 nm. b-g, high power micrographs of domain III in which the globules are more apparent. The bar represents 10 nm. h, schematic diagram of domain III.



Cell Adhesion

The cell attachment activity of domain III was tested using radiolabeled MMT cells (Fig. 6). The cells attached to recombinant domain III in a dose-dependent manner, with 40% of the cells attaching at a maximum dose of 2 µg of domain III/well. As a positive control, 100% of the cells attached to wells containing 1 µg of laminin (mouse).


Figure 6: Dose-dependent adhesion of mouse mammary tumor cells to domain III and laminin. [^3H]Thymidine-labeled MMT 060562 cells were added to 24-well tissue culture dishes (5 times 10^4 cells/well) coated with domain III or laminin as indicated. Attachment is expressed as a percent of total cells added. Mean basal level of attachment, defined as cell attachment to bovine serum albumin-coated wells, was subtracted from each observation. Each data point is an average of two independent observations.



To determine if the cell adhesive activity of domain III is due to cellular recognition of the RGDS sequence, attachment to domain III was assessed in the presence of increasing amounts of soluble RGDS, added to the medium at the time of plating the cells (Fig. 7). As little as 10 µg/ml RGDS inhibited cell attachment completely, while the control GRADSP peptide had very little effect.


Figure 7: RGDS mediated inhibition of cell attachment to domain III. Cells, preincubated with increasing amounts of RGDS or GRADSP, were allowed to attach to wells coated with 1 µg of domain III. Percent attachment was calculated as in Fig. 6, and each data point is an average of two observations.



To determine whether the RGDS-mediated attachment activity of domain III, demonstrated above, is present in intact native perlecan and, hence, biologically significant, attachment inhibition assays with intact perlecan were also performed (Fig. 8). MMT cells were incubated in domain III (1 µg/well)-coated wells in the presence of 100 µg/well soluble perlecan or laminin, as a control. Intact perlecan completely inhibited attachment to domain III, while laminin had no effect.


Figure 8: Intact perlecan, but not intact laminin (100 µg/well), inhibits cell adhesion to domain III. The results represent the mean of three measurements ± S.D.




DISCUSSION

Perlecan is known to support attachment of various types of cells (Clement and Yamada, 1990; Clement et al., 1989). More recently, murine perlecan has been shown to support adhesion of aortic endothelial cells involving integrins binding an RGD sequence (Hayashi et al., 1992). Domain III of perlecan contains the single RGD sequence present in murine perlecan. Since it is difficult to purify large quantities of perlecan and further purify proteolytically cleaved domains, we produced domain III as a recombinant protein and evaluated its cell attachment activity.

HT1080 cells transfected with the domain III construct, produced a 4-kb domain III transcript, as evidenced by Northern analysis on total RNA. The 4-kb message was translated into a 130-kDa secreted recombinant protein, whose apparent molecular mass increased to 160 kDa upon reduction. The 130-kDa size obtained for unreduced domain III on SDS-PAGE is close to the 126-kDa size predicted from its molecular mass. The increase in apparent molecular mass to 160 kDa upon reduction is due to the unfolding in its cysteine-rich EGF-like units. Even though recombinant domain III was derived from the core protein of a proteoglycan, the recombinant protein produced did not contain heparan sulfate side chains. The side chains are thought to be attached to domain I of perlecan, and although the first 14 amino acids of domain I are part of this construct, it does not contain the heparan sulfate attachment signal.

