Structure

and

function

multidomain
KONRAD
lnstitute
Biocenter

of laminin:

BECK,*

IRENE

HUNTER,T

AND JURGEN

for Biophysics,
University, A-4040 Linz, Austria,
of the University, CH-4056
Basel, Switzerland

is a large

(900

kDa)

mosaic

protein

composed

of many distinct
domains
with different
structures
and
functions.
Globular
and rodlike domains
are arranged
in an extended
four-armed,
cruciform
shape that is
well suited for mediating
between
distant
sites on cells
and other components
of the extracellular
matrix.
The
a-helical
coiled-coil
domain
of the long arm is involved
in the specific assembly
of the three chains (A, Bi, B2,
and possible
variants)
of laminin
and is the only domain composed
of multiple
chains.
It is terminated
by
a large globular
domain
composed
of five homologous
subdomains
formed
by the COOH-terminal
part of
the A chain.
Sites for receptor-mediated
cell attachment and promotion
of neurite outgrowth
reside in the
terminal
region of the long arm. A second cell attachment site, a cell signaling
site with mitogenic
action,
binding
sites for the closely
associated
glycoprotein
nidogen/entactin,
and regions
involved
in calciumdependent
aggregation
are localized
in the short arms.

These

domains,

which to a large extent

are composed

of Cys-rich
repeats with limited homology
to EGF, are
the most highly conserved
regions in laminins
of different origin.
At present,
most structural
and functional

data have been collected

mouse
form

tumor,
which
and
dissected

limited

proteolysis.

nins

from

different

for a laminin

expressed

by a

can be readily
isolated
in native
into
functional
fragments
by

Increasing

variations

species

information

on lami-

tissues
demonstrates
Isoforms
of laminm assembled
from different
chains
are focally and
transiently
expressed
and may serve distinct
functions
at early stages of development
even before being laid
down as major components
of basement
membranes.
-BECK,
K.; HUNTER,
I.; ENGEL, J. Structure
and
function
on laminin:
anatomy
of a multidomain
glycoprotein.
FASEB].
4: 148-160;
1990.

considerable

and

of structure.

Key Words: extracellular matrix - basement membrane

protein structure
coiled-coil - heparan sulfate proteoglycan epidennal growth factor

148

of a

glycoprotein

ABSTRACT
Laminin

anatomy

ENGEL

and tDepartment

of Biophysical

Chemistry,

LAMININS,
A FAMILY
OF LARGE
multidomain
glycoproteins of the extracellular
matrix (ECM),1 have attracted
much interest because of their importance
in the development and maintenance
of cellular organization.
Important cellular functions attributed
to laminin include

stimulation
of growth and differentiation,
neurite
outgrowth promotion,
and mediation
of cell communication. Laminin
is the first ECM protein
detected
during
embryogenesis;
it is present
at the two-cell stage in the
mouse embryo.
In later development
and in mature tissue it serves as a ubiquitous
and major noncollagenous
component
of basement
membranes.
It participates
in
the assembly
of this specialized
form of the ECM and
mediates
cell attachment
and maintenance
of the differentiated
state of epithelial
and endothelial
cell layers
that are intimately
associated
with their
basement

membranes.
There are a number

of excellent

recent

reviews

on

various
aspects
of laminin
and other ECM
proteins
(1-5) and on basement
membranes
(6). In this review
we focus on the structure
of laminin,
the progress
that
has been made in assigning
functions
to distinct
domains of the molecule,
and variations
of the laminin
structure
in tissue-specific
isoforms
and in phylogenetically distant
laminins.
SOURCES

OF

LAMININ

Laminin
was first isolated from the mouse EngelbrethHolm-Swarm
(EHS) tumor (7) and from the extracellular deposit
of murine
parietal
yolk sac (PYS) carcinoma
cells (8). Laminin
from these sources
can be
readily
extracted
and purified
in intact
form under
nonreducing
and nondenaturing
conditions.
In particular, EHS tumor tissue is a rich and convenient
source
of laminin
from which
it is extractable
by neutral
buffers containing
0.5 M NaC1 (9). By a superior

method,

with buffers

nm is extracted

containing

in the form

10 mM EDTA,

of its 1:1 complex

Abbreviations:
ECM,
extracellular
matrix;
EGF,
growth
factor;
EHS,
Engelbreth-Holm-Swarm;
HSPG,
sulfate
proteoglycan;
PYS, parietal
yolk sac; SDS-PAGE,
dodecyl
sulfate-polyacrylamide
gel electrophoresis.

0892-663819010004-0148/$01

lami-

with nido-

.50.

epidermal
heparan
sodium

FASEB

gen/entactin
(10). Consequently
most of the biochemical
and biophysical
work has been performed
with EHSlaminin,
which
thus became
the prototype
laminin.
Isolation
of laminins
from
normal
tissues
is often
difficult.
It has been achieved
in many cases only with
denaturing
and reducing agents (5, 11), but extraction
with EDTA appears
to be advantageous
(12). Nevertheless a few tissueand cell-specific
laminins
with distinctly different polypeptide
chains, chain compositions,
and structures
are known today. Although
data on these
so-called isoforms
and on laminin
variants
from phylogenetically
distant
species
are still fragmentary
(see
below),
there
is increasing
evidence
that the wellknown laminin
from EHS tumor is just one member
of
a protein
family.
The reader
should
remember
that

considerable
and

those

variations
that

have

may exist between

been

studied

GROSS
STRUCTURE
AND
EHS TUMOR
LAMININ

this laminin

in less detail.
SHAPE

OF

MOUSE

Rotary
shadowing
electron
microscopy
of laminin
revealed an unusual
cruciform
structure
(Fig. l#{192}),
with
three apparently
identical
short arms of 36 6 nm and
a long arm of 77 nm (13). Recent
studies
(14) have
shown, however, that one of the short arms is considerably longer (48 4 nm) than the other two (34 4
nm). The two smaller arms each contain
a central and
a terminal
globule separated
by rodlike regions, whereas
the longer arm contains
an additional
globular
region
(Fig. IA, Fig. 1G, arrows). The long arm of laminin
appears as a rather flexible rod with a large terminal
globule, which at the low resolution
of rotary shadowing
can
be resolved
into two closely spaced
smaller
globules.
Negative
staining
reveals that the globule
adjacent
to
the 3-nm thick rod is composed
of three, and the more
distant globule of two subdomains,
each 4 nm in diameter (Fig. 1B).
Electron
microscopy
thus revealed
a very extended
protein with a maximum
dimension
of 120 nm, consisting of globular
and rocilike elements.
Hydrodynamic
measurements
showed
that this shape
is essentially
preserved
in solution
(13).
FRAGMENTS

OF

LAMININ

Fragmentation
by limited
proteolysis
has been
instrumental
in the elucidation
of the domain
organization and detailed
structure
of large multidomain
proteins such as laminin
and fibronectin.
Laminin
has
been cleaved into a number
of distinct fragments
using
a variety
of enzymes.
Localizations
were derived
by

comparing the shapes of fragments with that of laminin

(Fig. 1), by detecting antigenic determinants
shared by
different
fragments
(15), and by comparing
partial
tein
sequences
with
cDNA-derived
sequences

pro(16,

17). Biochemical
and immunological
studies have enabled the assignment
of functions to distinct domains.
Table 1 is an overview of the important properties and
functions of several well-defined
fragments,
and their

LAM IN IN

C1-4
#{149}.

..

E4

P1
..,

I-*1
.

Figure
1. Electron
micrographs
of EHS-laminin
(A, B) and defined
laminin
fragments
(C-I) after rotary shadowing
(A, G-I) and negative staining
(B-F). Fragments
E3, T8, E8, and C8-9 originate
from the long arm of laminin.
Note that the second terminal
globule at the tip of the long arm is visible in intact laminin
only (double
arrow in A and B) but is absent
in fragments
T8, E8, and C8-9
(D-f). Fragment
C1-4 comprises
the entire short-arm
structures
of
laminin
and fragment
P1 the inner rodlike regions of the three short
arms. Note that one of the short arms is longer than the two others
and contains
a third globular
unit (arrow
in A and C). For further
information
molecule,

on fragments
see Table I and

and their
localization
Fig. 2. (bar: 50 nm).

in the

laminin

localization
within
the laminin
molecule
is shown in
Fig. 2.
The short arms of laminmn are remarkably
resistant
to proteases,
and several fragments
comprising
intact
or truncated
short arms have been detected
(14, 15).
Fragment
P1 is most resistant
to proteases
and comprises the inner region of the cross (Fig. iN). In fragment El, the outer part of one short arm has been removed in the form of fragment
E4 (Fig. 11) and the

other

short arms are often truncated.

