©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping of Network-forming, Heparin-binding, and 11 Integrin-recognition Sites within the -Chain Short Arm of Laminin-1 (*)

Holly Colognato-Pyke (1), Julian J. O'Rear (2), Yoshihiko Yamada (3), Salvatore Carbonetto (4), Yi-Shan Cheng (1), Peter D. Yurchenco (1)(§)

From the (1) Departments of Pathology and (2) Microbiology and Molecular Genetics, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, (3) NIDR, National Institutes of Health, Bethesda, Maryland 20892, and (4) Center for Neuroscience Research, McGill University, Montreal, Quebec H3A 2B2, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cell-interactive and architecture-forming functions are associated with the short arms of basement membrane laminin-1. To map and characterize these functions, we expressed recombinant mouse laminin-1 -chain extending from the N terminus through one third of domain IIIb. This dumbbell-shaped glycoprotein (r1(VI-IVb)`), secreted by mammalian cells, was found to possess three activities. 1) Laminin polymerization was quantitatively inhibited by recombinant protein, supporting an -chain role for a three-short arm interaction model of laminin self-assembly. 2) r1(VI-IVb)` bound to heparin, and the activity was localized to a subfragment corresponding to domain VI by I-heparin blotting. 3) PC12 rat pheochromocytoma cells adhered to, and rapidly extended branching neurites on, r1(VI-IVb)`, with adhesion inhibited by 1 and 1 integrin chain-specific antibodies. The ability of anti-laminin antibody to block PC12 cell adhesion to laminin was selectively prevented by absorption with r1(VI-IVb)` or -chain domain VI fragment. This active integrin-recognition site could furthermore be distinguished from a second cryptic 11-binding site exposed by heat treatment of fragment P1`, a short arm fragment lacking globules. Thus, a polymer-forming, a heparin-binding, and the active 11 integrin-recognition site are all clustered at the end of the -chain short arm, the latter two resident solely in domain VI.


INTRODUCTION

Laminin-1 (EHS-laminin) ()is a prototype for a growing family of closely related glycoproteins that appear to be essential components of basement membranes. This laminin contributes to the architecture of basement membranes in different tissues as well as provides information for cell adhesion, migration, and differentiation. Laminin-1 is composed of three distinct polypeptide chains, 1, 1, and 1 (formerly A, B1, and B2, respectively), joined in a multidomain structure possessing three short arms and one long arm (Burgeson et al., 1994). Each of these arms is subdivided into globular and rodlike domains. Studies involving in vitro self-assembly and the analysis of cell-formed basement membranes have shown that laminin-1 exists as a polymer, forming part of a basement membrane network (Yurchenco et al., 1985, 1992; Schittny and Yurchenco, 1990; Yurchenco and Cheng, 1993). Laminin-1 has been found to mediate a variety of cellular functions through integrin receptor-mediated recognition including cell adhesion, spreading, locomotion, neurite outgrowth, and differentiation (Edgar et al., 1984; Reh et al., 1987; Perris et al., 1989; Sonnenberg et al., 1990; Tomaselli et al., 1990; Calof and Lander, 1991; Sung et al., 1993; Glukhova et al., 1993; Clyman et al., 1994).

Several of these functions have been found to be associated with the short arms. First, the short arms have been found to participate in laminin polymerization (Schittny and Yurchenco, 1990; Yurchenco et al., 1992; Yurchenco and Cheng, 1993). A recently proposed three-arm interaction hypothesis of laminin polymerization (Yurchenco and Cheng, 1993) further holds that self-assembly is mediated through the end regions of each of the three short arms. A prediction of this model is that each short arm can independently and competitively inhibit laminin polymerization. However, it has not been possible to formally test this prediction using conventional biochemical techniques because of an inability to separate the - and -chains. Second, several heparin binding sites have been thought to reside in the short arms (Skubitz et al., 1991; Kouzi-Koliakos et al., 1989; Yurchenco et al., 1990), although the location of these sites has remained obscure. Third, the 11 integrin has been found to selectively interact with large short arm fragments containing all or most of the short arm domains (Hall et al., 1990; Tomaselli et al., 1990; Goodman et al., 1991).

Most functional activities of laminin appear to be dependent upon the conformational state of the glycoprotein. Specifically, self-assembly and its calcium dependence, nidogen (entactin) binding to laminin, 61 integrin recognition of the long arm, heparin binding to the proximal G domain (cryptic), and RGD-dependent recognition of the short -arm of laminin (cryptic) have all been found to be conformationally dependent (Yurchenco et al., 1985; Deutzmann et al. 1990; Goodman et al., 1987, 1991; Fox et al., 1991; Yurchenco and Cheng, 1993; Sung et al., 1993). Two consequences of improperly folded laminin, loss of normal functional activity and the activation of previously cryptic activities, argue that it is important to map and characterize biological activities using correctly folded glycoprotein.

In our present study we have combined several approaches to establish and map functions of the -chain short arm of laminin-1 (Fig. 1). These complementary approaches are ( a) recombinant technology, in which glycoprotein domains can be engineered and generated in quantities sufficient for biochemical and functional analysis; ( b) assays with a battery of defined fragments; and ( c) characterization of interactions using antibodies specific both for receptors and laminin domains. With these approaches, it has been possible to establish and map a polymerization activity, a heparin binding activity, and an 11 integrin recognition activity within the proximal moiety of the -chain short arm. Furthermore, a comparison between native laminin, recombinant glycoprotein, and fragments has led to the identification of an unrelated cryptic, recognition site for the 11 integrin, resolving an apparent discrepancy with an earlier study (Goodman et al., 1991). An abstract of this work was recently reported (Colognato-Pyke et al., 1994).


MATERIALS AND METHODS

DNA Constructions

A pCIS mammalian expression vector (Gorman et al., 1990) containing a full-length mouse laminin 1-chain cDNA was prepared as follows. An 0.7-kb mouse laminin 1 cDNA covering the first EcoRI fragment (bases 1-718) had a purified 3.0-kb laminin 1 cDNA (A-04), approximately base 950 to base 3943 (Sasaki et al., 1988), inserted in the sense orientation by an EcoRI partial digest. The missing bases between the 0.7- and 3.0-kb fragments were restored by inserting in the sense orientation a 1.0-kb cDNA beginning at base 719 using an EcoRI partial digest. The duplication caused by this insertion was removed by cleavage with MamI (base 1726), producing the 5` mouse laminin 1 cDNA, bases 1-3943. Next, a mouse placental library in ZAP (generously provided by Dr. Markku Kurkinen, Wayne State University, Detroit, MI) was screened with a laminin G-domain probe in order to retrieve the 3` portion of the laminin 1 cDNA. A cDNA covering bases 3944 ( EcoRI site) through 9503 (-chain C terminus) was rescued. The laminin 1 5` sequence was purified and inserted into Bluescript SK (Stratagene) by a complete XbaI digest and a partial EcoRI digest, and the XbaI cleavage site converted to a SalI cleavage site with a linker. The entire laminin 1 sequence contained in Bluescript SK was digested with SalI and inserted into the pCIS vector (Genentech, South San Francisco, CA) at the XhoI cleavage site in the polylinker region. The sequence encoding r1(VI-IVb)` (recombinant laminin 1-chain domains VI-IVb where prime (`) indicates extension into the adjacent domain) was generated by cleavage at base 2662 (corresponding to about one-third through domain IIIb of the -chain) with Tth111I, by blunting with the Klenow fragment of Escherichia coli DNA polymerase I, followed by insertion of a universal terminator sequence (dGCTTAATTAATTAAGC). Plasmid DNA was purified by CsCl-ethidium bromide equilibrium density gradient centrifugation.

