148 0892-663819010004-0148/$01 .50. FASEB

Structure and function of laminin: anatomy of amultidomain glycoprotein

KONRAD BECK,* IRENE HUNTER,T AND JURGEN ENGELlnstitute for Biophysics, University, A-4040 Linz, Austria, and tDepartment of Biophysical Chemistry,

Biocenter of the University, CH-4056 Basel, Switzerland

ABSTRACT

Laminin is a large (900 kDa) mosaic protein composedof many distinct domains with different structures andfunctions. Globular and rodlike domains are arrangedin an extended four-armed, cruciform shape that iswell suited for mediating between distant sites on cellsand other components of the extracellular matrix. Thea-helical coiled-coil domain of the long arm is involvedin the specific assembly of the three chains (A, Bi, B2,and possible variants) of laminin and is the only do-main composed of multiple chains. It is terminated bya large globular domain composed of five homologoussubdomains formed by the COOH-terminal part ofthe A chain. Sites for receptor-mediated cell attach-ment and promotion of neurite outgrowth reside in theterminal region of the long arm. A second cell attach-ment site, a cell signaling site with mitogenic action,binding sites for the closely associated glycoproteinnidogen/entactin, and regions involved in calcium-dependent aggregation are localized in the short arms.These domains, which to a large extent are composedof Cys-rich repeats with limited homology to EGF, arethe most highly conserved regions in laminins of differ-ent origin. At present, most structural and functionaldata have been collected for a laminin expressed by amouse tumor, which can be readily isolated in nativeform and dissected into functional fragments bylimited proteolysis. Increasing information on lami-nins from different species and tissues demonstratesconsiderable variations of structure. Isoforms of lami-nm assembled from different chains are focally andtransiently expressed and may serve distinct functionsat early stages of development even before being laiddown as major components of basement membranes.-BECK, K.; HUNTER, I.; ENGEL, J. Structure andfunction on laminin: anatomy of a multidomain glyco-protein. FASEB]. 4: 148-160; 1990.

Key Words: extracellular matrix - basement membrane pro-tein structure coiled-coil - heparan sulfate proteoglycan -epidennal growth factor

LAMININS, A FAMILY OF LARGE multidomain glycopro-teins of the extracellular matrix (ECM),1 have attractedmuch interest because of their importance in the devel-opment and maintenance of cellular organization. Im-portant cellular functions attributed to laminin includestimulation of growth and differentiation, neurite out-growth promotion, and mediation of cell communica-tion. Laminin is the first ECM protein detected duringembryogenesis; it is present at the two-cell stage in themouse embryo. In later development and in mature tis-sue it serves as a ubiquitous and major noncollagenouscomponent of basement membranes. It participates inthe assembly of this specialized form of the ECM andmediates cell attachment and maintenance of the differ-entiated state of epithelial and endothelial cell layersthat are intimately associated with their basementmembranes.

There are a number of excellent recent reviews onvarious aspects of laminin and other ECM proteins(1-5) and on basement membranes (6). In this reviewwe focus on the structure of laminin, the progress thathas been made in assigning functions to distinct do-mains of the molecule, and variations of the lamininstructure in tissue-specific isoforms and in phylogeneti-cally distant laminins.

SOURCES OF LAMININ

Laminin was first isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor (7) and from the extracel-lular deposit of murine parietal yolk sac (PYS) carci-noma cells (8). Laminin from these sources can bereadily extracted and purified in intact form undernonreducing and nondenaturing conditions. In partic-ular, EHS tumor tissue is a rich and convenient sourceof laminin from which it is extractable by neutralbuffers containing 0.5 M NaC1 (9). By a superiormethod, with buffers containing 10 mM EDTA, lami-nm is extracted in the form of its 1:1 complex with nido-

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

C1-4 P1#{149}. .. .., H

149

gen/entactin (10). Consequently most of the biochemicaland biophysical work has been performed with EHS-laminin, which thus became the prototype laminin.Isolation of laminins from normal tissues is oftendifficult. It has been achieved in many cases only withdenaturing and reducing agents (5, 11), but extractionwith EDTA appears to be advantageous (12). Neverthe-less a few tissue- and cell-specific laminins with dis-tinctly different polypeptide chains, chain compositions,and structures are known today. Although data on theseso-called isoforms and on laminin variants from phylo-genetically distant species are still fragmentary (seebelow), there is increasing evidence that the well-known laminin from EHS tumor is just one member ofa protein family. The reader should remember thatconsiderable variations may exist between this lamininand those that have been studied in less detail.

GROSS STRUCTURE AND SHAPE OF MOUSEEHS TUMOR LAMININ

Rotary shadowing electron microscopy of laminin re-vealed an unusual cruciform structure (Fig. l#{192}),withthree apparently identical short arms of 36 6 nm anda long arm of 77 nm (13). Recent studies (14) haveshown, however, that one of the short arms is consider-ably longer (48 4 nm) than the other two (34 4nm). The two smaller arms each contain a central anda terminal globule separated by rodlike regions, whereasthe longer arm contains an additional globular region(Fig. IA, Fig. 1G, arrows). The long arm of laminin ap-pears as a rather flexible rod with a large terminal glob-ule, which at the low resolution of rotary shadowing canbe resolved into two closely spaced smaller globules.Negative staining reveals that the globule adjacent tothe 3-nm thick rod is composed of three, and the moredistant globule of two subdomains, each 4 nm in diam-eter (Fig. 1B).