Fragment
E3 (Fig. 1C), comprising
the outer of the
two globules
at the end of the long arm (G4-G5
in
Fig. 2), is readily released during proteolysis
and is absent from fragments
derived
from the long arm. Fragments T8 (Fig. 1D) and E8 (Fig. 1E) of the long arm
consist of rodlike regions
of different
lengths
together
with the adjacent
globular
domain
(G1-G3
in Fig. 2),

149

TABLE

1. Properties of well-characterized fragments

of mouse EHS-laminin

M (kDa)6
SDS-PAGE

Fragments

native

red.

red.

NH2 terminal

aa

Shape

Short-arm
P1

290

280

El

450

400

E4

75

75

550

550

C1-4

nd

50
other

Predominant

Prominent

60

nd

Aperiodic

Mitogenic
Cell attachment
Binding of nidogen/entactin

Aperiodic

Mitogenic

18

If

Aperiodic

Inhibition
of Ca2-induced
aggregation
of larninin

21

Fig. IC

Aperiodic

Ca2-dependent

14

Fig.

IH

As C1-4,
but with one
truncated
arm

7 (B!)
a

Fig.

nd

Reference

structures

bands

functions

18
18, 19
10, 20

aggregation

Long arm
C8-9

340

E8

190 (A)
+170(BI-B2)

240
+

T8

nd

25K

50

140 (A)
80 (B1-B2)

55

cs-helical

(75%)

Cell attachment
(P. End, M. Bruch,
unpublished
results)

1540 (BI)
1329 (B2)
1886 (A)

Fig. IE

cr-helical

(45%)

Cell attachment
Promotion
of neurite
Outgrowth

nd

1679 (BI)
1473 (B2)
2009 (A)

Fig. ID

12 (B2)
10 (B!)

1679 (BI)
1473 (B2)

50

2666 (A) (E3)

140 (A)
45 (BI)
+ 35 (B2)
+

80 (A)
25 (Bl-B2)

26 (B1-B2)
+

E3, C3

Fig. IF

190 (A)
+ll0(B1)
+ 90 (B2)

50

nd

Short

rod

Fig. 1C

cr-helical

(30%)

Cell attachment

cs-helical

(100%)

Antibodies
against 25K
inhibit neurite outgrowth

il-structure

19, 22
22, 23

22

Binding of heparin and

and heparan sulfate

15, 20

The enzyme used for limited proteolysis is indicated by C, cathepsin G; E, pancreatic

elastase; P, pepsin; and T, trypsin. 25K is an endogenous
fragment (27). For protocols of preparation
and characterizations,
see refs 9, 14, 15, 27.
Molar masses of native fragments are from sedimentation
equilibrium.
Apparent molar masses for the constituent
chains were derived by SDS-PAGE.
Origin of chain segments are indicated by chain designations
in brackets. Bl-B2 indicates that segments of the Bi and B2 chains are disulfide linked.
Numbering
corresponds
to the original literature which might
differ from that of the data banks as numbering
starts after the presumptive
signal peptide sequences.
Data are according to refs 25 (E4, E3), 17 (E8),
27 (25K), and R. Deutzmann
(T8, unpublished
results). The B chains of E8, T8, and 25K, and the A chain of E3 Contain the COOH termini of the
laminin chains.
dA complex
pattern of bands is observed depending
on the time of exposure to the enzyme.

whose three subdomains

are clearly seen by negative
staining
(Fig. iD-iF).
Its sensitivity
to proteases
has
prevented
the isolation
of defined
fragments
from the
upper region of the long arm. Recently,
however (14),
cathepsin
G was found to cleave laminin
initially at two
sites only (Fig. 2, arrows),
thus making available
a fragment Ci-4 (Fig. 1G) comprising
the intact short arms,
and a fragment
C8-9 (Fig. iF) comprising
the entire
rodlike
region
of the long-arm
and globular
domains

G1-G3.
Laminin
is unevenly
glycosylated,
average
14 (24,
25) to 25% (26), and therefore
the molar masses of the
native fragments
deviate to different
extents from those
that may be predicted
from the sequence.
Glycosylation
also causes anomalous
migration
of the chains during
SDS-PAGE
giving apparent
molar masses.
Circular
dichroism
of intact laminin
revealed
about
25% a-helix,
which melted
out in a sharp transition
centered
on 59#{176}C
(15, 27). The conformation
of the
short-arm
fragments
cannot
be classified
in terms of
well-known
secondary
structure;
fragments
derived
from the long arm are highly a-helical
(Table 1) and
fragments
E3 and C3 have a 3-structure
(14, 15).
150

Vol. 4

Feb. 1990

A MODEL

OF

MOUSE

EHS

LAMININ

Mouse
EHS tumor laminin
consists of three different
polypeptide
chains-A
(440 kDa),
Bi, and B2 (each
about 220 kDa) -which
are disulfide linked to form the
characteristic
asymmetric
cross-structure
seen by electron microscopy
(Fig. IA). The recent cloning
and sequencing
of all three chains of mouse laminin
(17) as
well as chains from human
(28-30),
Drosophila (31, 32),
and rat s-laminin,
a tissue-specific
variant (11), have revealed a domain
organization
that fits well with the observed ultrastructure,
and have suggested
the arrangement of the three chains within the laminin
molecule
shown in Fig. 2.
All three chains contain
six domains,
1-VI (Fig. 2).
Additional
globular
and rodlike domains
(lila, IVa) are
found in the short-arm
region of the A chain. Domains
I and II, located at the COOH-terminal
ends of the B
chains and in a related
region of the A chain, contain
a series of heptad repeats (33) and are predicated
to be
a-helical.
The calculated
length of these domains,
assuming
a distance
of 0.15 nm/residue
in an a-helix, is

85 nm, which

The FASEB Journal

is in agreement

with the length

of the

BECK ET AL.

(A)

ifib

1-4

4
(Th
NH2 (Bi)

VI

ifi
U

In addition
to the six common
domains,
the Bi chain
contains
an additional
40-amino-acid-long
sequence
(domain
a) located between
domains
I and II. It contains 8 Gly and 6 Cys, and is likely to be highly folded
and stabilized
by disulfide
bonds.
It is incompatible
with a-helix
formation
and is presumed
to be looped
out of the long arm, although
no morphological
structure corresponding
to this domain has been detected
by
electron
microscopy.
Domain
G forms
the COOHterminal
region of the A chain with no counterpart
in
the Bi and B2 chains.
Its predicted
structure
fits well
with the large terminal
globule seen by electron
microscopy (Fig. IA). Domain
G consists of five homologous
repeats
(Gi-G5),
which
probably
represent
the five
subdomains
detected
by negative
staining
of intact

laminin

(Fig.

SEQUENCES

Short-arm

Gi

G2

G3

8-9

G4
LJ

3
Figure
2. Structural
model of laminin.
Designations
of domains
by
roman numerals
is according
to ref 17. Cys-rich
rod domains
in the
short arms are designated
by symbols
S and the triple coiled-coil
region (domain
I-LI) of the long arm by parallel
straight
lines. In
the Bl-chain,
the cr-helical
coiled-coil
domains
are interrupted
by a
small Cys-rich
domain
a. Interchain
disulfide
bridges are indicated

by thick bars. The primary cleavage sites of cathepsin G are marked

by arrows. Regions of the molecule corresponding
to fragments 1-4,
4, 8-9, 8, and 3 (see Table 1) are indicated.

long arm (77 nm) observed

Domains
III and V are

by electron

microscopy

(13).

rich in glycine and cysteine,

suggesting
that they contain
many turns, possibly
stabilized
by disulfide
bonds.
Each domain
consists
of
many homologous
repeats
of about
50 amino
acids,
with 8 Cys arranged
in regular
positions,
and have
some homology
to the 6 Cys motifs
of epidermal
growth factor (EGF) and transforming
growth factor a
(TGF-a) (17, 34). The repeats are probably
arranged
like beads on a string and are likely to correspond
to the
rodlike regions
of the short arms.
Domains
IV and VI are thought
to form the central
and terminal
globules
of the short arms. Domain
VI
contains
the NH2 terminus
of each chain.