Transfection and Cloning of Mammalian 293 Cells

Transfection of 293 cells (adenovirus-transformed primary embryonal kidney, human, ATCC CRL 1573) was carried out by calcium phosphate precipitation as modified for mammalian cell lines (Chen and Okayama, 1987). Plasmid DNA was co-transfected with JD214, a hygromycin resistance plasmid kindly provided by Dr. Joseph Dougherty (Robert Wood Johnson Medical School). Several days following transfection, r1(VI-IVb)`-transformed cells were selected for by the addition of 0.1 mg/ml hygromycin to the culture medium. Resistant colonies were isolated and grown in DMEM-F-12 serum-free medium (Life Technologies, Inc.) for 48 h. The harvested media were then screened for the presence of recombinant protein by Western analysis with a polyclonal antibody specific for a laminin short arm complex (anti-E1`).

Purification of Recombinant Glycoprotein

To obtain glycoprotein for biochemical and functional analysis, r1(VI-IVb)`-transformed 293 cells were expanded in number and grown in 150-cmtissue culture flasks (20 ml/flask), maintaining hygromycin throughout (1.8-day doubling time under these conditions). For a typical preparation, 30 flasks were seeded with cells at about 80% confluency, and the serum-containing maintenance medium was replaced by DMEM-F-12 serum-free medium. This medium was harvested after 48-72 h, placed on ice, adjusted to 1 m M PMSF and 2 m M EDTA, and centrifuged in the cold for 30 min at 10,000 rpm (Sorvall GSA rotor, DuPont) in order to remove cell debris. The medium was then passed through a heparin-Sepharose 4B affinity column (3.5 cm 2.5 cm inside diameter) equilibrated in 50 m M Tris-HCl, pH 8.0 (5 °C), 0.5 m M EDTA, and 0.5 m M PMSF. The bound protein was eluted with 1.0 M NaCl in the above buffer. Fractions containing the major protein peak were pooled and dialyzed overnight in the cold against 4 liters of 50 m M Tris-HCl containing 0.5 m M EDTA and 0.5 m M PMSF adjusted to pH 9.1 at 5 °C (equivalent to pH 8.5 at 25 °C). In the next step, the protein was bound to an HPLC TSKgel DEAE-5PW column (5 cm 0.5 cm inside diameter, glass; Toso-Haas, Philadelphia), at room temperature (25 °C) and eluted with a linear 0-0.5 M NaCl gradient. The salt concentration of the eluting solution was determined with a conductivity monitor attached to an in-line flow cell (Pharmacia Biotech. Inc., model 18-1500-00). The main eluted peak was pooled, dialyzed into 50 m M Tris-HCl, pH 8.0 (at 25 °C), 0.5 m M EDTA, and 0.5 m M PMSF, and then bound to a TSKgel heparin-5PW affinity column (5 cm 0.5 cm inside diameter, glass; Toso-Haas) equilibrated in the same buffer. A linear 0-1.0 M NaCl gradient was applied to the column and the recombinant glycoprotein eluted at 0.18 M NaCl. This purified glycoprotein was concentrated with Aquacide (Calbiochem) and dialyzed overnight against TBS-50, pH 7.4 (37 °C), 0.1 m M EDTA, and 0.5 m M PMSF. Approximately one milligram of purified recombinant protein was typically obtained from one liter of conditioned medium.

Preparation of Laminin-1 and Its Fragments

Laminin-1 was extracted in EDTA from lathyritic EHS tumor and purified as described by Yurchenco and Cheng (1993). Defined fragments (see Fig. 1) were prepared after digestion with elastase, cathepsin G, or pepsin. Elastase fragments E1` (short arm complex containing a nidogen fragment) (Yurchenco and Cheng, 1993), E8 (long arm and proximal G domain), E4 (-chain short arm domains VI and V), and E3 (distal G domain) were generated and purified by Sepharose CL-6B gel filtration (Pharmacia) and HPLC DEAE-5PW ion exchange (Toso-Haas). Fragment E1-X (Mann et al., 1988) was obtained from the larger fragment E1` (similar or identical to fragment E1X/Nd) as follows: fragment E1` was dialyzed into a dissociating buffer (2 M guanidine-HCl in 50 m M Tris-HCl, pH 7.4) in the cold. The protein was then chromatographed on a Sepharose CL-4B gel filtration column (90 cm 2.6 cm inside diameter) in the same buffer. The first (major) peak contained E1X and the second (minor) incompletely resolved peak contained E35 (see below) and a 90-100-kDa nidogen fragment (Mann et al., 1988). The first peak was pooled and dialyzed into TBS-50, pH 7.4, 0.1 m M EDTA. Elastase fragment E-35 was purified directly from E1` after dialysis into a urea dissociating buffer (8.0 M urea in 20 m M Tris-HCl, pH 7.4) by binding it to an HPLC heparin-5PW affinity column in the presence of the same buffer, followed by elution with a 0-1 M NaCl gradient. The protein was therefore separated from both E1X and the nidogen fragment (both nonbinding) in a single step, after which it was dialyzed into TBS-50, pH 7.4, 0.1 m M EDTA. Fragment E35, containing epitopes of -chain domain VI (Yurchenco and Cheng, 1993), has a molecular mass of 35 kDa by SDS-PAGE under reducing conditions (and an apparent molecular mass of 30 kDa under nonreducing conditions) and appears to be identical to fragment E30 described earlier (Mann et al., 1988). For preparation of fragment P1` (complex consisting of domain III of the -chain and the - and -chain short arms without the globular domains), laminin-nidogen complex was dialyzed into 10% acetic acid and digested with pepsin (1:15 enzyme substrate ratio) for 24 h at 15 °C, inhibited with pepstatin, dialyzed against 1 M calcium chloride in 50 m M Tris-HCl, pH 7.4, and purified by gel filtration through a Bio-Gel A-1.5 m (fine) column (2.6 90 cm inside diameter; Bio-Rad) in the same buffer. Crude fragment C8-9 (entire long arm and proximal G domain) (Bruch et al., 1989) was generated by incubating laminin with cathepsin G (Calbiochem) in TBS-50 containing 1 m M calcium chloride at an enzyme:substrate ratio of 1:300 for 5 h at 37 °C. Fragment C1-4 (complex containing all three short arms inclusive of globular domains) was prepared and solubilized as described previously (Yurchenco and Cheng, 1993). After removal of the C1-4 short arm complex, the supernatant, enriched in C8-9, but also containing some C8 (equivalent to E8) and C3 (equivalent to E3) was saved.