V8 protease digests of domain III release the same size 44- and 46-kDa fragments that have been previously shown to be derived from the EGF-rich regions in domain III of native perlecan (Ledbetter et al., 1987; Noonan et al., 1991). This indicates that the recombinant domain III is folded similarly, if not identically, to that domain in native perlecan. Interestingly, although the total number of cysteines in domain III is an even number, each of the four EGF subdomains in domain III contain an odd number of cysteines (Noonan et al., 1991). This suggests that the cysteine-cysteine pairing goes across the cysteine-free globules in domain III. Furthermore, in rotary shadow electron micrographs, the alternating globular units and cysteine-rich EGF units of recombinant domain III have a beaded appearance as in intact perlecan (Paulsson et al., 1987; Laurie et al., 1988). These observations suggest that the primary and secondary structure of recombinant domain III is similar, if not identical, to domain III of native perlecan. The recombinant protein supported attachment in a dose-dependent manner with 40% of the cells attached at the maximum dose. All of this attachment, however, could be abolished with the RGDS synthetic peptide. This indicates that the RGDS sequence is the only cell binding site in the recombinant protein. Hayashi et al.(1992) reported a considerably higher percent attachment of cells to the intact core protein, but this attachment could be reduced by only 40% in the presence of RGDS peptide. Taken together, these findings suggest that the RGDS-dependent site is in domain III and that the RGDS-independent cell adhesion sites in perlecan are in the other domains. To address the possibility that subtle differences in the folding of domain III is exposing a RGDS site which may be cryptic in native perlecan, we examined the ability of intact perlecan to inhibit cell attachment to recombinant domain III. Indeed, intact perlecan, but not laminin, could compete with domain III for cell surface receptors and block attachment to domain III. Although mouse laminin also supports integrin-mediated cell attachment, its RGDS sequence in the P1 region is cryptic and can be unmasked only by proteolysis (Aumailley et al., 1990). Most laminin-mediated adhesion is thought to occur through the E8 region (Deutzmann et al., 1990).

The role of extracellular matrix molecules in controlling cell differentiation and gene expression is well known. Cell surface integrin receptors, considered to be the major players in regulating these processes, affect cellular signaling pathways, such as, tyrosine phosphorylation, cytoplasmic alkalization, and intracellular Ca fluctuations (reviewed by Juliano and Haskill(1993)). In addition, integrin-mediated attachment of cells to the extracellular matrix may be essential for the survival of anchorage dependent cells (Meredith et al., 1993). Our study suggests that perlecan may be an extracellular matrix ligand in these phenomena.

The RGDS sequence in domain III of murine perlecan is also found in Caenorhabditis elegans (Rogalski et al., 1993). Therefore, this RGDS site is conserved in organisms from C. elegans to mouse, and perlecan presumably serves as a major integrin ligand in organisms up to rodents in the evolutionary hierarchy. The loss of this RGDS sequence in human perlecan (Kallunki and Tryggvason, 1992; Murdoch et al., 1992) raises the possibility of ligand degeneracy and the same cell signaling pathway being adequately induced by some other RGDS-containing ECM component.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM45380, FY09747, and P30 EY08098 and by Research to Prevent Blindness, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Doris and Jules Stein Research to Prevent Blindness Professor. To whom correspondence should be addressed: Dept. of Ophthalmology, University of Pittsburgh School of Medicine, Eye and Ear Institute, 203 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-647-5754; Fax: 412-647-5880.

(^1)
The abbreviations used are: EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s).


