Laminin:

multiple

forms,

multiple

receptors

A.M. Mercurio Laboratory

of Cancer

Biology,

Current

Deaconess Hospital, Harvard Massachusetts, USA Opinion

in Cell Biology

Introduction

an expanding

protein

School,

Boston,

1990, 2:845-849

122:373378) and during embryonic development (Klein et al, Cell 1988, 55:331-341). Similarly, the neurite-promoting factors secreted by Schwannoma cells are laminin molecules that lack a normal A chain (Edgar et al., J Cell Bioll988, 156:129+1306). These observations foreshadowed new data that convincingly demonstrate tissue-specific variation in the structure of basement membrane laminin. Improved extraction protocols were used to isolate intact mouse heart laminin [ 11 which has a much lower A chain content than EHS laminin. It also contains a disuffide-linked, 300kD polypeptide chain that is immunologically distinct from the A and B chains. The use of tissue-specific antibodies and molecular cloning has also revealed the existence of novel, tissue-specific basement membrane proteins. Merosin (estimated M, = 38OkD) is present in placenta, striated muscle, and peripheral nerve. Its deduced ammo acid sequence is homologous to the carboxy terminal region of laminin A chain and it complexes with larninin Bl and B2 chains [ 21. S-laminin, a homologue of laminin Bl, is found in synaptic basement membranes, as weU as glomeruli and blood vessels [3]. Unlike variants in fibronectin structure, which arise from alternative splicing (reviewed by Komblihtt and Gutrnan, Biol Rev Cambridge Philoqbic Sot 1988, 63:465-508), the laminin-like proteins identified to date represent distinct gene products [l-3].

laminin, the major glycoprotein component of basement membranes, is a potent modulator of cell function. The extensive list of its functions includes the ability to alter cell growth and motility, promote epithelial differentiation, modify leukocyte function, and stimulate neurite outgrowth (reviewed by Timpl, Eur J Biocbem 1989, 180:487-502); Beck et al, FtiEB J1990,4:148-160). The common denominator of these functions is that they are initiated by the interaction of laminin with specilic cellsurface receptors. Recent data, which I shall review here, indicate that diversity in the structure of laminin itself, as well as an array of receptors, contribute to this multitude of laminin-regulated cell functions.

Laminin:

Medical

family

Iarninin, initially characterized in extracts from the Englebreth-Holm-Swarm (EHS) murine tumor (Timpl et al, J Biol Chem 1979, 2549933-9937) and teratocarcinema cells (Chung et al., Cell 1979, 16:277-287), is comprised of three disuffide-linked polypeptide chains present in equal amounts: an A chain (M, = 400 kD) and two B chains (Bl, M, = 225 kD and B2, M, = 205 kD). The complete molecular structures of the Bl and B2 chains of mouse, human and Drosophila have been reported. The mouse A chain and pan of the human A chain have also been sequenced (reviewed by Kleinman and Weeks, Curr @in Cell Bioll989, 1:964-967; Timpl,

Taken together, the available data provide evidence of a structurally diverse family of laminin molecules. This diversity arises from variations in the expression of the laminin polypeptide chains that may be present in all basement membranes, as weU as from the tissue-specific expression of distinct proteins that are homologous to the laminin chains [l-3]. The continued use of tissuespecific antibodies, improved extraction protocols and molecular cloning should uncover more laminin-like proteins. A major challenge will be to correlate the structure and tissue-distribution of these novel proteins with specific cellular functions and receptors. S-laminin is most revealing in this regard because it appears to function in synapse formation or stabilization [3] and it contains a tripeptide (Leu-Arg-Glu) that mediates neuronal attachment [4].

1989).

A central consideration for this review is whether the protein structure of EHS laminin, by far the most studied, is reflective of all basement membrane laminin. This has not been an easy issue to resolve because laminin in most basement membranes is relatively insoluble and difficult to isolate in intact form. Previous studies using cells in culture provided the first indication that the structure of EHS tumor laminin is not universal. Specifically, A chain expression is diminished relative to B chain expression in many cultured cells (Kleinman et al, Dezj Biol 1987, Abbreviations CBP-carbohydrate-binding

@

protein;

Current

Biology

EHS-Englebreth-Holm-Swarm

Ltd

ISSN

0955-0674

[murine

tumor].