LAMI N IN

1B).
AND

CONFORMATION

structures

To examine
in more detail the relationship
between
the
A, BI, and B2 chains,
the amino acid sequences
of all
three chains were compared.
Alignment
of the shortarm sequences
is shown schematically
in Fig. 3.
The sequence
of the NH2-terminal
domain
VI is
significantly
conserved
in all three chains of EHS laminm, and highly homologous
regions have been found at
the NH2 termini
of all other laminins
sequenced
so far.
In particular,
the 6 or 8 Cys residues
are conserved;
a
sequence
WWQS
in the middle of the region and a pattern of Y(Y/F)YX8G
terminating
region VI are highly
preserved.
The latter motif is also found in a hypervariable region of some immunoglobulins.
The inner globular
regions (domain
IV) of the A and
B2 chains are homologous
and may be considered
to be
derived
from the Cys-rich,
so-called
EGF-like
repeats,
by a large insertion
between
the third and fourth Cys
of one of these motifs (34). Within
the A chain,
this
region occurs twice (IVa and IVb), corresponding
to
the two inner globules
seen on one short arm by electron microscopy
(Fig. IA, Fig. 1G). The large size of the
insertions
(180-200
amino acids, lacking Cys) explains
their appearance
as distinct domains
in electron
micrographs.
Domain
IV of the BI chain has a unique
sequence motif unrelated
to domain
IV of the B2 and A
chains. In the homology
scheme shown in Fig. 3, it appears
as an insertion
(IV) together
with the adjacent
Cys-rich
repeats.
There
are significant
homologies
between
the Cysrich, EGF-like
domains
III and V of all three chains,
and in a single chain
there are homologies
both within
and between
these domains.
The EGF-like
repeats
may
be classified
on the basis of the number
of residues
be-

tween the second

and third,
third
and fourth,
and
seventh and eighth Cys. These numbers
are very variable when the sequences
repeated
in the same chain are
compared,

but

responding

positions

species

well

are considered.

conserved

when

sequences

of the same chain

This

is clearly

in cor-

from different

demonstrated

by

IVb of the A chain

RYVVLPRP

PCHOC

and domain
IV of the B2 chain. The
partial sequence
of the core protein
of basement
membrane heparan
sulfate proteoglycan
(HSPG)
also contains tandem
repeats with 8 Cys (35) similar to region
IV and the neighboring
repeats of the A and B2 chains
of laminin
(Fig. 3).
Assuming
that the EGF-like
repeats
are arranged
linearly,
an average
translation
per domain
of 2.5 nm
follows from the number
of repeats
and the electron
microscopically
derived
dimensions
of the short-arm
rods.

This

an individual

mation

III1)IIlIIIECPAc

B2

,x

Long-arm

VVV

VV

IU i i i i

i i i

i i i#{149}

1U)

EJSCDDDC

{ ocoDI
LRtiDN

HSPG

3. Alignments
of sequence
regions
in the short-arm
structures of EHS-laminin.
Regions
with predicted
globular
conformation are indicated
by circles
and Cys-rich
EGF-like
repeats
by
squares.
For best alignment
of Cys-rich
repeats,
an insertion
in the
Figure

B! chain (dashed box) and a repeat and gap in the A chain were
introduced.
Cys-rich repeats with additional
insertions (see text)
are indicated by squares with half circles. Domains VI in all three
chains are related (hatched),
whereas
domain
IV (cross-hatched)
in
chain BI is unrelated to domains IV in A and B2. Cys-rich repeats
of heparan sulfate proteoglycan
(HSPG, 35) are aligned for comparison. Sequence data for mouse laminin are by Sasaki et al. (17).
Triangles mark putative sites of N-linked
glycosylation.
Sequence
regions for which functions have been proposed (Table 2) are indicated by their sequences in one letter code. Sequences that are probably involved in disulfide linkage of the three chains (Fig. 2) are indicated at the right-hand
side.

(34).
structure

(33).
Using
the long-arm

152

Vol. 4

of the

Feb. 1990

BI

chains

fragments

C8-9

and

E8 (Fig.

2),

we have recently
shown
that the a-helical
domains
I and II are involved in chain assembly (36). When the
A, Bi, and B2 chains present
in these fragments
are
separated
and unfolded
in urea, they reassemble
into
molecules
which
in their a-helix
content,
apparent
molar mass, chain composition,
and ultrastructural
appearance
are indistinguishable
from the native
fragments. The results indicate
that all three chains interact
to form a triple-coiled
coil, which can extend the length
of the long arm. The highly specific nature of this interaction suggests that it is the mechanism
by which laminm assembles
in vivo.
Figure
4 shows
a cross-section
through
a triple

coiled-coil,
as is suggested for laminin.
Burying the
hydrophobic
residues located in positions a and d in the
center of the structure,
thus shielding them from the
aqueous environment,
is energetically
favorable and is
the driving force for coiled-coil
formation.
Hydrophiic
amino acids are exposed on the surface, and the coiled-

coil is further
of mouse

EHS tumor
laminin,
Drosophila laminin,
and s-laminin
(11, 31). This
observation
suggests
that the order
of EGF-like
domains is of functional
importance
and argues against a
simple
structural
role as spacer elements.
The functional significance
of the short-arm
structures
is further
emphasized
by their high degree of conservation
(about
60% on average)
in laminins
that exhibit only 20% homology
in regions
of the long arm (ii, 31).
Within mouse EHS laminin,
the two EGF-like
repeats
adjacent
to each side of domain
IVa of the A chain are
similar
to those in the same position
around
domain
a comparison

with the predicted

dimension
of
assuming
an EGF-like
confor-

In contrast
to the short arms, the sequences
of the A,
Bi, and B2 chains assigned
to the rodlike region of the
long arm (domains
I and II) have little sequence
homology,
but all contain
a heptad
repeat
of the type
(a,b,c,d,e,f,g)n
where hydrophobic
amino acids are located preferentially
in positions
a and d, charged
residues normally
in positions
e and g, and polar residues
frequently
in b, c, and f. Such sequence
motifs are
characteristic
of proteins
in which
two or three
ahelices are wound around
each other to form a coiledcoil

agrees
well
domain,

residues

e and

stabilized

by ionic interactions

between

g.

To maximize coiled-coil interactions,

the alignment
of the A, Bi, and B2 chains shown in Fig. 5 is proposed.
The a-helical
domains
I and II of each chain are
bounded
by 1 COOH-terminal
and 2 NH2-terminal
Cys-residues.
These Cys are separated
by 566, 567, and
604 residues in the A, B2, and Bi chains, respectively.
A disulfide bridge between the COOH-terminal
Cysresidue of the BI and B2 chains has been established
(27), and it may be speculated that a free Cys-residue
in the corresponding
position of the A chain participates in the catalysis of its formation.
If domain a is

The FASEB Journal

BECK E AL.

[nm]

0.5

looped

out of the long

helical

domains

matched,

all

arm, then the length of the athree

chains
will be closely

with the NH2-terminal

in parallel

of

(Fig.

5), which

Cys-residues

is in agreement

aligned
with

their

proposed role in interchain

disulfide formation.
NH2terminal sequence analysis of the constituent
chains of
fragments
25K (a COOH-terminal
B1-B2 disulfidelinked fragment) and E8 (17, 27, 39) supports the proposed lateral arrangement
of the chains within the long
arm. Viewing the chains from the NH2-terminus
and
counting anticlockwise,
the proposed azimuthal
chain
arrangement,
based on maximizing
charge-charge
interactions
(J. Engel, unpublished
observations),
is
A-B2-B1 (Fig. 5, top left).
We have used the criteria of Parry (38) to examine
the stability of the coiled-coil
region. The analysis

based

on the preference

cific positions
which

F,
Figure
4. Projection
of a three-stranded
a-helical
coiled-coil,
one
heptad
each, viewed from the NH2 terminus.
A distance
of 1.0 nm
is assumed
between
the helix axes. Positions
of 3-carbon
atoms
within
a heptad
repeat
assuming
3.6 residues
per turn (a-g).
The
clustering
of hydrophobic
side chains (filled circles) and electrostatic
interactions
(dashed
circles) are schematically
indicated.
Side chain
in positions
b, c, and f are located
at the surface
and are normally
polar or charged
(open circles).

within

of certain

the heptad

is a measure

amino

acids

for spe-

gives rise to a factor

of the probability

of forming

coiled-coil structure.
The lowest probability
for coiledcoil is predicted
for all three chains near the NH2terminus of domain II, and this region is particularly
susceptible
to proteolysis
(14). The highest value of F#{176}
is found for the Bi chain near its COOH-terminus.
The entire B2 chain exhibits intermediate
values, and
the lowest values are found for the A chain. Thus,
although all three chains of laminin are believed to par-

V25K

B2dJ

Bi:

LQQSAA

KVESLI

B2:

NDILNN

NSVSSL

AHVHSN

PHQ

B2

EPA

SDI

LHREHG

Bi

Bi

uE8

:i

[j

100

200

300

Figure

5.

Putative

factor
arrangement

500

400
I

ii:
coiled-coil

SRARK

V//////DO.5

F1 x 108:
of the A, Bi, and

B2 chains

within

i..i>O2

the long

arm

I
of EHS

laminin.