Figure 1: Laminin-1 recombinant protein, fragments, and functions. Left panel, short arm -chain functions were localized using these reagents. r1(VI-IVb)` is shown in the shaded rectangle. Elastase fragments E1`, E1X ( dark line border), E35, and E4 all correspond to regions of the short arms. Pepsin fragment P1`, which is similar to E1` but lacks the globular domains (EGF-like rod domains remain attached to each other through disulfide bonds) is shown in dark gray. Long arm fragments are E8, E3, and cathepsin G fragment C8-9. Entactin/nidogen ( En/Nd), a sulfated glycoprotein, is shown bound to domain III of the -chain of laminin. Right panel, function map with the , , and -chains shown in shades of decreasing darkness. EGF repeats are indicated by bars in the rod domains of the short arm. Domains, based on sequence analysis, are indicated in small Roman numerals and letters. The locations of heparin-binding, polymer-forming, and the active 11 integrin-binding sites are shown in bold-face for the -chain short arm. The long arm functions of heparin binding ( heparin), 61 integrin-recognition site ( 61), and dystroglycan ( DG), mapped in other studies, are indicated with gray-shaded labels.



Antibodies

Polyclonal antibody specific for laminin was generated by immunizing a New Zealand White rabbit with laminin-1, followed by affinity purification on a Sepharose CL-4B column coupled to laminin-1. Anti-laminin was found to separately react with laminin fragments E1`, E1X, E35, P1`, E8, and E4 as determined by direct ELISA binding assay. Rabbit polyclonal antibody specific for fragment E1` was prepared in the same manner, with affinity purification on an E1` affinity column, and cross-absorption on E4, E8, and E3 Sepharose-CL-4B columns. Specificity of anti-E1` was determined by ELISA and immunoblots. Monoclonal antibody 3A3, specific for the 1 subunit of rat 11 integrin (Tomaselli et al., 1990), is a mouse IgG raised against PC12 cells. The function-blocking monoclonal antibody 3A3 has been used previously to inhibit PC12 cell attachment to laminin (Turner et al., 1989). Rabbit polyclonal anti-1 serum was raised against purified rat 1 integrin subunit and characterized as described previously (Tawil et al., 1990, 1993). Rabbit polyclonal antibody prepared against a synthetic peptide containing the laminin 1-chain domain VI sequence RPVRHAQCRVCDGNSTNPRERH was generously provided by Dr. Amy Skubitz (University of Minnesota) and characterized as described by Yurchenco and Cheng (1993).

Protein Determinations, SDS-PAGE, Protein Immunoblotting, Lectin Blotting, and N-terminal Sequencing

( a) Protein in solution was determined either by absorbance at 280 nm or colorimetrically (Schittny and Yurchenco, 1990). The absorbance (280 nm) extinction coefficients to convert absorbance units to protein alone used for laminin, E1`, P1, E8, and E3 are as described by Yurchenco and Cheng (1993). The extinction coefficient for r1(VI-IVb)` was determined by amino acid analysis to be 1.59 (and 1.17 by Lowry assay). The extinction coefficient for E1X was treated as equivalent to that of E1`. ( b) SDS-PAGE was carried out in 3.5-12% linear gradient gels (Laemmli, 1970) as described by Yurchenco et al. (1990) and stained with Coomassie Brilliant Blue R250. Molecular mass standards used were myosin (205 kDa), -galactosidase (116 kDa), phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), and carbonic anhydrase (31 kDa). ( c) For sequencing, proteins were separated by SDS-PAGE and electrophoretically transferred (Towbin et al., 1979) onto 0.45-mm polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). The band of interest was cut out and the N-terminal sequence determined by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University (New Haven, CT). ( d) Electrophoretic transfer of proteins onto 0.2-mm nitrocellulose membranes (Schleicher & Schuell) was performed by the Western blot method of Towbin et al. (1979). Immunoblots were prepared by incubating nitrocellulose sheets with 10 mg/ml antibody and allowing bound antibody to react with I-protein A; reacting bands were visualized by autoradiography. Lectin blots were prepared as described previously (Sung et al., 1993) using an enzyme immunoassay Glycan Differentiation Kit (Boehringer Mannheim). ( e) Elastase digestion of r1(VI-IVb)` was carried out at 25 °C at a 1:50 enzyme:substrate (mass) ratio.

Heparin Binding Assays

( a) Protein samples were applied to a heparin-5PW HPLC column in 50 m M Tris-HCl, pH 7.4, containing 1 m M CaCland eluted using a 0-1.0 M NaCl programmed linear gradient. The NaCl concentration of each fraction was determined by conductivity. ( b) Radioiodinated heparin was prepared following tyramine labeling as described by San Antonio et al. (1994) except that a 15-kDa gel filtration fraction of heparin was used. Protein samples to be analyzed for heparin binding were subjected to SDS-PAGE, electrophoretically transferred onto a nitrocellulose membrane, and incubated with I-heparin (10cpm/ml) in TBS-50 containing 0.05% Tween 20 for several hours, followed by several washes with buffer alone. After drying, autoradiograms were prepared.

Platinum/Carbon Replication and Electron Microscopy

Glycerol rotary shadowed replicas of proteins for electron microscopy were prepared as described previously (Yurchenco and Cheng, 1993). Electron micrographs are shown as standard, rather than contrast-reversed, images.

Sedimentation Assay for Inhibition of Laminin Polymerization

Samples (0.1-ml aliquots) containing I-laminin (0.25 mg/ml) and variable concentrations of unlabeled ligand were incubated in 50 m M Tris-HCl, pH 7.4, 90 m M NaCl (TBS) containing 1 m M CaClin 0.5-ml Eppendorf tubes at 37 °C for 3 h, then centrifuged at 10,500 rpm for 15 min to separate supernatant (soluble laminin) from pellet (polymerized laminin). Inhibition of polymer formation by various ligands was determined as described by Yurchenco et al. (1992).