Electron microscopy thus revealed a very extendedprotein with a maximum dimension of 120 nm, consist-ing of globular and rocilike elements. Hydrodynamicmeasurements showed that this shape is essentiallypreserved in solution (13).

FRAGMENTS OF LAMININ

Fragmentation by limited proteolysis has been in-strumental in the elucidation of the domain organiza-tion and detailed structure of large multidomain pro-teins such as laminin and fibronectin. Laminin hasbeen cleaved into a number of distinct fragments usinga variety of enzymes. Localizations were derived bycomparing the shapes of fragments with that of laminin(Fig. 1), by detecting antigenic determinants shared bydifferent fragments (15), and by comparing partial pro-tein sequences with cDNA-derived sequences (16,17). Biochemical and immunological studies have en-abled the assignment of functions to distinct domains.Table 1 is an overview of the important properties andfunctions of several well-defined fragments, and their

LAM IN IN

E4

I-*1

.

Figure 1. Electron micrographs of EHS-laminin (A, B) and definedlaminin fragments (C-I) after rotary shadowing (A, G-I) and nega-tive staining (B-F). Fragments E3, T8, E8, and C8-9 originatefrom the long arm of laminin. Note that the second terminal glob-ule at the tip of the long arm is visible in intact laminin only (doublearrow 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 oflaminin and fragment P1 the inner rodlike regions of the three shortarms. Note that one of the short arms is longer than the two othersand contains a third globular unit (arrow in A and C). For furtherinformation on fragments and their localization in the lamininmolecule, see Table I and Fig. 2. (bar: 50 nm).

localization within the laminin molecule is shown inFig. 2.

The short arms of laminmn are remarkably resistantto proteases, and several fragments comprising intactor truncated short arms have been detected (14, 15).Fragment P1 is most resistant to proteases and com-prises the inner region of the cross (Fig. iN). In frag-ment El, the outer part of one short arm has been re-moved in the form of fragment E4 (Fig. 11) and theother short arms are often truncated.

Fragment E3 (Fig. 1C), comprising the outer of thetwo globules at the end of the long arm (G4-G5 inFig. 2), is readily released during proteolysis and is ab-sent from fragments derived from the long arm. Frag-ments T8 (Fig. 1D) and E8 (Fig. 1E) of the long armconsist of rodlike regions of different lengths togetherwith the adjacent globular domain (G1-G3 in Fig. 2),

TABLE 1. Properties of well-characterized fragments of mouse EHS-laminin

150 Vol. 4 Feb. 1990 The FASEB Journal BECK ET AL.

Fragments native

M (kDa)6SDS-PAGE

- red. + red. NH2 terminal aa Shape Predominant Prominent functions Reference

Short-arm structures

P1 290 280 50+ other bands

nd Fig. IH Aperiodic MitogenicCell attachmentBinding of nidogen/entactin

1818, 1910, 20

El 450 400 d nd As C1-4,but with one

truncated arm

Aperiodic Mitogenic 18

E4 75 75 60 7 (B!) Fig. If Aperiodic Inhibition of Ca2-inducedaggregation of larninin

21

C1-4 550 550 a nd Fig. IC Aperiodic Ca2-dependent aggregation 14

Long arm

C8-9 340 190 (A)+170(BI-B2)

190 (A)+ll0(B1)

+ 90 (B2)

Fig. IFnd

cs-helical (75%) Cell attachment(P. End, M. Bruch,

unpublished results)E8 240 140 (A)

+ 80 (B1-B2)140 (A)

+ 45 (BI)+ 35 (B2)

1540 (BI) Fig. IE1329 (B2)1886 (A)

cr-helical (45%) Cell attachmentPromotion of neuriteOutgrowth

19, 22

22, 23

T8 nd 80 (A)25 (Bl-B2)

nd 1679 (BI) Fig. ID1473 (B2)2009 (A)

cr-helical (30%) Cell attachment

25K 50 26 (B1-B2) 12 (B2)+ 10 (B!)

1679 (BI) Short rod1473 (B2)

cs-helical (100%) Antibodies against 25Kinhibit neurite outgrowth

22

E3, C3 55 50 50 2666 (A) (E3) Fig. 1C il-structure Binding of heparin andand 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 endogenousfragment (27). For protocols of preparation and characterizations, see refs 9, 14, 15, 27. Molar masses of native fragments are from sedimentationequilibrium. Apparent molar masses for the constituent chains were derived by SDS-PAGE. Origin of chain segments are indicated by chain designationsin brackets. Bl-B2 indicates that segments of the Bi and B2 chains are disulfide linked. Numbering corresponds to the original literature which mightdiffer 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 thelaminin chains. dA complex pattern of bands is observed depending on the time of exposure to the enzyme.

whose three subdomains are clearly seen by negativestaining (Fig. iD-iF). Its sensitivity to proteases hasprevented the isolation of defined fragments from theupper region of the long arm. Recently, however (14),cathepsin G was found to cleave laminin initially at twosites only (Fig. 2, arrows), thus making available a frag-ment Ci-4 (Fig. 1G) comprising the intact short arms,and a fragment C8-9 (Fig. iF) comprising the entirerodlike region of the long-arm and globular domainsG1-G3.