1<0.2
Cys

pairs

at the

NH2

lOnm

terminus

of

domain
II are probably
involved
in interchain
disulfide
bonding
(see also Fig. 3). The established
disulfide
bridge near the COOH
termini
of the Bl and B2 chains is indicated
by a vertical
bar. Domain
a is looped
out. As originally
noted by Barlow
et al. (37), several phase
shifts have to be introduced
in the heptad repeats and these are indicated by displacements
between
blocks.
Proline
residues
that tend
to disrupt a coiled-coil structure are indicated by filled circles. Blocks are marked according to their coiled-coil potential as expressed by
the probability
factor F defined by Parry (38). Putative sites for N-glycosylation
are indicated by triangles.
For some sites (filled triangles), evidence for glycosylation was obtained (39). Sequence regions claimed
to be involved
in neurite
outgrowth
activity
are indicated

by horizontal bars and their first five residues (Table 2). The number of residues in the coiled-coil region is indicated (abscissa). NH2terminal sequences of fragments E8, 25K, and E3 are indicated. The length of coiled-coil regions and diameters
of domain
G are drawn
approximately
to scale.
LAMININ

153

ticipate
in coiled-coil
formation
(see above),
in homotypic interactions
the A chain would not be expected
to

adopt

this structure.

This is in agreement

with recent

observations
from this laboratory
(I. Hunter
andJ.
Engel, unpublished
results) that although
the Bi and B2
chains alone can form a coiled-coil,
the A chain can
participate
in coiled-coil
formation
only in the presence

of the B chains.
Domains
I and II of all three chains together
account
for a protein
molar mass of 192.8 kDa. Assuming
a
length of 77 nm for the long arm, a mass-per-length
ratio of 2500 Da/nm
follows, which is slightly higher
than expected
for a perfectly
packed
triple-coiled-coil
(2250 Da/nm)
and suggests
some nonhelical
interruptions, presumably
due to the variations
in the stability
of the coiled-coil
regions
described
above, and to the
presence
of a number
of proline
residues
(Fig. 5).
The COOH-terminal
region of the A chain adjacent
to the coiled-coil
domain
contains
five internal
homologous repeats
(G1 to G5), each consisting
of about 200
residues.
For human
laminin,
only regions
G2 to G5
have been sequenced
(30) and exhibit an overall identity of 74% with mouse EHS laminin.
This homology
is surprisingly
low when compared
with more than
90% identity
of other parts of mouse and human
laminm (28, 29).

cloning and sequencing

of laminin
chains has allowed,
through
the use of synthetic
peptides,
the possibility
of
defining
specific sequences
responsible
for the observed
functions
of laminin.
To date, only a limited number
of
synthetic
peptides have been studied.
Their localization

and possible function are summarized

in Table 2.
Laminin
has the ability to self-associate,
forming
large aggregated
structures (50). This activity is mediated by the globular
regions at the tips of the
(domain
VI), and is Ca2 dependent
(14,
Chelating
agents arrest the polymerization
of small polymers,
and may explain
why

readily

extractable

EDTA-containing

from
buffers

basement

short arms
21, 50, 51).
at the level
laminin
is

membranes

with

(10).

Laminin
forms a particularly
stable complex with
nidogen/entactin,
which can be extracted from mouse
EHS tumor
as a 1:1 complex
(10). Nidogen
has been
observed
to bind to the Cys-rich,
EGF-like
repeats
(domain
III) (10, 20) of both the B! and B2 chains

(M. Gerl, personal communication).

Nidogen has also
been demonstrated
to bind to type IV collagen and has
been proposed to mediate binding of laminin to this
collagen (20, 52). Direct interactions
of laminin with
collagen
IV have also been reported
(50, 53). Sites on
laminin

responsible

electron
short

for binding

microscopy
(domain

VI)

have been

to the terminal
and

long

localized

globules

(domain

G1-G5)

by

of the
arms

(50).

GLYCOSYLATION
Seventy-four

A major binding

potential

have been identified

N-glycosylation

in the three

sites

chains

NXS/T

and are indi-

cated in Fig. 3 and Fig. 5. The sites are unevenly

distributed
between
chains
and are concentrated
in the
long arm. In EHS laminin,
some 40 possible acceptor
sites are occupied
by an unusual
variety of oligosaccha-

ride (24-26). In contrast to earlier studies that reported

12-15% (w/w) glycosylation
(8, 24, 25), a value of
25-27%
(w/w) has recently
been reported
(26). Aligning the chains
in the long arm (Fig. 5) Asn(N)
of
NXT/S-motifs
occurs most often in positions
b, c, or f
of the heptad
repeats (Fig. 4), indicating
a localization
at the surface of the coiled-coil.
The only function
of
glycosylation
of laminin
described
so far is involvement

in tumor cell adhesion (40), whereas it is not needed for

chain assembly (41) or heparin binding, and does not
confer

stability

against

proteases

STRUCTURE-FUNCTION

(42).

RELATIONSHIPS

Laminin
exhibits
a variety
of biological
activities,
including
promotion
of cell attachment,
growth
and
differentiation
of a number
of cell types, and multiple
interactions
with other basement
membrane
components (1-6). Progress
toward assigning
these functions

to distinct domains of the molecule has come largely

from studies with proteolytic
fragments
and domainspecific

antibodies.

fragments

Table

1 provides

for which a function

a summary

has been assigned,

the localization
of these fragments
within
molecule
is shown in Fig. 2. In addition,

154

Vol. 4

Feb. 1990

of the

and

the laminin

the recent

terminal

globule

site for heparin

of the long

arm

was localized
(15) and

may

to the
also be

involved in the binding of basement membrane heparan

sulfate proteoglycans
(54). The five homologous regions
(Gi-G5)
constituting
the long-arm
globule contain
clusters of basic amino acids (17, 39) and probably represent the binding site for the polyanionic
heparin and
heparan sulfate proteoglycans.
The multidomain
nature
to mediate
the interactions

membrane
creating

components,
and

maintaining

of laminin
is ideally suited
of a variety
of basement

and thus plays a key role in

the

complex

3-dimensional

structure necessary for the correct functioning

of basement membranes.
In addition, its size and shape enable
it to span

surface
of effects

the basement

receptors

through

on cellular

membrane

which

and

contact

cell-

it can exert a variety

processes.

Laminin possesses two cell attachment

sites, one located on the short-arm
fragment
P1 (19, 55) and the
other of high affinity (56) on the long-arm
fragment,
E8
(19, 57). The short-arm
site is not active, however,
in
the complete
short-arm
fragment
E1-4 that encompasses P1, or in intact laminin
(56). It thus appears
to
be a cryptic
site that may be activated
by proteolysis
during
tissue
remodeling
and tumor
cell invasion.
Studies
with synthetic
peptides
(Table
2) have identified
a sequence
YIGSR
present
in domain
III of the Bi
chain
with cell binding
activity,
but only at very high
molar
concentrations
when
compared
with
laminin.
The major
cell attachment
site of laminin
is apparently
located
within
the long-arm
fragment,
E8. This
is in

good
tions

The FASEB Journal

agreement
with immunoultrastructural
(58) indicating
that the inner regions

observaof the short

BECK ET AL.

TABLE
Chain

2. Functional peptides synthesized according to sequence segments in mouse EHS-Iaminin

Domain

Bi

aa-residues

Peptide

442-447
(H:
5:

D:
B!

IV

64 1-660

Proposed

III

902-906

B!

III

925-933

function

Reference

LGTIPG
LGTIPG
RGTVPG
LGTLNN)

Binding to a cellular elastin receptor of

which a 67-kDa component
is homologous
to a laminin receptor protein

RYVVLPRPVCFEKGMNYTVR

Binding

RYVVLPRPVCFEKGTNYTVR
5: RVLVFPRPVCLEPGLSYKLK
D: RQVVALNEVCLEAGKVYKFR)

B!

chains

of heparin;

promotes

cell adhesion

(H:

of murine melanoma
cells, fibrosarcoma,
glioma, pheochromocytoma,
and aortic
endothelial
cells

PDSGR
(H: PDSGR
s: PGSQR
D: VASGL)

Similar to (CDPG)YIGSR
(see below);
proposed to act synergistically
with
YIGSR

CDPGYIGSR
CDPGYIGSR
5: CRAGYTGLR
D: CQEGYSGSR)

(H:

Promotion
of cell attachment,
chemotaxis,
neuronal attachment
but not neurite
outgrowth

44, 45

Promotes melanoma
cell migration
Inhibits formation of lung tumors

44, 46
44, 47

1542-155!