Cell Culturing, Cell Adhesion, and Neurite Outgrowth of PC12 Cells

PC12 rat pheochromocytoma cells were generously provided by Dr. Arthur Lander (Massachusetts Institute of Technology, Cambridge, MA) and maintained at 37 °C, 5% COin DMEM (Life Technologies, Inc.) containing 4.5 g/liter glucose supplemented with heat-inactivated 10% horse serum and 5% fetal calf serum (Upstate Biotechnology Inc.). In order to quantitate adhesion, cells were incubated overnight in the presence of 10 µCi/ml [ methyl-H]thymidine (Amersham Corp.). Half-area 96-well tissue culture plates (Costar Corp., Cambridge, MA) were coated with proteins in TBS containing 1 m M CaCl. Heat denaturation of protein substrates for cell adhesion assays was accomplished by incubating protein solutions, at desired substrate coating concentrations in coating buffer, at 80 °C for 10 min according to the method of Goodman et al. (1991). Following overnight incubation of substrates in the cold, the plates were washed with Dulbecco's phosphate-buffered saline (D-PBS; Life Technologies, Inc.) three times then blocked in D-PBS containing 0.5% bovine serum albumin for 2 h at 37 °C. Cells were harvested from maintenance culture flasks with 0.1% trypsin and 0.5 m M EDTA, washed once in adhesion medium (DMEM containing 0.5% bovine serum albmin), and resuspended in adhesion medium (cell suspensions were approximately 6 10cells/ml). Fifty µl of the suspension were added to each well. For antibody inhibition studies, the cell suspension was mixed with antibody prior to initiation of adhesion. When an experiment called for preincubation of antibody with soluble ligand, the antibody was mixed with ligand 30 min at room temperature prior to initiation of adhesion. Cells were incubated with substrate for 70 min at 37 °C, followed by three washes with D-PBS to remove unattached cells. Attached cells were solubilized overnight in 2% SDS, then transferred to 5 ml of Ecoscint A (National Diagnostics, Atlanta, GA) to determine radioactivity by scintillation counter (model LS-6000IC, Beckman Instruments). In order to prime PC12 cells for neurite outgrowth studies, the cells were cultured for 5-7 days in the presence of 50 ng/ml nerve growth factor (2.5S NGF; Sigma). These cells were then cultured on precoated six-well plates (Corning) for up to 24 h at 37 °C, 5% COin a defined medium (Bottenstein and Sato, 1979) supplemented with 50 ng/ml 2.5S NGF. At various intervals, the plates were placed on an inverted phase microscope (Olympus) fitted with a 35-mm camera and inspected, and five representative fields were photographed at low magnification.


RESULTS

Expression and Characterization of r1(VI-IVb)`

A cytomegalovirus promoter-based mammalian expression vector (pCIS) containing the 5` region of laminin-1 -chain cDNA was transfected into human 293 cells. This sequence encompasses domains VI, V, IVb, and the first third of domain IIIb, contains 11 consensus N XT/S glycosylation sites and has a protein mass of 94.2 kDa (following cleavage of the signal sequence). Secreted protein from hygromycin-selected clones was detected by Western blot as a single reactive band with an Mof 120 kDa (data not shown). This recombinant protein, r1(VI-IVb)`, was harvested from medium and purified to homogeneity as previously described. Purified r1(VI-IVb)` was evaluated by SDS-PAGE under reducing conditions, and a single band at 120 kDa was observed (Fig. 2 a, lane 1). The difference between the apparent molecular mass of 120 kDa and the predicted protein mass of 94 kDa was attributed to carbohydrate. Under nonreducing conditions, r1(VI-IVb)` migrated faster and appeared as a doublet of near-identical apparent mass. This was interpreted as evidence for intrachain disulfide bonds (Fig. 2 a, lane 2). The presence of carbohydrate on the recombinant glycoprotein was confirmed using a glycoconjugate analysis kit in which specific lectins detect and identify terminal sugar residues. Neither laminin nor r1(VI-IVb)` reacted with either Galanthus nivalis agglutinin (which recognizes terminal mannoses characteristic of ``high mannose'' N-linked oligosaccharides) or with peanut agglutinin (which recognizes the Gal (1, 2, 3) - N-GalNAc core disaccharide of O-linked oligosaccharides). Datura stramonium agglutinin (which recognizes Gal- (1, 2, 3, 4) GlcNAc found in complex and hybrid N-linked oligosaccharide structures) reacted with both native laminin and recombinant glycoprotein, as did Maackia amurensis agglutinin (which recognizes sialic acid terminally linked (2, 3) to galactose). The only difference in lectin specificities between the native and recombinant glycoprotein was seen with Sambucus nigra agglutinin which like M. amurensis agglutinin reacts with sialic acid, but is specific for the (2, 3, 4, 5, 6) linkage to galactose.


Figure 2: SDS-PAGE and electron microscopy. Panel a, purified r1(VI-IVb)` was electrophoresed in acrylamide gels under reducing ( lane 1) and nonreducing ( lane 2) conditions. The difference between the apparent molecular mass of 120 kDa and the predicted protein mass of 94 kDa is attributed to carbohydrate. Panels b, c, and d. Glycerol rotary shadow (8° angle) of r1(VI-IVb)` ( Panels b and c) and laminin ( Panel d). Recombinant 1(VI-IVb)` is seen as a dumbbell-shaped molecule encompassing domains VI, V, and IVb, with the short protruding stem representing the C-terminal portion which extends into domain IIIb. The corresponding region of laminin is encircled ( Panel d), with the third included globule of the -chain indicated by the arrow.



Electron microscopy of glycerol rotary shadowed platinum/carbon replicas was performed to analyze the morphology of r1(VI-IVb)` and compare it to that of native laminin-1 (Fig. 2 b-d). Micrographs of r1(VI-IVb)` revealed a dumbbell-shaped molecule, 20-25 nm in length, matching the morphology seen in the corresponding region of intact laminin. The -chain short arm in laminin can be recognized by its length (50 nm compared to 35-37 nm for the other short arms) and its three globules, one near the intersection of the cross (Fig. 2 d, arrow) (Yurchenco and Cheng, 1993). The N-terminal moiety of this arm, corresponding to r1(VI-IVb)`, is indicated with a circle (Fig. 2 d). Globular domains VI and IVb are clearly visible, and sometimes a short rodlike extension can be identified at the end of one globule, probably corresponding to the one-third of domain IIIb predicted from the cDNA construct. The rod (domain V) usually appears slightly bowed, similar to that observed in intact laminin.