Laminin is unevenly glycosylated, average 14 (24,25) to 25% (26), and therefore the molar masses of thenative fragments deviate to different extents from thosethat may be predicted from the sequence. Glycosylationalso causes anomalous migration of the chains duringSDS-PAGE giving apparent molar masses.

Circular dichroism of intact laminin revealed about25% a-helix, which melted out in a sharp transitioncentered on 59#{176}C(15, 27). The conformation of theshort-arm fragments cannot be classified in terms ofwell-known secondary structure; fragments derivedfrom the long arm are highly a-helical (Table 1) andfragments E3 and C3 have a 3-structure (14, 15).

A MODEL OF MOUSE EHS LAMININ

Mouse EHS tumor laminin consists of three differentpolypeptide chains-A (440 kDa), Bi, and B2 (eachabout 220 kDa) -which are disulfide linked to form thecharacteristic asymmetric cross-structure seen by elec-tron microscopy (Fig. IA). The recent cloning and se-quencing of all three chains of mouse laminin (17) aswell as chains from human (28-30), Drosophila (31, 32),and rat s-laminin, a tissue-specific variant (11), have re-vealed a domain organization that fits well with the ob-served ultrastructure, and have suggested the arrange-ment of the three chains within the laminin moleculeshown in Fig. 2.

All three chains contain six domains, 1-VI (Fig. 2).Additional globular and rodlike domains (lila, IVa) arefound in the short-arm region of the A chain. DomainsI and II, located at the COOH-terminal ends of the Bchains and in a related region of the A chain, containa series of heptad repeats (33) and are predicated to bea-helical. The calculated length of these domains, as-suming a distance of 0.15 nm/residue in an a-helix, is85 nm, which is in agreement with the length of the

(A)

ifib

4(Th

NH2 (Bi)

VI V, ifi

U

a

8-9

Gi G2 G3 G4

LJ

3

LAMI N IN

Figure 2. Structural model of laminin. Designations of domains byroman numerals is according to ref 17. Cys-rich rod domains in theshort arms are designated by symbols S and the triple coiled-coilregion (domain I-LI) of the long arm by parallel straight lines. Inthe Bl-chain, the cr-helical coiled-coil domains are interrupted by asmall Cys-rich domain a. Interchain disulfide bridges are indicatedby thick bars. The primary cleavage sites of cathepsin G are markedby 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 by electron microscopy (13).Domains III and V are rich in glycine and cysteine,

suggesting that they contain many turns, possibly sta-bilized by disulfide bonds. Each domain consists ofmany homologous repeats of about 50 amino acids,with 8 Cys arranged in regular positions, and havesome homology to the 6 Cys motifs of epidermalgrowth factor (EGF) and transforming growth factor a(TGF-a) (17, 34). The repeats are probably arrangedlike beads on a string and are likely to correspond to therodlike regions of the short arms.

Domains IV and VI are thought to form the centraland terminal globules of the short arms. Domain VIcontains the NH2 terminus of each chain.

In addition to the six common domains, the Bi chaincontains an additional 40-amino-acid-long sequence(domain a) located between domains I and II. It con-tains 8 Gly and 6 Cys, and is likely to be highly foldedand stabilized by disulfide bonds. It is incompatiblewith a-helix formation and is presumed to be loopedout of the long arm, although no morphological struc-

1-4 ture corresponding to this domain has been detected byelectron microscopy. Domain G forms the COOH-terminal region of the A chain with no counterpart inthe Bi and B2 chains. Its predicted structure fits wellwith the large terminal globule seen by electron micros-copy (Fig. IA). Domain G consists of five homologousrepeats (Gi-G5), which probably represent the fivesubdomains detected by negative staining of intactlaminin (Fig. 1B).

SEQUENCES AND CONFORMATION

Short-arm structures

To examine in more detail the relationship between theA, BI, and B2 chains, the amino acid sequences of allthree chains were compared. Alignment of the short-arm sequences is shown schematically in Fig. 3.

The sequence of the NH2-terminal domain VI issignificantly conserved in all three chains of EHS lami-nm, and highly homologous regions have been found atthe NH2 termini of all other laminins sequenced so far.In particular, the 6 or 8 Cys residues are conserved; asequence WWQS in the middle of the region and a pat-tern of Y(Y/F)YX8G terminating region VI are highlypreserved. The latter motif is also found in a hypervari-able region of some immunoglobulins.