845

846

Cell-to-cell

contact

Laminin

receptors

and extracellular

matrix

The initial isolation of laminin receptors was by lamininsepharose chromatography of cell extracts. The predominant protein that bound to such columns exhibited an Mr of 67-69kD and was thought to be ‘the laminin receptor’ (Rao et al, Biocbem Biopkys Res Commun 1983, 111:804-808; Malinoff and Wicha, J Cell Bioll983, 96:1475-1479; Lesot and von der Mark, EMBO J 1983, 2:861-+X65). Since then, a wealth of specific integrin antibodies and improved conditions for binding integrins to laminin columns have established the central importance of integrins as laminin receptors. At the same time, continued studies on non-integrin laminin -binding proteins have stimulated much discussion about their possible role in laminin adhesion.

lntegrin

laminin

(reviewed by Kleinman and Weeks, 1989), and it has been used to identify several non-integrin binding proteins [ 161.

receptors

Current data indicate that cell adhesion to laminin is mediated by multiple members of the integrin family, in particular the pl or very late antigen (VLA) integrins (Horwitz et al, J Cell BioIl985,lOl:2l342l44; Hemler, Annu Rev Immunof 1990,8:365400). Of the six known j31 integrins, four (alp1 [5,6], a2pl [T-91, a3j31 [lo], a6pl [11,12] have been implicated as laminin receptors. Ln addition, a new pl integrin, possibly a7p1, may also function as a laminin receptor (S Goodman and K von der Mark, unpublished) [13]. These data are collated from studies on many different cells: each cell type only uses a subset of pl integrins to adhere to kuninin. With the possible exception of OrbpI, most integrin laminin receptors are promiscuous and can also bind collagen, for example, alp1 [ 5,6], a2pl [7-91, a3pl [ 101, and fibronectin, for example, a2pl [ 141, a3pl (Horwitz et al, 1985) [lo]. Iaminir-binding function may not be the sole domain of fll integrins, but may be shared by other j3 integrin fan-& lies. Thus, the a6p4 integrin has been implicated as a carcinoma laminin receptor [9] and the vitronectin receptor (a-$3) may bind to an RDG-containing site in laminin [ 11,151. In both cases, however, the other integrins function in conjunction with pl integrins. The past year has seen a significant increase in the use of proteolytic fragments of laminin to obtain more precise data on integrin-binding specificity (Fig. 1). The major cell-binding site in laminin is located in the long arm of its cruciform structure (~8 fragment) [ 151. The other cellbinding site, located in the short arms and core (El and Pl fragment) is cryptic and only exposed upon proteolysis [ 151. The a?$1 [lo] and a6pl [5,11] integrins bind largely to E8, and alp1 binds to El 151. Because the El binding site is cryptic, the physiological significance of its integrin binding is unclear. The binding sites of a2j31 and a6p4 are not known. Another approach to studying laminin functional domains has been the use of synthetic peptides that correspond to specific laminin sequences. This has identified laminin domains that are active in several processes including neurite outgrowth and tumor cell metastasis

Fig. 1. Schematic of Englebreth-Holm-Swarm picting major protease fragments with cell The binding specificities of integrin laminin in boxes. See text for references.

(EHS) laminin deattachment activity. receptors are noted

Although the emphasis in laminin adhesion research has been on characterizing integrin receptors and de&-ring their ligand specificities, the need to understand their functional significance in mediating cellular interactions with laminin should be borne in mind. In this regard, some recent papers have provided insight into integrin function and have defined new areas for future studies [12,14,17-191. There are two important questions that have emerged from these papers. First, how do cells regulate integrin function? The expression of a laminin-binding integrin on the cell surface is not always sufficient for that integrin to be functional [12]. Moreover, the ligand specificity of an integrin can be cell-type-dependent [8,9,14]. Possible fac-