IlIb

1118-1128

GTFALRGDNGQ

Promotes adhesion of endothelial

stimulate process extension of
neuroblastoma
cells

2091-2 108

SRARKQAASIKVAVSADR

Promotes neurite outgrowth of some but

not all neuronal cells; promotes adhesion
of neuronal and nonneuronal
cells;
stimulates collagenase activity

For
sequence

43

44

B2

Drosophila)

RNIAEIIKDA
(H: RDIEEIMKDI
D: RELKDEVQ.NI)

58a

Stimulates

neurite

outgrowth

48

cells; can

49

49

localization,
see Fig. 3 and Fig. 5.
bThe homologous
sequences at corresponding
sites of other laminins (H, human;
s, s-laminin;
D,
that have not been tested for their activity are indicated in parentheses.
The sequence of the synthetic peptide corresponds
to the partial
reported in (37); in the complete sequence (17), N is replaced by D.

arms are confined

to the
branes,
while the long-arm

interior
of basement
epitopes
are located

basement
membrane
surface.
Laminin is a potent stimulator

memat the

A general problem
associated
with studies using
thetic peptides
is that they represent
only a small

of the domains
of neurite

outgrowth

likely

from which they are derived

therefore

to have

the correct

and are un-

structure.

Many

the functions
mapped
shown
to be conformation

rounded by laminin-producing
Schwann cells. Domainspecific antibodies
have implicated
sequences
within
fragment E8 as important
for this activity, and a synthetic peptide corresponding
to a defined sequence of
the B2 chain (Table 2) has been reported to have neu-

mitogenic
function
of fragment
P1 is lost upon reduction (P. End, unpublished
results),
and cell adhesion

rite

to have

sequences
must also consider
the structural
requirements of such sequences.
To achieve this, studies (44)
were performed
recently
with a cyclized
form of the

some activity (49).

Laminin has a mitogenic effect on a number of cells
in culture (55), which in dose response and time depen-

peptide YIGSR.
Homology searches have not been helpful in suggesting other possible functions of distinct regions of lami-

dence is comparable
to that of EGF (18). This function
has been localized
to fragment
P1, which consists
of
Cys-rich
repeats
(domain
III) and is not related
to its

nm. The only strong homology

with a protein
not belonging
to the laminin
family
is the one with HSPG
(35;
see Fig. 3). The weak homology
of domains
III and V
with EGF and EGF-motif
containing
proteins
has been

outgrowth-promoting

tide with a sequence

of the A chain

cell attachment

(Table

activity

(48).

from the a-helical

2) has also been

properties

(18). Synthetic

Another

pep-

rodlike
shown

region

peptides

cor-

neurite

abolished

outgrowth
upon

ture studies

specific
domains
have
dependent.
In particular,

of

(22, 23), a function

that may be involved in the growth
and regeneration
of peripheral
neurons,
which are sur-

and

to

synpart

activities

denaturation

aimed

been
the

of fragment
E8 are
57). Therefore,
fudelineating
functional

(20,

at precisely

responding
to domain
III sequences,
including
the pentapeptide
YIGSR,
which represents
part of the Cysrich repeat with closest homology
to EGF, had no mito-

mentioned.
Weak homologies
are also observed between domains I and II and many other proteins
con-

genic activity

homology

LAMININ

(18).

taining

coiled-coil

exists

structures.

between

Perhaps

domains

GI

an

interesting

to G5

and

sex

155

steroid-binding
protein
(codes G08607
and S00077 of
the National
Biomedical
Research
Foundation
protein
sequence
data base).
In agreement
with its numerous
biological
activities,
a variety of cell-surface
receptors
necessary
to mediate
laminin
activity
have been identified
(2, 59). In the
present context the fast-growing
field of laminin
receptors cannot be reviewed,
and only a few examples
will
be mentioned.
A 67-kDa protein is particularly
well expressed
in metastatic
cells (60), which could be eluted
from a laminin
affinity
column
by the domain
IIIderived,
synthetic
peptide
YIGSR
(45). Recent studies
have questioned
this proposed
binding
site, as purified
67-kDa
receptor
tron
microscopy

was

shown

to bind

by rotary

specifically

shadowing

to the

elec-

top of the

long arm (domain

II) near
the center
of the cross
(M. E. Sobel and V. Castronovo,
personal
communication). A laminin
binding
protein
from skeletal muscle,
aspartactin
(66 kDa), was reported
to bind to fragment
E3 (G4-G5,
61).
Cell-surface
receptors
of the integrin
type (62),
several
of which bind through
the specific
sequence
RGD on their ligands,
have been implicated
in laminin
binding.
Binding
of laminin
to an integrin
receptor
isolated from rat RuGli
cells and human
placenta
required divalent
cations,
but was not inhibited
by RGDcontaining
peptides
(63). This integrin
is believed
to
bind to the fragment
8 region. An RGD sequence
detected in the G3 domain
of human
laminin
is probably
not functional
(20) and is not conserved
in mouse laminm. Another
integrin-type
receptor
was proposed
to be
responsible
for the cell attachment
activity of fragment
P1 and was found to be RGD dependent
(20). Mouse
laminin
contains
an RGD sequence
in domain
Ilib of
the A chain, and a synthetic
peptide containing
this sequence (Table 2) has been found to promote
cell adhesion (49). In intact basement
membranes,
however,
the
short-arm
structures
containing
the RGD sequence
are
buried
whereas
the long-arm
terminal
domains
are
likely to contact
cells (58).
ISOFORMS
OF

AND

STRUCTURAL

VARIANTS

LAMININ

The identification
of distinct
but related
pepsin fragments in tissue digests as compared
with digests of EHS
laminin
(64), antigenic
differences
between
laminins
derived
from tumor
cells and normal
tissues (12, 65),
and the absence or reduced
expression
of the 440-kDa
A chain in a number
of cell types in culture (5, 6, 30),
has led to the suggestion
that structural
variants
of
laminin
may exist. In addition,
variation
in the levels
of mRNA
for the A, BI, and B2 chains of laminin
in
different
tissues of the same species (66, 67) indicates
the formation
of tissue-specific
isoforms,
possibly with
different
functional
properties.
Laminin
appears
to be developmentally
regulated.
During
mouse
embryogenesis,
the B chains
are detected at the 2-4 cell stage, whereas
the 440-kDa
A
chain is not detected
until the 16-cell stage (68), which

156

Vol. 4

Feb. 1990

is coincident
with the ultrastructural
appearance
of a
distinct
basement
membrane.
During
normal
kidney
development,
antibodies
against
EHS laminin
could
detect only B chains in undifferentiated
mesenchyme,
but on conversion
to a polarized
epithelium,
A chain
epitopes
were expressed
(69). Whether
these laminin
variants
consist
of B chains
only or have in addition
a
modified
A chain,
antigenically
unrelated
to the A
chain
of EHS
laminin,
is presently
unknown.
Isolation
and
characterization
of laminin
from

Schwannoma
cells, which lack the 440-kDa
A chain,
revealed
a Y-shaped
molecule
with one long and two
short arms (Table 3). Although
only B chains (200-220
kDa) could be detected
by SDS-PAGE
under reducing
conditions,
the Schwannoma
laminin
had an apparent
molecular
mass of about 850 kDa under nonreducing
conditions,
indicating
that it was not formed
solely
from the Bi and B2 chains and suggesting
the existence
of a variant (shorter)
A chain. Such variant
forms have
recently
been described
in laminin
from mouse heart
(300 kDa) (12) and 3T3-Li
cells (180 kDa) (75). In
3T3-L1 cells, as shown previously
for cells synthesizing
the 440-kDa
A chain, B1-B2 disulfide-linked
dimers are
formed as intermediates
in laminin
assembly,
but these
can be secreted
only in the form of a ternary
complex
with the 180-kDa
A chain (75). Merosin
first described
as an 80-kDa
protein
present
in basement
membranes
of human
muscle
and peripheral
nerves
(76) has been
identified
on the basis
of a 40%
homology
as a fragment
originating
from the COOH-terminal
portion
of
another
tissue-specific
A chain variant (71). This new A

chain was shown to be associated

with apparently
mal Bi and B2 chains to a molecule
with a shape

norsimi-

lar

to EHS
laminin
(71).
All isoforms
investigated
so far by electron
microscopy
(12, 22, 70) have a long-arm
terminal
globule,
which
in mouse
EHS laminin
is derived
entirely
from
the A chain.
Available
evidence
structural
variants
of laminin

suggests
therefore
that
are of the type A(B)2.

Whether
the A-chain
variants
detected
so far are
related to the 440-kDa
A chain, possibly splicing variants, or are novel subunits,
awaits the sequencing
of
these chains.
Additional
structural
information
has come from
studies of laminins
of different
species origin. The gross
shape
of laminins
originating
from different
phyla
ranging
from Cnidaria
to mammals
has been investigated in some detail by electron
microscopy
(Table 3).
All of the laminins
studied exhibit an asymmetric
crossstructure
with one long arm bearing
a terminal
globule
and three short arms each with terminal
and centrally
located globules.
The structure
of these molecules
thus
closely resembles
that of mouse EHS tumor
laminin.
The dimensions
of the short arms are well preserved
(Table 3) but the length of the long arm varies considerably
with species. Two globular
domains
at the tip
of the long arm, connected
by a short, 5-10 nm long
rod, may be related
to the subdomains
G1-G3
and
G4-G5
seen in negatively
stained
preparations
of
mouse tumor laminin
(Fig. 1B). The preparation
of a

The FASEB Journal

BECK ET AL.