The inner globular regions (domain IV) of the A andB2 chains are homologous and may be considered to bederived from the Cys-rich, so-called EGF-like repeats,by a large insertion between the third and fourth Cysof one of these motifs (34). Within the A chain, thisregion occurs twice (IVa and IVb), corresponding tothe two inner globules seen on one short arm by elec-tron microscopy (Fig. IA, Fig. 1G). The large size of theinsertions (180-200 amino acids, lacking Cys) explainstheir appearance as distinct domains in electron micro-graphs. Domain IV of the BI chain has a unique se-quence motif unrelated to domain IV of the B2 and Achains. In the homology scheme shown in Fig. 3, it ap-pears as an insertion (IV) together with the adjacentCys-rich repeats.

There are significant homologies between the Cys-rich, EGF-like domains III and V of all three chains,and in a single chain there are homologies both withinand between these domains. The EGF-like repeats maybe classified on the basis of the number of residues be-tween the second and third, third and fourth, andseventh and eighth Cys. These numbers are very vari-able when the sequences repeated in the same chain arecompared, but well conserved when sequences in cor-responding positions of the same chain from differentspecies are considered. This is clearly demonstrated by

U

152 Vol. 4 Feb. 1990 The FASEB Journal BECK E AL.

RYVVLPRP

B2 III1)IIlIIIECPAc,x VV

i i i I i iU

VVVi i i i i#{149}.

1U)

LRtiDN

EJSCDDDC

DIioco

HSPG {Figure 3. Alignments of sequence regions in the short-arm struc-tures of EHS-laminin. Regions with predicted globular conforma-tion are indicated by circles and Cys-rich EGF-like repeats bysquares. For best alignment of Cys-rich repeats, an insertion in theB! chain (dashed box) and a repeat and gap in the A chain wereintroduced. Cys-rich repeats with additional insertions (see text)are indicated by squares with half circles. Domains VI in all threechains are related (hatched), whereas domain IV (cross-hatched) inchain BI is unrelated to domains IV in A and B2. Cys-rich repeatsof heparan sulfate proteoglycan (HSPG, 35) are aligned for com-parison. Sequence data for mouse laminin are by Sasaki et al. (17).Triangles mark putative sites of N-linked glycosylation. Sequenceregions for which functions have been proposed (Table 2) are indi-cated by their sequences in one letter code. Sequences that are prob-ably involved in disulfide linkage of the three chains (Fig. 2) are in-dicated at the right-hand side.

a comparison of the BI chains of mouse EHS tumorlaminin, Drosophila laminin, and s-laminin (11, 31). Thisobservation suggests that the order of EGF-like do-mains is of functional importance and argues against asimple structural role as spacer elements. The func-tional significance of the short-arm structures is furtheremphasized by their high degree of conservation (about60% on average) in laminins that exhibit only 20% ho-mology in regions of the long arm (ii, 31).

Within mouse EHS laminin, the two EGF-like repeatsadjacent to each side of domain IVa of the A chain aresimilar to those in the same position around domain

IVb of the A chain and domain IV of the B2 chain. Thepartial sequence of the core protein of basement mem-brane heparan sulfate proteoglycan (HSPG) also con-tains tandem repeats with 8 Cys (35) similar to regionIV and the neighboring repeats of the A and B2 chainsof laminin (Fig. 3).

Assuming that the EGF-like repeats are arrangedlinearly, an average translation per domain of 2.5 nmfollows from the number of repeats and the electron

PCHOC microscopically derived dimensions of the short-armrods. This agrees well with the predicted dimension ofan individual domain, assuming an EGF-like confor-mation (34).

Long-arm structure

In contrast to the short arms, the sequences of the A,Bi, and B2 chains assigned to the rodlike region of thelong arm (domains I and II) have little sequence ho-mology, but all contain a heptad repeat of the type(a,b,c,d,e,f,g)n where hydrophobic amino acids are lo-cated preferentially in positions a and d, charged resi-dues normally in positions e and g, and polar residuesfrequently in b, c, and f. Such sequence motifs arecharacteristic of proteins in which two or three a-helices are wound around each other to form a coiled-coil (33).

Using the long-arm fragments C8-9 and E8 (Fig. 2),we have recently shown that the a-helical domainsI and II are involved in chain assembly (36). When theA, Bi, and B2 chains present in these fragments areseparated and unfolded in urea, they reassemble intomolecules which in their a-helix content, apparentmolar mass, chain composition, and ultrastructural ap-pearance are indistinguishable from the native frag-ments. The results indicate that all three chains interactto form a triple-coiled coil, which can extend the lengthof the long arm. The highly specific nature of this inter-action suggests that it is the mechanism by which lami-nm assembles in vivo.

Figure 4 shows a cross-section through a triplecoiled-coil, as is suggested for laminin. Burying thehydrophobic residues located in positions a and d in thecenter of the structure, thus shielding them from theaqueous environment, is energetically favorable and isthe driving force for coiled-coil formation. Hydrophiicamino acids are exposed on the surface, and the coiled-coil is further stabilized by ionic interactions betweenresidues e and g.