REFERENCES

  1. Aumailley, M., Gerl, M., Sonnenberg, A., Deutzmann, R., and Timpl, R. (1990) FEBS Lett. 262, 82-86 [CrossRef][Medline] [Order article via Infotrieve]
  2. Battaglia, C., Mayer, U., Aumailley, M., and Timpl, R. (1992) Eur. J. Biochem. 208, 359-366 [Abstract]
  3. Chakravarti, S., Tam, M. F., and Chung, A. E. (1990) J. Biol. Chem. 265, 10597-10603 [Abstract/Free Full Text]
  4. Chomcznski, P, and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  5. Clement, B., and Yamada, Y. (1990) Exp. Eye Res. 187, 320-323
  6. Clement, B., Segui-Real, B., Hassell, J. R., Martin, G. R., and Yamada, Y. (1989) J. Biol. Chem. 264, 12467-12471 [Abstract/Free Full Text]
  7. Deutzmann, R., Aumailley, M., Wiedemann, H., Pysny, W., Timpl, R., and Edgar, D. (1990) Eur. J. Biochem. 191, 512-522
  8. Farquhar, M. G. (1981) in Cell Biology of the Extracellular Matrix (Hay, E. D., ed) pp. 335-378, Plenum, New York
  9. Graf, J., Iwamoto, Y., Sasaki, M., Martin, G. R., Kleinman, H. K., Robey, F. A., and Yamada, Y. (1987) Cell 48, 989-996 [Medline] [Order article via Infotrieve]
  10. Hassell, J. R., Blochberger, T. C., Rada, J. A., Chakravarti, S., and Noonan, D. (1993) in Advances in Molecular and Cell Biology, Vol 6: The Extracellular Matrix (Kleinman, H., ed) pp. 69-113, J. A. I. Press, Greenwich, CT
  11. Hayashi, K., Madri, J. A., and Yurchenco, P. D. (1992) J. Cell Biol. 119, 945-95 [Abstract]
  12. Heremans, A., De Cock, B., Cassiman, J.-J., van der Berghe, H., and David, G. (1990) J. Biol. Chem. 265, 8716-8724 [Abstract/Free Full Text]
  13. Heuser, J. (1989) J. Elect. Microsc. Tech. 13, 244-263 [Medline] [Order article via Infotrieve]
  14. Juliano, R. L., and Haskill, S. (1993) J. Cell Biol. 120, 577-585 [Medline] [Order article via Infotrieve]
  15. Kallunki, P., and Tryggvason, K. (1992) J. Cell Biol. 116, 59-571
  16. Klein, D. J., Brown, D. M., Oegema, T. R., Brenchley, P. E., Anderson, J. C., Dickenson, M. A., Horigan, E. A., and Hassell, J. R. (1988) J. Cell Biol. 106, 963-970 [Abstract]
  17. Laurie, G. W. (1985) Dev. Biol. 108, 299-309 [Medline] [Order article via Infotrieve]
  18. Laurie, G. W., Bing, J. T., Kleinman, H. K., Hassell, J. R., Aumailley, M., Martin, G. R., and Feldman, R. J. (1986) J. Mol. Biol. 189, 205-216 [Medline] [Order article via Infotrieve]
  19. Laurie, G. W., Inoue, S., Bing, J. T., and Hassell, J. R. (1988) Am. J. Anat. 181, 320-326 [Medline] [Order article via Infotrieve]
  20. Ledbetter, S. R., Tyree, B., Hassell, J. R., and Horigan, E. A. (1985) J. Biol. Chem. 260, 8106-8113 [Abstract/Free Full Text]
  21. Ledbetter, S. R., Fisher, L. W., and Hassell, J. R. (1987) Biochemistry 26, 988-995 [Medline] [Order article via Infotrieve]
  22. Meredith, J. E., Fazell, B., and Schwartz, M. A. 1993. J. Mol. Biol. Cell 4, 953-961
  23. Murdoch, A. D., Dodge, G. R., Cohen, I., Tuan, R. S., and Iozzo, R. V. (1992) J. Biol. Chem. 267, 8544-8557 [Abstract/Free Full Text]
  24. Noonan, D. M., Horigan, E., Ledbetter, S., Vogeli, G., Sasaki, M., Yamada, Y., and Hassell, J. R. (1988) J. Biol. Chem. 263, 16379-16387 [Abstract/Free Full Text]
  25. Noonan, D., Fulle, A., Valente, P., Cai, S., Horigan, E., Sasaki, S., Yamada, Y., and Hassell, J. R. (1991) J. Biol. Chem. 266, 22939-22947 [Abstract/Free Full Text]
  26. Patthy, L., and Nikolics, K. (1994) Neurochem. Int. 24, 301-316 [Medline] [Order article via Infotrieve]
  27. Paulsson, M., Yurchenco, P. D., Ruben, G. C., Engel, J., and Timpl, R. (1987) J. Mol. Biol. 197, 297-313 [Medline] [Order article via Infotrieve]
  28. Rogalski, T. M., Williams, B. D., Mullen, G. P., and Moerman, D. G. (1993) Genes & Dev. 7, 1471-1484
  29. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  30. Timpl, R. (1993) Experentia 49, 417-428 [Medline] [Order article via Infotrieve]
  31. Ushkaryov, Y. A., Petrenko, A. G., Geppert, M., and Südhof, T. C. (1992) Science 257, 50-56 [Medline] [Order article via Infotrieve]
  32. Yurchenco, P. D., Cheng, Y.-S., and Ruben, G. C. (1987) J. Biol. Chem. 262, 17668-17676 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.