TABLE

3. Properties of laminins pur!fied from different

Source

Mouse
Mouse

sources

Shape

EHS tumor
heart

Chains

Three short arms (36, 36, and 48 nm);

one long arm (77 nm); see Fig. !

440 kDa (A)

220 kDa (B!,

Similar to EHS laminin

differences in short-arm

Prominent
unrelated

but possible
structures

laminin,

Rat Schwannoma

Human

cells

placenta

Y-shaped

Similar

with two short and one long arm

to EHS laminin;

long arm,

83 nm

Sea urchin

embryo

350 kDa (A)

195, 185 kDa (B)
240 kDa (M chain)

Anthomedusa
Length
reduction.

measurements

to A and

Three short arms (35, 35, and 48 nm);

one unusually long arm (113 nm)
The extended short arm is found for a

480 kDa (A?)

269 kDa (B!, B2?)

of molecules
globules

only

14

12

22,

70

71
immunologically

B chains

400 kDa

exhibits
capsules

300-kDa chain immunologically

to A and B chains of EHS
220 kDa (B)

Three short arms (30, 30, and 36 nm);

one long arm (84 nm)

subpopulation

Leech ganglion

13,

B2)

220 kDa, 180 kDa; probably a mixture

of
a short A-chain variant and B chains of
normal size

related

Drosophila K, cells

Reference

(A)

72

215, 185 kDa (BI,

B2)
73

and

two inner

Three short arms (36, 36, and 53 nm);

one long arm (94 nm)

340 kDa (A?)

220 kDa (B?)

73, 74

Three short arms (36 nm);

one long arm (97 nm)

340 kDa (A?)

260 kDa (B?)

73

were performed

after rotary

shadowing

electron

proteolytic
fragment
from sea urchin laminin,
which is
indistinguishable
by electron
microscopy
from fragment E8 of mouse tumor laminin
(73), indicates
that
there
may
be similarities
between
these
COOHterminal
regions in both laminins.
Thus, modifications
leading
to the elongation
of the long arm in many species may be restricted
to the proximal
part of this arm.
Such length variations
may be required
to span basement membranes
of different
thicknesses.
OUTLOOK

microscopy.

Apparent

molar

masses

were determined

by SDS-PAGE

after

ing to the mouse

laminin
sequences
(Table
2) are promising, but the required
doses are many orders
of magnitude higher
on a molar
basis than for native
laminin
or
its active
fragments.
Small
synthetic
peptides
of random
conformation
are unlikely
to exhibit
functions
such
as cell attachment
and
neurite
outgrowth
of
peripheral
neurons
that are abolished
by denaturation
of intact
laminin.
A related
problem
is the correct
formation
of disulfide
bonds
in synthetic
peptides
that
resemble
disulfide-rich
regions
of, for example,
the
short-arm
structures
of laminin.
It is well known
that
for many
hormones
and other
biologically
active
molecules,
the correct
disulfide
linkage
is required
for the

A key problem
for future research
on laminin
will be
the further
elucidation
of structure/function
relationships.
Assignment
of functions
to regions
in the
molecule
by the proteolytic
fragmentation
approach
has been very successful
and will probably
continue
to
be so. Its limitations
are that defined fragments
cannot
be prepared
from all parts of laminin
and the fact that
small fragments
do not retain their native conformation. The alternative
approach
of testing the activity of
peptides
synthesized
according
to sequence
regions suspected to be involved in functions
has become popular

and in vivo may be useful.

A more conventional
method
that has been applied
to laminin
in only a few cases is
inhibition
by monoclonal
antibodies
directed
to regions
proposed
to be involved
in functions.
Work on the expression
of distinct
regions
of laminin
is being
pursued
in several
laboratories.
If properly

in the last few years,

and was motivated
in part by the
possible
pharmacological
value of such peptides
as, for
example,
antimetastatic
drugs.
A major
breakthrough
of this strategy
was the localization
of the fibronectin
cell binding
site to the tripeptide
RGD
(62). Some
of
the results
obtained
with peptides
synthesized
accord-

folded and disulfide stabilized

domains
can be prepared
by suitable expression
systems, these could be tested for
functions.
Also, 3-dimensional
structures
could then be
determined
at atomic
resolution
by nuclear
magnetic
resonance
and X-ray crystallography.
Laminins
are a
protein
family of utmost
biological
importance,
and

1.AMININ

native conformation
and for functionality.
It is therefore required
to confirm the results obtained
with peptides by independent
methods.
Site-directed
mutagenesis and functional
tests of the modified
laminins
in vitro

157

efforts should
be made to understand
the functional
variations
of the various isoforms
on a structural
basis.
In addition,
there are many
domains
in laminin
to
which
functions
have been assigned
on the basis of
weak arguments
or for which no functions
have yet
been found. Many exciting
discoveries
are expected.

We thank

Ms.

C. Fauser

for

expert

technical

assistance

chain
has a unique
basement
membrane
J. Biol. C/tern. 263,

18. Panayotou,
G., End, P., Aumailley,
M., Timpl,
R.,
gel,
(1989) Domains
of laminin
with growth-factor
Cell 56, 93-101
19. Aumailley,
M., Nurcombe,
V., Edgar,
D., Paulsson,

J.

(Be 1143/1-1).

20. Timpl,
Edgar,

VI, and proteoglycans.

Methods Enzymol.

G. R., Timpl,
proteins:

R., and

molecular

tein C/tern. 39, 1-50

6. Timpl, R., and Dziadek,
pathology

Kuhn,

R. A. (1986) Structures

M. (1986) Structure,
of basement

27.

Robey,
G. R.

P., Rennard,
S. I.,
(1979) Laminin-a
j Biol. C/tern. 254,

28.

Vergnes,J.
P., Braginof a basement
memin culture
by a mouse

Cell 16, 277-287

and Fujiwara,
S. (1987)
Basement membranes.
Methods Enzymol. 145, 363-391
10. Paulsson,
M., Aumailley,
M., Deutzmann,
R., Timpl,
R.,
Beck, K., and Engel, J. (1987) Laminin-nidogen
complex.
Extraction
with chelating
agents
and structural
characterization.

Biochern. 166,

11-19

11. Hunter,
D. D., Shah, V., Merlie,
J. P., and Sanes, J. R. (1989)
A laminin-like
adhesive
protein
concentrated
in the synaptic
cleft of the neuromuscular
junction.
Nature (London) 338,
229-234
12. Paulsson,
M., and Saladin,
K. (In press) Mouse
heart laminin:
purification
of the nature
protein
and structural
comparison

with EHS tumor laminin. j Biol. C/tern.

13. Engel, J., Odermatt,
E., Engel, A., Madri, J. A., Furthmayr,
H., Rohde, H., and Timpl, R. (1981) Shapes, domain organization
and flexibility
of laminin
proteins
of the extracellular

14.

15.

16.

17.

158

fibronectin,
two multifunctional
matrix. J. Mol. Biol. 150, 97-120
Bruch, M., Landwehr,
R., and Engel, J. (1989) Dissection
of
laminin
by cathepsin
G into its long arm and short arm structures and localization
of regions
involved
in calcium
dependent
stabilization
and self-association.
Eur j Biochern. 185, 271-279
Ott, U., Odermatt,
E., Engel, J., Furthmayr,
H., and Timpi,
R.
(1982) Protease
resistance
and conformation
of laminin.
Eur. j
Biochern. 123, 63-72
Hartl, L., Oberb#{228}umer,I., and Deutzmann,
R. (1988) The N
terminus
of laminin
A chain is homologous
to the B chains.
Eur.
J. Biochern. 173, 629-635
Sasaki,
M., Kleinman,
H. K., Huber,
H., Deutzmann,
R.,
Yamada, Y. (1988) Laminin,
a multidomain
protein.
The A

Vol. 4

Feb. 1990

and

26.

development,
mt. Rev. Exp.

membranes.

glycoprotein
from basement
membranes.
9933-9937
8. Chung,
A. E.,Jaffe,
R., Freeman,
I. L.,
ski, J. E., and Carlin,
B. (1979) Properties
brane
related glycoprotein
synthesized
embryonal
carcinoma-derived
cell line.
9. Timpl,
R., Paulsson,
M., Dziadek,
M.,

25.

K. (1988) Basement
and function.
Adv. Pro-

structure

Pathol. 29, 1-112

7. Timpl, R., Rohde, H., Gehron
Foidart,
J. M., and Martin,

J.

complex.