To maximize coiled-coil interactions, the alignmentof the A, Bi, and B2 chains shown in Fig. 5 is proposed.The a-helical domains I and II of each chain arebounded by 1 COOH-terminal and 2 NH2-terminalCys-residues. These Cys are separated by 566, 567, and604 residues in the A, B2, and Bi chains, respectively.A disulfide bridge between the COOH-terminal Cys-residue of the BI and B2 chains has been established(27), and it may be speculated that a free Cys-residuein the corresponding position of the A chain partici-pates in the catalysis of its formation. If domain a is

0 [nm] 0.5

I I

+

Figure 4. Projection of a three-stranded a-helical coiled-coil, oneheptad each, viewed from the NH2 terminus. A distance of 1.0 nmis assumed between the helix axes. Positions of 3-carbon atomswithin a heptad repeat assuming 3.6 residues per turn (a-g). Theclustering of hydrophobic side chains (filled circles) and electrostaticinteractions (dashed circles) are schematically indicated. Side chainin positions b, c, and f are located at the surface and are normallypolar or charged (open circles).

B2dJ Bi

Bi: LQQSAAB2: NDILNN

V25KKVESLINSVSSL

Bi PHQ

B2 EPA

A SDI

A : AHVHSN LHREHG

a :iuE8 [j

0

E

I -SRARK v

100 200 300 400 500I I I I I

Iii:coiled-coil factor F1 x 108: V//////DO.5 i..i>O2 I 1

154 Vol. 4 Feb. 1990 The FASEB Journal BECK ET AL.

ticipate in coiled-coil formation (see above), in homo-typic interactions the A chain would not be expected toadopt this structure. This is in agreement with recentobservations from this laboratory (I. Hunter andJ. En-gel, unpublished results) that although the Bi and B2chains alone can form a coiled-coil, the A chain canparticipate in coiled-coil formation only in the presenceof the B chains.

Domains I and II of all three chains together accountfor a protein molar mass of 192.8 kDa. Assuming alength of 77 nm for the long arm, a mass-per-lengthratio of 2500 Da/nm follows, which is slightly higherthan expected for a perfectly packed triple-coiled-coil(2250 Da/nm) and suggests some nonhelical interrup-tions, presumably due to the variations in the stabilityof the coiled-coil regions described above, and to thepresence of a number of proline residues (Fig. 5).

The COOH-terminal region of the A chain adjacentto the coiled-coil domain contains five internal homolo-gous repeats (G1 to G5), each consisting of about 200residues. For human laminin, only regions G2 to G5have been sequenced (30) and exhibit an overall iden-tity of 74% with mouse EHS laminin. This homologyis surprisingly low when compared with more than90% identity of other parts of mouse and human lami-nm (28, 29).

GLYCOSYLATION

Seventy-four potential N-glycosylation sites NXS/Thave been identified in the three chains and are indi-cated in Fig. 3 and Fig. 5. The sites are unevenly dis-tributed between chains and are concentrated in thelong arm. In EHS laminin, some 40 possible acceptorsites are occupied by an unusual variety of oligosaccha-ride (24-26). In contrast to earlier studies that reported12-15% (w/w) glycosylation (8, 24, 25), a value of25-27% (w/w) has recently been reported (26). Align-ing the chains in the long arm (Fig. 5) Asn(N) ofNXT/S-motifs occurs most often in positions b, c, or fof the heptad repeats (Fig. 4), indicating a localizationat the surface of the coiled-coil. The only function ofglycosylation of laminin described so far is involvementin tumor cell adhesion (40), whereas it is not needed forchain assembly (41) or heparin binding, and does notconfer stability against proteases (42).

STRUCTURE-FUNCTION RELATIONSHIPS

Laminin exhibits a variety of biological activities, in-cluding promotion of cell attachment, growth anddifferentiation of a number of cell types, and multipleinteractions with other basement membrane compo-nents (1-6). Progress toward assigning these functionsto distinct domains of the molecule has come largelyfrom studies with proteolytic fragments and domain-specific antibodies. Table 1 provides a summary of thefragments for which a function has been assigned, andthe localization of these fragments within the lamininmolecule is shown in Fig. 2. In addition, the recent

cloning and sequencing of laminin chains has allowed,through the use of synthetic peptides, the possibility ofdefining specific sequences responsible for the observedfunctions of laminin. To date, only a limited number ofsynthetic peptides have been studied. Their localizationand possible function are summarized in Table 2.

Laminin has the ability to self-associate, forminglarge aggregated structures (50). This activity is medi-ated by the globular regions at the tips of the short arms(domain VI), and is Ca2 dependent (14, 21, 50, 51).Chelating agents arrest the polymerization at the levelof small polymers, and may explain why laminin isreadily extractable from basement membranes withEDTA-containing buffers (10).

Laminin forms a particularly stable complex withnidogen/entactin, which can be extracted from mouseEHS tumor as a 1:1 complex (10). Nidogen has beenobserved 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 alsobeen demonstrated to bind to type IV collagen and hasbeen proposed to mediate binding of laminin to thiscollagen (20, 52). Direct interactions of laminin withcollagen IV have also been reported (50, 53). Sites onlaminin responsible for binding have been localized byelectron microscopy to the terminal globules of theshort (domain VI) and long (domain G1-G5) arms(50).