Laminin:

tom that regulate integrin function include the association of their cytoplasmic domains with the cytoskeleton [ 121 and their ability to cluster at sites of adhesion (Isenberg et al., J Cell Biol 1987, 10411655-1663; Mueller et al., J Cell Bioll989, 109:34553464). Such processes may be related to the phosphorylation state of specific integrin subunits (Chatila et al, J Cell Biol1989, 109:3435-3444; Dahl and Grabel, J Cell Biol lOS:lSl-190) [12] and cytoskeletal proteins (Danilov and Juliano, J Cell Biol1989, 108:19251933). Alternative mRNA splicing of integrin cy toplasmic domains could provide a molecular basis for cell-type differences in integrin specificity [20]. The formation of functional complexes comprised of integrins and other surface molecules also constitutes a potentially important mode of regulating integrin function (see, for example, Cheresh et al, J Cell Bio11987,105:116~1174). Second, what is the functional role of integrins in laminin adhesion? In simple terms, integrins can be viewed as receptors that attach cells to a laminin substratum. Several lines of evidence, however, indicate a more dynamic functional role. Adhesion is a complex process that involves reorganization of the cytoskeleton, membrane spreading, and the formation of stable contacts (see, for example, Mueller et al, J Cell Biol1989, 109:3455-3464). Jntegrin interactions with laminin also indicate many other cellular responses (including motility, neurite outgrowth, and differentiation). Thus, integrins may communicate or ‘signal’ the extracellular environment to the inside of the cell, a function implicit in their original definition (Hynes, Cell 1987,48:54+554). The ability of some integrins, especially fibronectin receptors, to function as transmembrane signalling receptors has been documented (Wright et al, J Cell Bioll984, 99:336339) [ 17-191. Clearly, the exciting task ahead will be to ascribe signalling functions

multiDIe

forms,

inultiDle

receDtokMercurie

to some of the many laminin receptor integrins and to correlate these with specific functional responses.

Non-integrin

laminin

binding

proteins

In addition to the integrins, most cell types express a complement of proteins that bind laminin with high affinity. Although such proteins have been studied extensively, confusion still exists concerning their possible function in mediating cellular interactions with laminin. As many abundant cytoplasmic proteins can bind laminin (HJ Woo and A Mercurio, unpublished), as well as collagen (Nagata et al, J Cell Bioll986,103:223229), caution must be exerted in ascribing surface functions to nonintegrin proteins. Recent studies, however, suggest that some surface laminin-binding proteins could function in laminin interactions. The major laminin -binding protein expressed on the macrophage surface is identical to carbohydrate-binding protein (CBP) 35 [21], a well characterized soluble or S-type lectin @a and Wang, J Biol Cbem 1988, 263:600+6011). This lectin exhibits specificity for both galactose and poly-N-acety-lactosamine-type oligosaccharides which contain the repeating [3Ga@l, 4GlcNAcPl], disaccharide [22,23]. It is intriguing to note that EHS laminin is heavily glycosylated (17-27% Asn-linked oligosaccharide; Knibbs et al, Biochenzisty 1989, 28:637%392; Fujiwara et al., Bicxbem J 1988, 252:453-461) and is one of the few known glycoproteins to express an abundance of polylactosamine-type carbohydrates [ 231. Although a functional role for CBP 35 in laminin adhesion remains to be established, the importance of laminin carbohydrate in cellular interactions is supported by studies using unglycosylated laminin [24].

lntegrins

1

Fig. 2. Hypothetical model of cell adhesion to laminin. Putative signalling functions regulated by integrin binding to laminin and possible stabilization of adhesion by lectins are depicted. See text for references.

84p

848

Cell-to-cell

contact

and extracellular

matrix

Also, it is likely that other non-integrin la$nin-binding proteins are lectins as evidenced by the fact that the 67 kD laminin-binding protein is similar, if not identical, to the elastin-binding protein, a galactose-specific lectin [ 251. An obvious question that arises is why cells would use both integrin and non-integrin molecules to interact with laminin. One possible rationale is that integrins confer specticity and signalling functions through relatively low aflinity interactions. Non-integrin proteins, such as CBPs or other surface molecules, may stabilize or strengthen integrin-initiated interactions. In fact, the involvement of distinct signalling and stabilizing receptors is well documented for T-cell adhesion (reviewed by Springer, Nuture 1990, 346:425-434).

adhesive site in the synaptic Cell 1989, 599OF913. The primaty sequence t.eu-Arg-Glu. Neurons fotm it blocks neuron

5. l

lamina

protein

of the adhesive site of Slaminin adhere to the immobilized peptide adhesion to S-laminin.

HALL DE, RIXXARDT LF, CROWU3’ SONNENIXRG A, D.WXY CH: The

S-laminin.

is identified as and in soluble

E, HOLIIT

alpI attachment

B, MOEZZI

and abpl to distinct

hetercdimers mediate cell laminin. J Cell Biol 1990, 110:2175-2184.

