From the
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
Laminin-1 (EHS
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
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,
In our
present study we have combined several approaches to establish and map
functions of the
The electron micrographs
strongly suggested that r
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`, E
In the present study, we identified and localized three
functions of the N-terminal moiety of the
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
The
-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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-chain extending from the N terminus through one third
of domain IIIb. This dumbbell-shaped glycoprotein (r
1(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) r
1(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, r
1(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 r
1(VI-IVb)` or
-chain domain VI
fragment. This active integrin-recognition site could furthermore be
distinguished from a second cryptic
1
1-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
1
1 integrin-recognition site are all clustered at the end of
the
-chain short arm, the latter two resident solely in domain VI.
-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).
- 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
1
1 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).
6
1 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.
-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
1
1 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
1
1 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).
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 r
1(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-cm
tissue 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 E
35 (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 E
35, 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.
r
1(VI-IVb)` is shown in the shaded rectangle. Elastase
fragments E1`, E1X ( dark line border), E
35, 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
1
1 integrin-binding sites are
shown in bold-face for the
-chain short arm. The long arm
functions of heparin binding ( heparin),
6
1
integrin-recognition site (
6
1), 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
1
1 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 r
1(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 (
10
cpm/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 CaCl
in
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
10
cells/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% CO
in 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.
Expression and Characterization of
r
A cytomegalovirus
promoter-based mammalian expression vector (pCIS) containing the 5`
region of laminin-1 1(VI-IVb)`
-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 M
of 120 kDa (data not
shown). This recombinant protein, r
1(VI-IVb)`, was harvested from
medium and purified to homogeneity as previously described. Purified
r
1(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, r
1(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 r
1(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 r
1(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
r
1(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 r
1(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.
1(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 E
35)
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 r
1(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 r
1(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
r
1(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 E
35, apparently because it was blocked at the N
terminus.
Figure 3:
Elastase digest of r1(VI-IVb)`.
A, r
1(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, r
1(VI-IVb) was nearly
completely digested, revealing the same 35-kDa band ( arrow)
found in elastase fragment E1` (subfragment E
35). The lane at the
far right of Panel A shows laminin digested for 6 h.
B, SDS-PAGE (reducing conditions, Coomassie Blue-stained) of
r
1(VI-IVb)` ( lane 1), and r
1(VI-IVb)` after
digestion with elastase ( lane 2). Recombinant fragment
E
35 ( 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 r
1(VI-IVb)` was found to block
laminin self-assembly in a concentration-dependent manner. Furthermore,
r
1(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
E
Heparin was found to bind r35
1(VI-IVb)`, a
property exploited in developing a purification protocol. When bound to
an HPLC heparin-5PW affinity column, r
1(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 E
35 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. r
1(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, 10
cpm/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
PC12 rat pheochromocytoma cells have previously
been shown to adhere to laminin primarily through the -Chain Short
Arm
1
1
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, r
1(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
r
1(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, r
1(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 r
1(VI-IVb)`, like adhesion on E1`
and laminin, is mediated through the
1
1 integrin and that the
1
1 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, r
1(VI-IVb)`, E1`, E8, or P1`. PC12
cells adhered strongly to laminin, r
1(VI-IVb)`, and E1`, while
adhering poorly or not at all to E8 or P1`. B,
1
1
integrin mediates PC12 adhesion to r
1(VI-IVb)`.Cells
were incubated in wells coated (0.1 µ
M) separately with
laminin, r
1(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
1
1 integrin mediates PC12 adhesion to
r
1(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, r
1(VI-IVb)`, and fragments over a
period of about 24 h. These cells extended long branching cellular
processes on r
1(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, r
1(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
The binding of
cells to r1
1 Integrin-recognition
Site by Laminin Antibody Absorption
1(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 E
35 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.
35, and
r
1(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 r
1(VI-IVb)`, 82 and 87%, respectively, of cell binding was
restored. When antibody was absorbed with subfragment E
35 (domain
VI, corresponding to the N-terminal moiety of r
1(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, E
35 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
1
1 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 E
35. 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
E
35 and E1X (or another fragment) would have restored
activity.
Figure 8:
Domain localization of the 1
1
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 r
1(VI-IVb)`, fragment E1`, fragment E1X, fragment E
35,
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 E
35 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
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
1 Integrin-binding Site in P1`
Exposed by Heat Denaturation
1(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 r
1(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
r
1(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 r
1(VI-IVb)` lost its
ability to support cell adhesion following heat treatment. In contrast
to the observations with laminin, E1` and r
1(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 r
1(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 1
1-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
1
1 recognition site on whole laminin (not
shown). These results provide evidence that the heat-activated
1
1 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.
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
1
1
integrin-recognition site was mapped to the same domain VI. Finally, a
cryptic
1
1 integrin activity, not present in the recombinant
molecule, was identified elsewhere in the EGF-repeat regions of the
short arms.
Structure of r
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(VI-IVb)
1-chain short arm,
r
1(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 r
1(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
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 Short Arm Binds to
Heparin
-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).
-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
PC12 pheochromocytoma cells have proven to
be a useful cell line to evaluate the 1
1 Integrin-recognition Site and Neuronal
Interactions
1
1 integrin in laminin
because the
1
1 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
1
1 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
E
35 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
1
1 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).
1
1 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
1
1 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
1
1 integrin is reported to
recognize a conformational triple helical epitope containing
Arg
of the
(IV) chain and Asp
of 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
1
1 integrin and a good substrate for the
3
1
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
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
1 Recognition Site
in the Short Arms of Laminin
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
1
1
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
6
1 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.