A major binding site for heparin was localized to theterminal globule of the long arm (15) and may also beinvolved in the binding of basement membrane heparansulfate proteoglycans (54). The five homologous regions(Gi-G5) constituting the long-arm globule containclusters of basic amino acids (17, 39) and probably rep-resent the binding site for the polyanionic heparin andheparan sulfate proteoglycans.

The multidomain nature of laminin is ideally suitedto mediate the interactions of a variety of basementmembrane components, and thus plays a key role increating and maintaining the complex 3-dimensionalstructure necessary for the correct functioning of base-ment membranes. In addition, its size and shape enableit to span the basement membrane and contact cell-surface receptors through which it can exert a varietyof effects on cellular processes.

Laminin possesses two cell attachment sites, one lo-cated on the short-arm fragment P1 (19, 55) and theother of high affinity (56) on the long-arm fragment, E8(19, 57). The short-arm site is not active, however, inthe complete short-arm fragment E1-4 that encom-passes P1, or in intact laminin (56). It thus appears tobe a cryptic site that may be activated by proteolysisduring tissue remodeling and tumor cell invasion.Studies with synthetic peptides (Table 2) have identifieda sequence YIGSR present in domain III of the Bichain with cell binding activity, but only at very highmolar concentrations when compared with laminin.The major cell attachment site of laminin is apparentlylocated within the long-arm fragment, E8. This is ingood agreement with immunoultrastructural observa-tions (58) indicating that the inner regions of the short

LAMININ 155

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

Chain Domain aa-residues Peptide Proposed function Reference

Bi V 442-447(H:

5:

D:

LGTIPGLGTIPGRGTVPGLGTLNN)

Binding to a cellular elastin receptor ofwhich a 67-kDa component is homologousto a laminin receptor protein

58a

B! IV 64 1-660(H:

5:

D:

RYVVLPRPVCFEKGMNYTVRRYVVLPRPVCFEKGTNYTVRRVLVFPRPVCLEPGLSYKLKRQVVALNEVCLEAGKVYKFR)

Binding of heparin; promotes cell adhesionof murine melanoma cells, fibrosarcoma,glioma, pheochromocytoma, and aorticendothelial cells

43

B! III 902-906(H:

s:

D:

PDSGRPDSGRPGSQRVASGL)

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

44

B! III 925-933(H:

5:

D:

CDPGYIGSRCDPGYIGSRCRAGYTGLRCQEGYSGSR)

Promotion of cell attachment, chemotaxis,neuronal attachment but not neuriteoutgrowth

Promotes melanoma cell migrationInhibits formation of lung tumors

44, 45

44, 4644, 47

B2 I 1542-155!(H:D:

RNIAEIIKDARDIEEIMKDIRELKDEVQ.NI)

Stimulates neurite outgrowth 48

A IlIb 1118-1128 GTFALRGDNGQ Promotes adhesion of endothelial cells; canstimulate process extension ofneuroblastoma cells

49

A I 2091-2 108 SRARKQAASIKVAVSADR Promotes neurite outgrowth of some butnot all neuronal cells; promotes adhesionof neuronal and nonneuronal cells;stimulates collagenase activity

49

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

arms are confined to the interior of basement mem-branes, while the long-arm epitopes are located at thebasement membrane surface.

Laminin is a potent stimulator of neurite outgrowth(22, 23), a function that may be involved in the growthand regeneration of peripheral neurons, which are sur-rounded by laminin-producing Schwann cells. Domain-specific antibodies have implicated sequences withinfragment E8 as important for this activity, and a syn-thetic peptide corresponding to a defined sequence ofthe B2 chain (Table 2) has been reported to have neu-rite outgrowth-promoting activity (48). Another pep-tide with a sequence from the a-helical rodlike regionof the A chain (Table 2) has also been shown to havesome activity (49).

Laminin has a mitogenic effect on a number of cellsin culture (55), which in dose response and time depen-dence is comparable to that of EGF (18). This functionhas been localized to fragment P1, which consists ofCys-rich repeats (domain III) and is not related to itscell attachment properties (18). Synthetic peptides cor-responding to domain III sequences, including the pen-tapeptide YIGSR, which represents part of the Cys-rich repeat with closest homology to EGF, had no mito-genic activity (18).

A general problem associated with studies using syn-thetic peptides is that they represent only a small partof the domains from which they are derived and are un-likely therefore to have the correct structure. Many ofthe functions mapped to specific domains have beenshown to be conformation dependent. In particular, themitogenic function of fragment P1 is lost upon reduc-tion (P. End, unpublished results), and cell adhesionand neurite outgrowth activities of fragment E8 areabolished upon denaturation (20, 57). Therefore, fu-ture studies aimed at precisely delineating functionalsequences must also consider the structural require-ments of such sequences. To achieve this, studies (44)were performed recently with a cyclized form of thepeptide YIGSR.