Ann. NY

Acad. Sci. In press

23. Edgar,
D., Timpi,
R., and Thoenen,
H. (1984) The heparinbinding
domain
of laminin
is responsible
for its effects on neurite outgrowth
and neuronal
survival.
EMBOJ.
3, 1463-1468
24. Arumugham,
R. G., Hsieh, T. C. -Y., Tanzer, M. L., and Lame,

145, 3-78

R. (1989) Structure
and biological
activity
of basement
membrane
proteins.
Eur. j Biochern. 180, 487-502
3. Martin,
G. R., and Timpi,
R. (1987) Laminin
and other basement membrane
components.
Annu. Rev. Cell Biol. 3, 57-85
4. Paulsson,
M. (1987)
Noncollagenous
proteins
of basement
membranes.
Collagen Relat. Res. 7, 443-461

Eur.

and

fraghigh

R., Aumai!ley,
M., Gerl, M., Mann,
K., Nurcombe,
V.,
D., and Deutzmann,
R. (1989) Structure and function of

the laminin-nidogen

2. Timpl,

and molecular

M.,

106, 1299-1306

1. Engel, J., and Furthmayr,

H. (1987) Electron
microscopy
and
other physical
methods
for the characterization
of extracellular
matrix
components:
laminin,
fibronectin,
collagen
IV, collagen

membrane

and Enactivity.

21. Schittny,
J. C., and Yurchenco, P. D. (In press) Terminal short
arm domains
in the basement
membrane
protein
laminin
are
critical
for its self-assembly.
j Cell Biol.
22. Edgar,
D., Timpl,
R., and Thoenen,
H. (1988) Structural
requirements
for the stimulation
of neurite
outgrowth
by two variants of laminin
and their inhibition
by antibodies.
j Cell Biol.

REFERENCES

5. Martin,

with the
B chains.

Timpl, R. (1987) The cellular interactions

of laminin
ments. Cell adhesion correlates with two fragment-specific
affinity binding sites. J. Biol. C/tern. 262, 11532-11538

and

M. Bruch
and M. Paulsson
for valuable
discussions
and communicating
unpublished
results. Original
work from this laboratory
reported
herein has been supported
by The Swiss National
Science
Foundation
(grant
no. 31-9088.87
to J. E.). The present
work by
K. B. is supported
by the
Deutsche
Forschungsgemeinschaft

globular
domain
and homology
proteoglycan
and the laminin
16536-16544

of othe asparigine-linked

sugar chains

of laminin.
Biochirn. Biophys. Acta 883, 112-126
Fujiwara,
S., Shinkai,
H., Deutzmann,
R., Paulsson,
M., and
Timpl,
R. (1988)
Structure
and
distribution
of N-linked
oligosaccharide
chains
on various
domains
of mouse
tumour
laminin.
Biochem. j 252, 453-461
Knibbs, R. N., Perini, F, and Goldstein,
I. J. (1989) Structure
of the major concanavalin
A reactive oligosaccharides
of the cxtracellular
matrix
component
laminin.
Biochemistry
28,
6379-6392
Paulsson,
M., Deutzmann,
R., Timpl,
R., Dalzoppo,
D., Odermatt, E., and Engel,
(1985) Evidence
for coiled-coil
a-helical
regions
in the long arm of laminin.
EMBOJ
4, 309-316
Pikkarainen,
T, Eddy, R., Fukushima,
Y., Byers, M., Shows,
T., Pihlajaniemi,
T., Saraste, M., and Tryggvason,
K. (1987)
Human
laminin
B1 chain.
A multidomain
protein
with gene
(LAMB1) locus in the q22 region of chromosome
7. j Biol.

J.

C/tern. 262,

10454-10462

29. Pikkarainen,
T., Ka!lunki,
Human
laminin
B2 chain.

T,
and
Tryggvason,
K. (1988)
Comparison
of the complete
amino
acid sequence with the B! chain reveals variability
in sequence
homology
between
different
structural
domains.
j BioL C/tern.
263, 6751-6758
30. Olsen, D., Nagayoshi,
T, Fazio, M., Peltonen, J., Jaakkola,
S.,
Sanborn,
D., Sasaki,
T., Kuivaniemi,
H., Chu, M. -L., Deutzmann,
R., Timpl,
R., and Uitto, J. (1989) Human
laminin:
cloning and sequence
analysis of cDNAs encoding
A, BI and B2
chains,
and expression
of the corresponding
genes in human
skin and cultured
cells. Lab. Invest. 60, 772-782
31. Montell,
D. J., and Goodman,
C. S. (1988) Drosophila substrate
adhesion
molecule:
sequence
of laminin
B1 chain reveals domains of homology with mouse.
Cell 53, 463-473

32. Chi, H. -C., Hui,

drosophila
laminin

C. -F. (1989) Primary

structure
of the
B2 chain and comparison
with human,
and drosophila
laminin
BI and B2 chains. j BioL C/tern.

33.

34.

35.

36.

mouse,
264, 1543-1550
Parry, D. A. D. (1987) Fibrous
protein
structure and sequence
analysis.
In Fibrous Protein Structure (Squire,
J. M., and Vibert,
P. J., eds) pp. 141-17!, Academic,
London
Engel, J. (1989) EGF-like
domains
in extracellular
matrix
proteins: localized
signals
for growth
and differentiation?
FEBS
Let:. 251, 1-7
Noonan,
D. M., Horigan,
E. A., Ledbetter,
S. R., Vogeli, G.,
Sasaki,
M., Yamada,
Y., and Hassell, J. R. (1988) Identification
of cDNA
clones encoding
different
domains
of the basement
membrane
heparan
sulfate
proteoglycan.
J. Biol. C/tern. 263,
16379-16387
Hunter,
I., Schulthess,
T., Bruch,
M., Beck, K., and Engel, J.
(In press) Evidence
for a specific mechanism
of laminin assem-

bly. Eur. j

The FASEB Journal

Biochem.

BECK Er AL.

37. Barlow,
D. P., Green,
N. M., Kurkinen,
M., and Hogan,
B. L. M. (1984) Sequencing
of lam
B chain cDNAs
reveals
C-terminal
regions of coiled-coil
alpha helix. EMBOJ
3, 23552362
38. Parry, D. A. D. (1982) Coiled-coils
in a-helix-containing
proteins: analysis
of the residue
types within the heptad
repeat and
the use of these data in the prediction
of coiled-coils
in other
proteins.
Biosci. Rep. 2, 1017-1024

mm

39. Deutzmann,

40.

41.

42.
43.

44.

R., Huber,

H., Schmetz,

K. A., Oberb#{228}umer,I.,

and Hartl,
L. (1988) Structural
study of long arm fragments
of
laminin.
Evidence
for repetitive
C-terminal
sequences
in the Achain, not present
in the B-chains.
Eur. j Bloc/tern. 177, 35-45
Dennis,
J. W., Waller, C. A., and Schirrmacher,
V. (1984)
Identification
of asparagine-linked
oligosaccharides
involved
in
tumor
cell adhesion
to laminin
and type IV collagen.
j Cell
Biol. 99, 1416-1423
Wu, C., Friedman,
R., and Chung,
A. E. (1988) Analysis
of the
assembly
of laminin
and the laminin
entactin
complex
with
laminin
chain
specific
monoclonal
and polyclonal
antibodies.
Biochemistry
27, 8780-8787
Howe,
C. C. (1984) Functional
role of laminin
carbohydrate.
Mol. Cell. Biol. 4, 1-7
Charonis,
A. S., Skubitz,
A. P. N., Koliakos,
G. G., Reger,
L. A., Dege, J., Vogel, A. M., Wohihueter,
R., and Furcht,
L. T. (1988) A novel synthetic
peptide
from the BI chain
of
laminin
with heparin-binding
and cell adhesion-promoting
activities. j Cell Biol. 107, 1253-1260
Kleinman,
H. K., Graf,J., Iwamoto, Y., Sasaki, M., Schasteen,

C. S., Yamada,

Y., Martin,

G. R., and Robey,

F A. (1989)

Identification
of a second
active site in laminin
for promotion
of cell adhesion
and migration
and inhibition
of in vivo melanoma lung colonization.
Arch. Biochern. Biophys. 272, 39-45