The electron micrographs strongly suggested that r1(VI-IVb)` has a structure folded similar to native laminin, and this conclusion was further supported by limited proteolytic digestion. Elastase cleaves after common residues (uncharged, nonaromatic residues, e.g. Ser, Leu, Gly, Val), but only in a very limited number of sites in laminin because most potential sites are inaccessible to enzyme as a result of conformation (Yurchenco et al., 1993). Therefore, elastase should cleave at the same sites in the recombinant molecule only if it is folded in the same way as laminin. The short arm elastase fragment complex E1` contains a 35-kDa subfragment (designated here as E35) noncovalently attached to the rest of the complex, which is observed as a distinct species by SDS-PAGE and reacts with antibody to a synthetic peptide in -chain domain VI (Yurchenco and Cheng, 1993). A time course elastase digestion of r1(VI-IVb)` was carried out over a 22-h period and analyzed in Western blots (Fig. 3, Panel A). Two major products were identified, one of apparent M= 35,000, and the other of apparent M= 105,000 (Fig. 3, Panel B). At least one of the digestion products migrates somewhat anomalously, since the combined apparent molecular masses of 35 and 105 kDa exceed the original molecular mass of 120 kDa. Of the two digested products, only the 35-kDa band of r1(VI-IVb)` was found to react in immunoblots with an -chain domain VI-specific antibody. Furthermore, a second slightly smaller antibody-positive degradation product could also be identified in the recombinant digest (Fig. 3) and in preparations of E1` (Fig. 4). The antibody nonreactive band of r1(VI-IVb)`, thought to correspond to the more C-terminal moiety of the protein, was evaluated by N-terminal sequence analysis to identify the site of elastase cleavage. Eighteen sequential cycles of peptide release and analysis revealed the following sequence: S-H-R-N-L-R-D-L-D-P-I-V-T-R-R-W-W-W. This sequence corresponds to residues 225-242 in the 1-chain of mouse laminin-1 (Sasaki et al., 1988). Thus the cleavage site lies between residues 224 and 225, present at the end of domain VI near its junction with domain V. No sequence was obtained from the domain VI antibody-reactive recombinant E35, apparently because it was blocked at the N terminus.


Figure 3: Elastase digest of r1(VI-IVb)`. A, r1(VI-IVb)` was digested with elastase at 25° (enzyme-to-substrate mass ratio of 1:50). Aliquots removed at various time points were analyzed by SDS-PAGE under reducing conditions, transferred to nitrocellulose, incubated with anti-1-VI antibody, and visualized by autoradiography. By 22 h, r1(VI-IVb) was nearly completely digested, revealing the same 35-kDa band ( arrow) found in elastase fragment E1` (subfragment E35). The lane at the far right of Panel A shows laminin digested for 6 h. B, SDS-PAGE (reducing conditions, Coomassie Blue-stained) of r1(VI-IVb)` ( lane 1), and r1(VI-IVb)` after digestion with elastase ( lane 2). Recombinant fragment E35 ( arrow) and a recombinant fragment of 105-kDa apparent mass ( arrowhead) were the sole observed products of digestion. The 105-kDa fragment of r(VI-IVb)` was sequenced, revealing cleavage between amino acid residues 224 and 225 (see ``Results'').




Figure 4: Laminin polymerization inhibition. r1(VI-IVb)` and elastase fragments E1`, E4, and E3 were each incubated with I-labeled laminin at different ligand-to-laminin molar ratios. Aliquots were incubated for 3 h at 37 °C and then centrifuged in order to separate polymerized laminin (pellet) from soluble laminin (supernatant). Recombinant glycoprotein inhibited polymerization with the same concentration-dependence at fragment E4.



Laminin Polymerization

The contribution of the now isolated -chain short arm in the formation of a laminin network was examined using a quantitative polymerization inhibition assay (Fig. 4). Recombinant r1(VI-IVb)` was found to block laminin self-assembly in a concentration-dependent manner. Furthermore, r1(VI-IVb)` exhibited a molar inhibition capacity indistinguishable with another laminin single chain short arm moiety, the -chain fragment E4. A control laminin fragment (E3) did not inhibit self-assembly. These data supported the hypothesis of parallel separate contributions from the individual short arms for self-assembly in which the end of each short arm can competitively disrupt the central bond of the polymer formed by a contribution from each short arm (Yurchenco and Cheng, 1993).

Heparin Binding and the Domain VI Fragment E35

Heparin was found to bind r1(VI-IVb)`, a property exploited in developing a purification protocol. When bound to an HPLC heparin-5PW affinity column, r1(VI-IVb)` eluted at a NaCl concentration of 0.18 M (Fig. 5, left). Fragment E1` behaved similarly, eluting at 0.17 M NaCl. In contrast, the distal long arm fragment E3 eluted at 0.27 M, indicating a higher relative affinity for heparin. Fragment E1X, which unlike E1` lacks the -chain domain VI outer globule, did not bind heparin. Fragment E1` was further analyzed to determine which domain(s) possessed this heparin binding activity (Fig. 5, right). Under nonreducing conditions, a 35-kDa fragment and a 90-100-kDa nidogen fragment dissociate from the rest of the large complex while the remainder of the short arm complex (E1X) remains linked together through disulfide bonds (Mann et al.,1988; Yurchenco and Cheng, 1993). Only the E35 band (35 kDa apparent molecular mass as determined in reduced gels) was found to bind both to I-heparin and to an antibody specific for -chain domain VI. Fragment E1X, corresponding to the rest the short arm complex, failed to bind heparin both by blot assay (Fig. 5) and by heparin affinity chromatography. Therefore domain VI of the -chain was identified as a second heparin binding site in laminin. This binding site is of lower affinity relative to that present in fragment E3. Since heparin does not bind to fragment E1X, or -chain fragments E4 (Yurchenco et al., 1990) and E10 (Mann et al., 1988), which collectively account for the rest of the short arms, we concluded that -chain domain VI is the only heparin site present in the short arms.


Figure 5: Left panel, heparin binding to r1(VI-IVb)`. The recombinant glycoprotein was bound to an HPLC heparin-5PW affinity column in 50 m M Tris, 1 m M CaCl, pH 7.4, and eluted with a 0-1 M NaCl gradient. r1(VI-IVb) eluted from the column similar to fragment E1` and earlier than long arm fragment E3. In contrast, fragment E1X did not bind to the column. Right panel, heparin binding to E1`. Fragment E1` was subjected to SDS-PAGE under nonreducing conditions and electro-blotted onto nitrocellulose membranes. Bands consist of disulfide-linked three chains of E1` ( upper arrow), a noncovalently attached 35-kDa fragment ( lower arrow) and a 90-100-kDa nidogen fragment. The blots were incubated with one of three reagents: Amido black ( lane 1), I-labeled heparin ( lane 2, 10cpm/ml), or an antibody ( anti-1-VI) for a sequence found in laminin 1-chain domain VI ( lane 3), followed by autoradiography for the latter two. Molecular mass standard positions are marked on left ( lanes 1 and 2) and right ( lane 3). The heparin binding site was localized to the same 35-kDa fragment.