Homology searches have not been helpful in suggest-ing other possible functions of distinct regions of lami-nm. The only strong homology with a protein not be-longing to the laminin family is the one with HSPG (35;see Fig. 3). The weak homology of domains III and Vwith EGF and EGF-motif containing proteins has beenmentioned. Weak homologies are also observed be-tween domains I and II and many other proteins con-taining coiled-coil structures. Perhaps an interestinghomology exists between domains GI to G5 and sex

156 Vol. 4 Feb. 1990 The FASEB Journal BECK ET AL.

steroid-binding protein (codes G08607 and S00077 ofthe National Biomedical Research Foundation proteinsequence data base).

In agreement with its numerous biological activities,a variety of cell-surface receptors necessary to mediatelaminin activity have been identified (2, 59). In thepresent context the fast-growing field of laminin recep-tors cannot be reviewed, and only a few examples willbe mentioned. A 67-kDa protein is particularly well ex-pressed in metastatic cells (60), which could be elutedfrom a laminin affinity column by the domain III-derived, synthetic peptide YIGSR (45). Recent studieshave questioned this proposed binding site, as purified67-kDa receptor was shown by rotary shadowing elec-tron microscopy to bind specifically to the top of thelong arm (domain II) near the center of the cross(M. E. Sobel and V. Castronovo, personal communica-tion). A laminin binding protein from skeletal muscle,aspartactin (66 kDa), was reported to bind to fragmentE3 (G4-G5, 61).

Cell-surface receptors of the integrin type (62),several of which bind through the specific sequenceRGD on their ligands, have been implicated in lamininbinding. Binding of laminin to an integrin receptor iso-lated from rat RuGli cells and human placenta re-quired divalent cations, but was not inhibited by RGD-containing peptides (63). This integrin is believed tobind to the fragment 8 region. An RGD sequence de-tected in the G3 domain of human laminin is probablynot functional (20) and is not conserved in mouse lami-nm. Another integrin-type receptor was proposed to beresponsible for the cell attachment activity of fragmentP1 and was found to be RGD dependent (20). Mouselaminin contains an RGD sequence in domain Ilib ofthe A chain, and a synthetic peptide containing this se-quence (Table 2) has been found to promote cell adhe-sion (49). In intact basement membranes, however, theshort-arm structures containing the RGD sequence areburied whereas the long-arm terminal domains arelikely to contact cells (58).

ISOFORMS AND STRUCTURAL VARIANTSOF LAMININ

The identification of distinct but related pepsin frag-ments in tissue digests as compared with digests of EHSlaminin (64), antigenic differences between lamininsderived from tumor cells and normal tissues (12, 65),and the absence or reduced expression of the 440-kDaA chain in a number of cell types in culture (5, 6, 30),has led to the suggestion that structural variants oflaminin may exist. In addition, variation in the levelsof mRNA for the A, BI, and B2 chains of laminin indifferent tissues of the same species (66, 67) indicatesthe formation of tissue-specific isoforms, possibly withdifferent functional properties.

Laminin appears to be developmentally regulated.During mouse embryogenesis, the B chains are de-tected at the 2-4 cell stage, whereas the 440-kDa Achain is not detected until the 16-cell stage (68), which

is coincident with the ultrastructural appearance of adistinct basement membrane. During normal kidneydevelopment, antibodies against EHS laminin coulddetect only B chains in undifferentiated mesenchyme,but on conversion to a polarized epithelium, A chainepitopes were expressed (69). Whether these lamininvariants consist of B chains only or have in addition amodified A chain, antigenically unrelated to the Achain of EHS laminin, is presently unknown.

Isolation and characterization of laminin fromSchwannoma cells, which lack the 440-kDa A chain,revealed a Y-shaped molecule with one long and twoshort arms (Table 3). Although only B chains (200-220kDa) could be detected by SDS-PAGE under reducingconditions, the Schwannoma laminin had an apparentmolecular mass of about 850 kDa under nonreducingconditions, indicating that it was not formed solelyfrom the Bi and B2 chains and suggesting the existenceof a variant (shorter) A chain. Such variant forms haverecently been described in laminin from mouse heart(300 kDa) (12) and 3T3-Li cells (180 kDa) (75). In3T3-L1 cells, as shown previously for cells synthesizingthe 440-kDa A chain, B1-B2 disulfide-linked dimers areformed as intermediates in laminin assembly, but thesecan be secreted only in the form of a ternary complexwith the 180-kDa A chain (75). Merosin first describedas an 80-kDa protein present in basement membranesof human muscle and peripheral nerves (76) has beenidentified on the basis of a 40% homology as a frag-ment originating from the COOH-terminal portion ofanother tissue-specific A chain variant (71). This new Achain was shown to be associated with apparently nor-mal Bi and B2 chains to a molecule with a shape simi-lar to EHS laminin (71).

All isoforms investigated so far by electron micros-copy (12, 22, 70) have a long-arm terminal globule,which in mouse EHS laminin is derived entirely fromthe A chain. Available evidence suggests therefore thatstructural variants of laminin are of the type A(B)2.Whether the A-chain variants detected so far arerelated to the 440-kDa A chain, possibly splicing vari-ants, or are novel subunits, awaits the sequencing ofthese chains.