45. Graf, J., Iwamoto,

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

Y., Sasaki,

M., Martin,

G. R., Kleinman,

H. K., Robey, F. A., and Yamada,

Y. (1987) Identification
of an
amino
acid sequence
in laminin
mediating
cell attachment,
chemotaxis
and receptor
binding.
Cell 48, 989-996
Iwamoto,
Y., Graf,J.,
Sasaki,
M., Kleinman,
H. K., Greatorex,
D. R., Martin,
G. R., Robey, F A., and Yamada,
Y. (1988) Synthetic
pentapeptide
from the BI chain
of laminin
promotes
B16F1O melanoma
cell migration.
J. Cell. P/tysioL 134, 287-291
Iwamoto,
Y., Robey,
F A., Graf, J., Sasaki,
M., Kleinman,
H. K., Yamada,
Y., and Martin,
G. R. (1987) YIGSR,
a synthetic laminin
pentapeptide,
inhibits
experimental
metastasis
formation.
Science 238, 1132-1134
Liesi, P., N#{228}rv#{228}nen,
A., Soos, J., Sariola, H., and Snounou,
G.
(1989) Identification
of a neurite
outgrowth-promoting
domain
of laminin
using synthetic
peptides.
FEBS Lett. 244, 141-148
Sephel,
G. C., Tashiro,
K. -I., Sasaki,
M., Greatorex,
D.,
Martin, G. R., Yamada, Y., and Kleinman,
H. K. (1989) Laminm A chain
synthetic
peptide
which
supports
neurite
outgrowth.
Bloc/tern. Biophys. Res. Comrnun. 162, 821-829
Charonis,
A. S., Tsilibary,
E. C., Yurchenco,
P. D., and Furthmayr, H. (1985) Binding
of laminin
to type IV collagen:
a morphological
study. J. Cell BIoL 100, 1848-1853
Paulsson,
M. (1988) The role of Ca2 binding
in the selfaggregation
of laminin-nidogen
complexes.
j BioL C/tern. 263,
5425-5430
Aumailley,
M., Wiedemann,
H., Mann,
K., and Timpl,
R.
(1989) Binding
of nidogen
and the laminin-nidogen
complex
to
basement
membrane
collagen
type IV. Eur J. Biochern. 184,
241-248
Laurie,
G. W., Bing, J. T., Kleinman,
H. K., Hassell,
J. R.,
Aumailley,
M., Martin,
G. R., and Feldmann,
R. J. (1986)
Localization
of binding
sites for laminin,
heparan
sulfate
proteoglycan
and fibronectin
on basement
membrane
(type IV)
collagen.
j MoL BioL 189, 205-216
Frenette, 0. P., Ruddon, R. W., Krzesicki, R. F., Naser, J. A.,
and Peters,
B. P. (1989) Biosynthesis
and deposition
of a noncovalent laminin-heparan
sulfate proteoglycan
complex and
other basal lamina
components
by a human
malignant
cell line.
J. Biol. Chem. 264, 3078-3088
Kleinman,
H. K., Cannon,
F B., Laurie,
G. W., Hassell, J. R.,
Aumailley,
M., Terranova,
U. P., Martin,
G. R.. and Dubois-

LAMININ

Dale, M. (1985)
27, 317-325

56. Nurcombe,

Biological

activities

V., Aumailley,

J. Cell Bloc/tern.

of laminin.

M., Timpl,

R., and Edgar,

D. (1989)

The high-affinity
binding
of laminin
to cells. Assignation
of a
major cell-binding
site to the long arm of laminin
and of a latent
cell-binding
site to its short arms. Eur j Bioc/tern. 180, 9-14

57. Goodman,
S. L., Deutzmann,
R., and von der Mark, K. (1987)
Two distinct cell-binding domains in laminin can independently
promote nonneuronal
cell adhesion and spreading. j Cell BioL
105, 589-598
58. Schittny,
J. C., Timpl, R., and Engel, J. (1988) High resolution
immunoelectron
microscopic
localization
of functional
domains
of laminin,
nidogen,
and
heparan
sulfate
proteoglycan
in
epithelial
basement
membrane
of mouse
cornea
reveals different topological
orientations.
j Cell BIoL 107, 1599-1610

58aMecham,

R. P., Hinek,

A., Griffin,

G. L., Senior,

R. M., and

Liotta,
L. A. (1989) The elastin
receptor
shows structural
and
functional
similarities
to the 67-kDa
tumor
cell laminin
receptor. j BioL C/tern. 264, 16652-16657
59. Edgar,
D. (1989) Neuronal
laminin
receptors.
Trends Neurosci.

12, 248-251
60. von der Mark, K., and K#{252}hl,
U. (1985) Laminin
tor. Bioc/tirn. Biophys. Acta. 823, 47-160
61. Hall, D. E., Frazer,
(1988) Isolation
and

K. A., Hann,
characterization

tein from rat and chick muscle.

62. Rouoslahti,

E. (1988)

Fibronectin

and its recep-

B. C., and Reichardt,

L. F
of a laminin
binding
proj CelL BioL 107, 687-697
and its receptors.
Annu. Rev.

Biochern. 57, 375-413

63. Gehlsen,
K. R.,
(1988) The human

L., Engvall,
E., and Ruoslahti,
E.
receptor
is a member of the integrin

Dillner,
laminin

family of cell adhesion receptors. Science 241, 1228-1229. With

a correction: Gehlsen, K. R., Engvall, E., Dillner, L. Ruoslahti,
E., and Goodman,
gin. Science 245,

64. Risteli,

5. (1989)

RuGli

cell line

not of human

L., and Timpl,

R. (1981) Isolation

and characterization

of pepsin fragments
of laminin
from human
placental
basement
membranes.
Bloc/tern. J. 193, 749-755
65. Wewer,
U. M., Tichy,
D., Damjanov,
A., Paulsson,

Damjanov,

ori-

342-343

I. (1987) Distinct

and
M.,

renal
and

antigenic characteristics
of murine
Develop. BioL 121, 397-407

parietal
yolk sac laminin.
66. Boot-Handford,
R. P., Kurkinen,
M., and Prockop,
D. J. (1987)
Steady
state levels of mRNAs
coding
for the type IV collagen
and laminin
polypeptide
chains of basement
membranes
exhibit
marked
tissue-specific
stoichiometric
variations
in the rat.
j Biol. C/tern. 262, 12475-12478
67. Kleinman,
H . K., Ehihara,
I., Killen, P. D., Sasaki,
M., Cannon, F B., Yamada,
Y., and Martin,
0. R. (1987) Genes for

basement

membrane

proteins

are coordinately

expressed

in

differentiating
F9 cells but not in normal
adult murine
tissues.
Dcv. BioL 122, 373-378
68. Cooper,
A. R., and MacQueen,
H. A. (1983) Subunits
of lasninm are differentially
synthesized
on mouse eggs and early embryos. Dev. Biol. 96, 467-471
69. Klein,
G., Langegger,
M., Timpl,
R., and Ekblom,
P. (1988)

Role of laminin A chain in development

of epithelial cell polarity. Cell 55, 331-34!
70. Davis, G. E., Mathorpe,
M., Engvall,
E., and Varon,
S. (1985)
Isolation
and characterization
of rat Schwannoma
neuritepromoting
factor: evidence that the factor contains laminin. J.
Neurosci. 5, 2662-2671
71. Ohno,

M.,

Martinez-Hernandez,

N. A. (1985) Comparative
placental

membrane

with

A., Ohno,

study
laminin

N., and

Kefa!ides,

found

in normal

of laminin
of neoplastic

meat Mernbranes (Shibata,

S., ed) pp. 3-11, Elsevier
(Mishima, Japan) Amsterdam
72. Fessler,

J. H.
tion. j
73. Beck,

origin.

In Base-

Science

Publ.

L. I., Campbell,
A. G., Duncan,
K. 0., and Fessler,
(1987) Drosophila laminin:
characterization
and localiza-

Cell Biol. 105, 2383-2391

K.,

McCarthy,

R. A., Chiquet,

M.,

Masuda-Nakagawa,

L., and Schlage, W. K. (1989) Structure of the basement membrane protein laminin: variations on a theme. In Cytos/celetal and
Extracellular Proteins: Structure, Interactions and Assembly (Aebi, U.,
and

Engel,

J.,

eds)

pp.

102-105,

Springer-Verlag,

Berlin

74. Chiquet,
tachment
sprouting

M., Masuda-Nakagawa,
to an endogenouis
by leech neurons.
j

L., and
laminin-like

Beck, K. (1988) Atprotein

initiates
Cell Biol. 107, 1189-1198

75. Aratani,
Y., and Kitagawa,
Y. (1988) Enhanced
synthesis
and
secretion
of type IV collagen
and entactin
during
adipose
conversion
of 3T3-Lll
cells and production
of unorthodox
laminin
complex.
J. BioL Chern. 263, 16163-16169

160

Vol. 4

Feb. 1990

76. Leivo, I., and Engvall, E. (1988) Merosin,

a protein specific for
basement
membranes
of Schwann
cells, striated
muscle
and
trophoblast,
is expressed
late in nerve and musde
development.
Proc. NatL Acad. Sci. USA 85, 1544-1548
77. Engvall,
E., Leivo,
I., Ehring,
K., and Ruoslahti,
E. (1989)
Merosin
is a tissue restricted
basement
membrane
component
and a member
of a family of laminin-like
proteins.].
Cell Biol.

109, 4a

The FASEB Journal

BECK Er AL.