Integrin Recognition of the -Chain Short Arm

PC12 rat pheochromocytoma cells have previously been shown to adhere to laminin primarily through the 11 integrin (Turner et al., 1989; Tawil et al., 1990); furthermore, this cell interaction activity has been thought to reside somewhere in the short arms of the laminin molecule (Tomaselli et al., 1990). PC12 adhesion to the -short arm of laminin was evaluated in a cell adhesion assay as a function of substrate coat concentration (Fig. 6 A). PC12 cells readily adhered to laminin, r1(VI-IVb)`, and short arm complex E1`, but not to a smaller short arm fragment P1` (which lacks globular domains) and only at very low levels to distal long arm fragment E8. In a separate experiment (data not shown), PC12 cells were found not to adhere to fragments C8-9, E4, or E3. In order to determine whether r1(VI-IVb)` was recognized by the same integrin as intact laminin, cell adhesion was evaluated with integrin-blocking reagents specific for the 1 (monoclonal antibody 3A3) and 1 integrin chains. PC12 adhesion to intact laminin, r1(VI-IVb)` and fragment E1` was almost completely inhibited using either monoclonal antibody 3A3 or 1-specific antibody (Fig. 6 B). Therefore, we concluded that PC12 adhesion on r1(VI-IVb)`, like adhesion on E1` and laminin, is mediated through the 11 integrin and that the 11 integrin is the primary laminin-binding integrin present in PC12 cells.


Figure 6: A, substrate dependence of PC12 cell adhesion. Cells labeled overnight with [H]thymidine were plated onto wells previously coated separately with laminin, r1(VI-IVb)`, E1`, E8, or P1`. PC12 cells adhered strongly to laminin, r1(VI-IVb)`, and E1`, while adhering poorly or not at all to E8 or P1`. B, 11 integrin mediates PC12 adhesion to r1(VI-IVb)`.Cells were incubated in wells coated (0.1 µ M) separately with laminin, r1(VI-IVb)`, and E1` in the presence of either the anti-1 3A3 monoclonal antibody (1:50) or anti-1 (1:50) rabbit antiserum or control rabbit IgG (10 µg/ml). The average and S.E. of the mean of eight replicates is shown, and adhesion on laminin alone is defined as 100%. PC12 adhesion was inhibited on all three substrates by both antibodies. Thus 11 integrin mediates PC12 adhesion to r1(VI-IVb)`, E1`, and laminin.



Neurite Outgrowth

Recombinant -short arm glycoprotein was evaluated for its ability to support neurite outgrowth (Fig. 7). NGF-primed PC12 cells were maintained on substrates of intact laminin, r1(VI-IVb)`, and fragments over a period of about 24 h. These cells extended long branching cellular processes on r1(VI-IVb)` as well as on intact laminin and E1`, but not on P1`, E8, or E4. The neurite processes developed at equivalent rates on these substrates as judged by photographs taken at 0.5, 2, 6, 9, and 24 h (early time points not shown), such that by 6 h and later many processes extended more than 10 times their cell body length. Neurite sprouts could first be detected (about one-half to one cell length) by 0.5-h incubation. Long arm fragment E8 provided only minimal support for neurite outgrowth, while short arm fragments E4 and P1` did not support any significant neurite outgrowth. All substrates which supported adhesion also supported neurite growth.


Figure 7: Neurite outgrowth of PC12 cells. PC12 cells primed for 5 days in the presence of NGF were plated onto tissue culture dishes coated separately at 1 µ M with laminin, E1`, P1`, E8, E4, or r1(VI-IVb)`. Cells were incubated 20 h at 37 °C and examined for the extension of neurites. PC12 cells grown on a substrate of laminin, r1(VI-IVb), or E1` produced complex networks of neurites, with many neurites extending more than 10 times their cell body length. In contrast, E8 supported neurite formation only marginally, while substrates P1` and E4 did not support any neurite outgrowth.



Domain Mapping of the 11 Integrin-recognition Site by Laminin Antibody Absorption

The binding of cells to r1(VI-IVb)` and E1`, but not to P1` (Fig. 6 A) suggested the site was domain VI or domain IVb of the 1-chain. However, the conditions used to purify domain VI fragment E35 from E1` (8 M urea) inactivate cell binding activity, so this domain could not be assessed directly for integrin recognition activity. We circumvented this problem by using an antibody-absorption cell adhesion assay to more precisely map the binding site. This assay took advantage of a polyclonal antibody specific for intact laminin-1 which inhibited PC12 adhesion to laminin, an effect which could be reversed by coincubation with one of several soluble fragments or recombinant glycoprotein. In this approach, antibody is competitively prevented from binding to specific regions of laminin by absorption, i.e. opening up topographical ``windows'' on laminin that permit cell interactions if the ``window'' contains the active site. The fragments in solution were used at concentrations which were found to block antibody binding to epitopes in that region, as determined by both direct and competitive ELISA (data not shown). Furthermore, soluble ligand alone did not interfere with adhesion of cells to immobilized substrate, even at concentrations as high as 2 mg/ml (not shown). It is likely that the cell surface receptors bind cooperatively and simultaneously to multiple ligands immobilized on plastic, resulting collectively in a high affinity interaction, yet these same receptors are competed only by individual ligand molecules in solution, each a low affinity interaction.

PC12 cells were evaluated for adhesion to laminin both in the presence of ligand solutions alone and ligand solutions plus anti-laminin antibody (Fig. 8). As expected, E1`, E35, and r1(VI-IVb)` did not in themselves inhibit or otherwise perturb PC12 adhesion to immobilized laminin under the conditions of the assay. Anti-laminin antibody completely blocked cell adhesion to intact immobilized laminin, and when PC12 cells were incubated with antibody plus ligand in solution, only ligands containing -chain domain VI restored adhesion. When laminin antibody was separately absorbed with E1` or r1(VI-IVb)`, 82 and 87%, respectively, of cell binding was restored. When antibody was absorbed with subfragment E35 (domain VI, corresponding to the N-terminal moiety of r1(VI-IVb)`), 66% of cell binding (80% of E1` value) was restored. In contrast, when antibody was absorbed with E1X (short arm complex which is contiguous to, and noncovalently attached to, E35 in E1`), no cell binding (10%) above background was restored. Fragments E4, P1`, and E8 also failed to restore binding. These data provided evidence that the 11 integrin recognition of laminin is mediated through domain VI of the laminin -chain. If the active site resided in a different domain, i.e. in E1X (or a site contained in another fragment), then E1X (or another fragment) would have restored cell binding rather than E35. If one site resided in domain VI, and a second site resided elsewhere in E1X (regardless of whether the second site had the same or a different integrin-binding motif), then both E35 and E1X (or another fragment) would have restored activity.