Additional structural information has come fromstudies of laminins of different species origin. The grossshape of laminins originating from different phylaranging from Cnidaria to mammals has been inves-tigated in some detail by electron microscopy (Table 3).All of the laminins studied exhibit an asymmetric cross-structure with one long arm bearing a terminal globuleand three short arms each with terminal and centrallylocated globules. The structure of these molecules thusclosely 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 con-siderably with species. Two globular domains at the tipof the long arm, connected by a short, 5-10 nm longrod, may be related to the subdomains G1-G3 andG4-G5 seen in negatively stained preparations ofmouse tumor laminin (Fig. 1B). The preparation of a

TABLE 3. Properties of laminins pur!fied from different sources

1.AMININ 157

Source Shape Chains Reference

Mouse EHS tumor Three short arms (36, 36, and 48 nm);one long arm (77 nm); see Fig. !

440 kDa (A)220 kDa (B!, B2)

13, 14

Mouse heart Similar to EHS laminin but possibledifferences in short-arm structures

Prominent 300-kDa chain immunologicallyunrelated to A and B chains of EHSlaminin, 220 kDa (B)

12

Rat Schwannoma cells Y-shaped with two short and one long arm 220 kDa, 180 kDa; probably a mixture ofa short A-chain variant and B chains ofnormal size

22, 70

Human placenta Similar to EHS laminin; long arm, 83 nm 350 kDa (A)195, 185 kDa (B)240 kDa (M chain) immunologicallyrelated to A and B chains

71

Drosophila K, cells Three short arms (30, 30, and 36 nm);one long arm (84 nm)

400 kDa (A)215, 185 kDa (BI, B2)

72

Sea urchin embryo Three short arms (35, 35, and 48 nm);one unusually long arm (113 nm)The extended short arm is found for asubpopulation of molecules only andexhibits two inner globules

480 kDa (A?)269 kDa (B!, B2?)

73

Leech ganglion capsules Three short arms (36, 36, and 53 nm);one long arm (94 nm)

340 kDa (A?)220 kDa (B?)

73, 74

Anthomedusa Three short arms (36 nm);one long arm (97 nm)

340 kDa (A?)260 kDa (B?)

73

Length measurements were performed after rotary shadowing electron microscopy. Apparent molar masses were determined by SDS-PAGE afterreduction.

proteolytic fragment from sea urchin laminin, which isindistinguishable by electron microscopy from frag-ment E8 of mouse tumor laminin (73), indicates thatthere may be similarities between these COOH-terminal regions in both laminins. Thus, modificationsleading to the elongation of the long arm in many spe-cies may be restricted to the proximal part of this arm.Such length variations may be required to span base-ment membranes of different thicknesses.

OUTLOOK

A key problem for future research on laminin will bethe further elucidation of structure/function relation-ships. Assignment of functions to regions in themolecule by the proteolytic fragmentation approachhas been very successful and will probably continue tobe so. Its limitations are that defined fragments cannotbe prepared from all parts of laminin and the fact thatsmall fragments do not retain their native conforma-tion. The alternative approach of testing the activity ofpeptides synthesized according to sequence regions sus-pected to be involved in functions has become popularin the last few years, and was motivated in part by thepossible pharmacological value of such peptides as, forexample, antimetastatic drugs. A major breakthroughof this strategy was the localization of the fibronectincell binding site to the tripeptide RGD (62). Some ofthe results obtained with peptides synthesized accord-

ing to the mouse laminin sequences (Table 2) are prom-ising, but the required doses are many orders of magni-tude higher on a molar basis than for native laminin orits active fragments. Small synthetic peptides of ran-dom conformation are unlikely to exhibit functionssuch as cell attachment and neurite outgrowth ofperipheral neurons that are abolished by denaturationof intact laminin. A related problem is the correct for-mation of disulfide bonds in synthetic peptides thatresemble disulfide-rich regions of, for example, theshort-arm structures of laminin. It is well known thatfor many hormones and other biologically active mol-ecules, the correct disulfide linkage is required for thenative conformation and for functionality. It is there-fore required to confirm the results obtained with pep-tides by independent methods. Site-directed mutagene-sis and functional tests of the modified laminins in vitroand in vivo may be useful. A more conventional methodthat has been applied to laminin in only a few cases isinhibition by monoclonal antibodies directed to regionsproposed to be involved in functions.

Work on the expression of distinct regions of lamininis being pursued in several laboratories. If properlyfolded and disulfide stabilized domains can be preparedby suitable expression systems, these could be tested forfunctions. Also, 3-dimensional structures could then bedetermined at atomic resolution by nuclear magneticresonance and X-ray crystallography. Laminins are aprotein family of utmost biological importance, and

158 Vol. 4 Feb. 1990 The FASEB Journal BECK Er AL.

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

We thank Ms. C. Fauser for expert technical assistance andM. Bruch and M. Paulsson for valuable discussions and com-municating unpublished results. Original work from this laboratoryreported herein has been supported by The Swiss National ScienceFoundation (grant no. 31-9088.87 to J. E.). The present work byK. B. is supported by the Deutsche Forschungsgemeinschaft(Be 1143/1-1).

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