Figure 8: Domain localization of the 11 recognition site by antibody absorption. Aliquots of PC12 cells in suspension were mixed with either 10 µg/ml rabbit polyclonal anti-laminin or nonimmune rabbit IgG control prior to incubation with immobilized laminin substrate (0.05 µ M). For different aliquots of cells, the suspensions were also mixed with 50 µg/ml of either r1(VI-IVb)`, fragment E1`, fragment E1X, fragment E35, fragment P1`, or fragment E8 as indicated. The average (± S.E., n = 8) cell adhesion is shown relative to IgG alone (100% relative = 85% absolute adhesion). Recombinant 1(VI-IVb)`, fragment E1`, and fragment E35 were able to selectively remove a population of antibodies which had previously blocked adhesion to native laminin, restoring PC12 adhesion in the presence of anti-laminin. Fragments E1X and P1` were unable to remove the adhesion-blocking antibody population, while fragment E8 had minimal activity.



A Cryptic 11 Integrin-binding Site in P1` Exposed by Heat Denaturation

Laminin substrates which had been heated at 80 °C prior to coating were assayed for their ability to support PC12 adhesion (Fig. 9). PC12 cell adhesion to both intact laminin and r1(VI-IVb)` was found to be stable to heat treatment, while the activity of fragment E1` was destroyed by heat treatment. Fragment E1`, in contrast to laminin and r1(VI-IVb)`, contains an internal cleavage at the end of domain VI, near the junction with domain V, in which the two segments are held together as a single species by noncovalent bonds (Yurchenco and Cheng, 1993). The difference between these proteins suggested that this internal cleavage was required for heat inactivation. This hypothesis was tested with r1(VI-IVb)` by treating it with elastase to generate the same internal cleavage (see Fig. 3). The elastase-treated recombinant glycoprotein retained its ability to support cell adhesion. However, like its E1` counterpart, elastase-treated r1(VI-IVb)` lost its ability to support cell adhesion following heat treatment. In contrast to the observations with laminin, E1` and r1(VI-IVb)`, cell adhesion to pepsin fragment P1`, normally absent, was activated by the same heat treatment.


Figure 9: Alteration of cell recognition by heat treatment of substrate. Panel A, tissue culture wells were coated with various laminin substrates (0.5 µm) including elastase-digested recombinant glycoprotein, before and after heat treatment. The average adhesion (±S.E., n = 8) is shown, and 100% adhesion is defined on laminin alone. Adhesion of PC12 cells to laminin or r1(VI-IVb)` was unaffected by heating, in contrast to adhesion to E1` or E8. However, if r1(VI-IVb)` was first treated with elastase (in order to produce a cleavage at the junction between domains VI and V), it then exhibited the same heat sensitivity as E1`, which possesses this same cleavage (see Fig. 5). In addition, cell adhesion to P1` increased after treatment with heat, interpreted as reflecting the exposure of a cryptic binding site. Panel B, adhesion of PC12 cells to tissue culture wells coated with 0.5 µ M heat-denatured P1` was completely inhibited by the presence of monoclonal antibody 3A3 (anti-1), while GRGDS peptide had no effect. Average percent adhesion is shown relative to adhesion on laminin alone (defined as 100%) for both panels (± S.E., n = 8).



The cell adhesion activity observed on heat-treated P1` was blocked with monoclonal antibody 3A3, but not by RGD peptide. These results are interpreted as evidence that there exists a cryptic 11-binding site in P1` and that this binding site is exposed in fragment P1` by heat denaturation. Furthermore, heat-activated P1` was unable to competitively absorb out antibody against the active 11 recognition site on whole laminin (not shown). These results provide evidence that the heat-activated 11 recognition site in P1` is not exposed in the native laminin molecule. We have further found that P1`, when maintained in the cold for periods of weeks, can become similarly activated.


DISCUSSION

In the present study, we identified and localized three functions of the N-terminal moiety of the 1-chain (A chain) short arm of laminin. First, the short arm was shown to possess an active self-assembly site equivalent in its polymer-inhibiting activity to the N-terminal moiety of the 1 short arm. Second, a novel heparin binding site was identified in this arm and was mapped to domain VI, the N-terminal globule. Third, the active 11 integrin-recognition site was mapped to the same domain VI. Finally, a cryptic 11 integrin activity, not present in the recombinant molecule, was identified elsewhere in the EGF-repeat regions of the short arms.

Structure of r1(VI-IVb)

The generation of recombinant mammalian basement membrane proteins that are structurally correct and functionally active has depended on their expression in eukaryotic cells which carry out necessary post-translational modifications of disulfide isomerization and glycosylation. Both insect and mammalian cells have proved useful in this regard; however, proteins produced by the latter contain the typical complex oligosaccharide found in native molecules. Nidogen (entactin), sparc, and fibulin-2 have all been expressed in and secreted by mammalian cells and appear to possess normal structure and activity (Fox et al., 1991; Nischt et al., 1991; Pan et al., 1993). The recombinant N-terminal moiety of the 1-chain short arm, r1(VI-IVb)`, possessed the same globule and rod morphology as the equivalent region of native laminin, contained the same restricted cleavage site, and possessed the expected complex carbohydrate.

Laminin Polymerization

Laminin-1, and probably other full chain length laminins as well, self-assemble into a reversible polymeric network that is found in basement membranes and may form an important part of the architectural scaffolding (Yurchenco et al., 1985, 1992; Schittny and Yurchenco, 1990; Yurchenco and Cheng, 1993). It has further been suggested that network formation may be thermodynamically facilitated by laminin attachment to its receptors in the lipid bilayer, resulting in a cell surface-stabilized polymer. The short arms of laminin-1 have previously been shown to be critical for this self-assembly and a model has recently been proposed describing the polymerization process as one mediated through the N-terminal domains of all three short arms. A prediction of this model is that each short arm separately should be able to inhibit formation of the laminin polymer. Previously, the 1-chain arm could not be independently tested because this arm was not available as an isolated entity, and generation of r1(VI-IVb)` solved this problem. The recombinant glycoprotein specifically inhibited laminin polymerization and therefore confirmed the prediction, supporting and strengthening the three-arm polymer hypothesis.

Domain VI of the Laminin-1 -Chain Short Arm Binds to Heparin

The short arms have previously been reported to bind to heparin; however, considerable uncertainty has existed over the location and number of binding sites (Kouzi-Koliakos et al., 1989; Yurchenco et al., 1990). In this study we have established that -chain domain VI contains the sole short arm heparin binding site. While conformation can play a role in heparin-protein interactions (Sung et al., 1993), probably by clustering reactive groups on the surface of an interacting protein, electrostatic interactions between the sulfate/carboxylic acid groups of heparin and the basic amino acids of proteins are essential for binding (Margalit et al., 1993). We note that the domain VI globule of the -chain is by far the most basic of all the mouse laminin-1 short arm globule domains (net charges calculated from each domain Arg, Lys, Glu, and Asp (Sasaki and Yamada, 1987; Sasaki et al., 1987, 1988) are (+9) for 1-VI, (-8) for 1-VI, (-9) for 1-VI, (-6) for 1-IVb, (-6) for 1-IVa, (-12) for 1-IV, and (-6) for 1-IV).

What is the function of this site? Interactions between heparin and/or heparan sulfates and laminin are thought to be important for both cell surface and basement membrane anchorage. Heparin can modulate laminin polymerization and the heparan sulfates of perlecan can bind to laminin, likely stabilizing the network (reviewed in Yurchenco and O'Rear, 1993). These glycosaminoglycan chains may be anchored at both sites of laminin, contributing to the charge-sieve barrier in microvascular basement membranes, immobilizing and activate growth factors, and decreasing basement membrane thrombotic potential. It is interesting to note that the two heparin binding sites of laminin-1 are located at the extreme ends of the -chain. Since each heparin-binding domain is located near an integrin-binding site, it raises the possibility that dual recognition by both integrin and cell surface heparin sulfate might play a biological role in some cells and/or under certain circumstances. Although heparin had no effect on PC12 cell adhesion, a ``coreceptor'' activity might operate in other more complex cells. For example, neural crest cells have been found to lose adhesion to calcium-activated laminin when substrate is coated in the presence of heparin (Lallier and Bronner-Fraser, 1992).

The 11 Integrin-recognition Site and Neuronal Interactions

PC12 pheochromocytoma cells have proven to be a useful cell line to evaluate the 11 integrin in laminin because the 11 integrin is by far the dominant receptor in this cell that can react with laminin, removing the background of other receptors, and the cells exhibit a specific biological response to this recognition if first activated with NGF, i.e. they rapidly extend neuritic processes. In this study, PC12 rat pheochromocytoma cells were found to interact with the -chain of laminin-1 through the 11 integrin. Furthermore, several lines of evidence demonstrate that this interaction is mediated by a single locus, domain VI. In particular, 1) cells adhered only to proteins containing globular domains VI and IVb of the -chain; 2) domain VI fragment E35 absorbed out an anti-laminin adhesion-blocking activity, whereas adjacent fragment E1X, containing domain IVb but not VI, did not; and 3) an elastase cleavage in distal domain VI rendered the 11 integrin recognition susceptible to heat inactivation. The last observation not only reveals that the integrin-recognition site is conformationally dependent, but also suggests that the recognition site may lie very close to the cleavage site. In one study (Lallier et al., 1994), however, a completely different localization was proposed, i.e. the long arm fragment E8. This conclusion, unlike the short arm associations made by other investigators (Hall et al., 1990; Tomaselli et al., 1990), is not supported by any of the data in this study. One notes, however, that the E8 conclusion was dependent on analyzing interactions of an avian integrin with a mammalian substrate, and it may be that recognition between molecules of widely different species can be different than those determined with closely related species (in this case, a rat integrin with a mouse substrate).

The 11 integrin has been reported to play an important role in neural crest cell adhesion to, and migration on, laminin (Duband and Thiery, 1988; Lallier and Bronner-Fraser, 1992; Lallier et al., 1994). This integrin is expressed in the central nervous system, sympathetic and spinal sensory ganglia, skeletal, smooth, and cardiac muscle, and capillary endothelia of the developing avian embryo, but eventually becomes restricted to the muscles and endothelium in the adult (Duband et al., 1992; Glukhova et al., 1993). The 11 integrin can assemble into unique clathrin-associated point contacts (Tawil et al., 1993) and recognizes laminin-1 as well as collagen types I and IV. In the case of collagen type IV, the 11 integrin is reported to recognize a conformational triple helical epitope containing Argof the (IV) chain and Aspof the two (IV) chains (Eble et al., 1993). It seems unlikely that the specific recognition sequence in laminin-1 is identical since it possesses a completely different structure with no known homologies to the collagens. The 1-chain of laminin has a partially overlapping distribution compared to the short arm integrin, with prominent expression in developing central nervous system (neuroretina, olfactory bulbs, cerebellum), meninges, kidney, testis, and diverse microvessels, but not in muscle (Vuolteenaho et al.,1994; Engvall et al., 1990). The 2 laminin chain (merosin, Am chain), on the other hand, is found in mesenchymally derived tissues and is more widely distributed (Vuolteenaho et al., 1994). The laminin containing this chain (laminin-2) appears to be a poor substrate for the 11 integrin and a good substrate for the 31 integrin, the reverse of what is observed with laminin-1 (Tomaselli et al., 1993; Pfaff et al., 1994). This difference may be important for cell-matrix communication, permitting cells to receive distinctly different integrin-dependent signals simultaneously or separately from the two laminin isoforms.

Identification of a Cryptic 11 Recognition Site in the Short Arms of Laminin

A recurrent theme in the study of laminin and nidogen is that most of their functions have been found to be conformationally dependent and often heat-sensitive. It was reported in one study (Goodman et al., 1991) that the 1 integrin can bind to fragment P1, a smaller pepsin fragment which consists mostly of domain III from each short arm. We considered the possibility that this particular site might be cryptic in laminin. Indeed, heat treatment of P1`, a larger pepsin fragment containing all of the domains found in P1, resulted in the activation of a previously latent 11 integrin-dependent cell adhesion activity, an activity was also found to develop upon long-term storage of P1`. Our study has shown that this activity is not present in native laminin, and it seems unlikely that it has biological relevance in intact laminin. Thus another example has been provided that the folded state of a complex matrix glycoprotein such as laminin is required for function, and that it is important to determine whether activities attributed to various peptide sites are actually functional in native protein.

Geography of Laminin Functions

An inspection of a map of laminin functions (Fig. 1) reveals that the sites are clustered into relatively small regions. With the exception of the nidogen-binding site, these functions have peripheral locations. The two main integrin-recognition sites are located at opposite poles in domains VI and G of the laminin cross (although the 61 integrin activity requires a coiled-coil in domain I, it appears to be predominantly dependent on determinants present in G subdomains 1-3; Sung et al., 1993) with both located near heparin-binding sites. Collectively, these data raise the possibility that adjacent cell, heparin and self-assembly sites can act cooperatively, and that an important role of each arm is to project these functions to away from the center in accessible geometries.


FOOTNOTES

*
This study was supported in part by National Institutes of Health Grant R01-DK36425. 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.

§
To whom correspondence should be addressed: Dept. of Pathology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Tel.: 908-235-4674; Fax: 908-235-4825.

The abbreviations used are: EHS, Engelbreth-Holm-Swarm; DMEM, Dulbecco's modified Eagle's medium; PMSF, phenylmethylsulfonyl fluoride; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; D-PBS, Dulbecco's phosphate-buffered saline; NGF, nerve growth factor; kb, kilobase(s).


ACKNOWLEDGEMENTS

-We wish to thank Michael Ward for his techni-cal assistance and Dr. C. Gorman and Genentech, Inc. (South San Francisco, CA), for kindly providing the cytomegalovirus expression vector, pCIS.


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