H,

integrin sites on

Both the al pl and a6pl integrins mediate choriocarcinoma cell attachment to laminin. alal binds to the El fragment of laminin and a6pl binds to EB. ale1 also functions as a collagen IV receptor.

6. l

Antim laminin

Emerging

basal

DC, FLIER y\. CARBONETTO S: Identification surface protein involved in PC1 2 cell-substratum and neurite growth on laminin and collagen. TURNER

1989, 9~3287-3296. to a 2COkD integrin and several coilagens,

subunit but not

(al) blocks to fibronectin.

PC12

of a ceU adhesion J Neztrosci adhesion

to

7.

concepts

L~NG~INO LR, GEHL~EN KR WAYTVER E, CARTER WG, ENGVAU E, Ru0xw1-1 E: Endothelial cells use a2/pl integrin as a laminin receptor. J Cell Biol 109:2455-2462. The a2pl integrin. a known collagen receptor on some cell types. is shown to function as the major laminin receptor on endothelial cells. l

The following key concepts are emerging, based on the data highlighted in this review (see Fig. 2). First, the specificity of cellular interactions with laminin is determined by structural diversity in laminin itself, as weU as by an increasing number of integrins. Second, the ability of integrins to function as laminin receptors is not constitutive but is regulated by a variety of cell-type-specific factors. Third, one of the major functions of integrins may be to communicate or signal the presence of laminin on the cell surface to the inside of the cell, thereby initiating a multitude of responses. Finally, the interaction of cells with laminin may be stabilized or strengthened by non-integrin molecules such as lectins.

8.

Eucts

l

gen receptor

ME: The human integrin VLA-2 on some cells and a coUagen/laminin

MJ, HEMIER

is a coUareceptor

on others. Pnx Natl Acud Sci USA 1989, 86:990&9910. VL&2 (a2pl) functions as laminin/coUagen receptor on melanoma cells. but only as a collagen receptor on platelets and libroblasts.

9.

Lo’12 MM,

l

noma

KORZJWIS

cells

use

volvement

CA, MERCURIO AM: Human colon carcireceptors to adhere to laminin: inan a201 integrins. Cell Reguhfion 1990,

multiple

of a6p4

1~249257. Invasive colon carcinoma cells both a6&i and a2pl integrins is not evident in these cells.

adhere atidty to laminin. Such cells use to attach to laminin. The a6pl integrin

10.

GEHLSEN KR, DICURSON K, ARGRAVES WS, ENGVAU E, Ruoxwn E: Subunit structure of a laminin binding integrin and localization of its binding site on laminin. J Biol Gem 198?, 264:19Q34-19Q38. The a3pl integrin is implicated as a laminin receptor on osteosarcoma cells and its laminimbinding site is proximal to the carbovterminus of the Bl chain (E8 fragment). l

Annotated reading 0 l e

references

Of interest Of outstanding

1.

PAUL%XN tion of

l

and recommended

11.

interest

M,

SALADIN

l

K:

Mouse

the native protein Englebreth-Helm-Swarm

with

heart

laminin.

and structural laminin. / Biol

264:1872618732. Mouse heart laminin, isolated by an EDTA-containing buffer, exhibits a low proportion of A chain relative to Bl and B2 chains. It also contains an additional, disullide-linked 3OOkD poiypeptide distinct from A and B chains.

2.

EHRIC

l

Merosin, a tissue-specific is a laminin-like protein. 87:326+3268.

K,

&rvo

I, ARGRNES

WS,

Ruosl~rm

E,

basement

membrane

Pm

Acud

Nat1

Sci

ENCULL

E:

protein, (ISA

1990,

The deduced amino acid sequence of merosin, a novel tissue-specific basement membrane protein, is homologous to the carboxytenninal part of laminin A chain. Isolated merosin is associated with laminin Bl and B2 chains.

3.

Humit

l

hesive protein neuromuscular

S-laminin,

DD,

SHAH V, MERUE JP, SANES JR: A laminin-like

concentrated in the synaptic cleft junction. Nature 1989, 338:22%234.

a glycoprotein

ina, is a novel

selectively

homologue

4.

HUNIXR

l

SANEs JR: Primary

DD,

associated

of lamlnin

PORTER

BE,

sequence

with

synaptic

of

basal

adthe lam-

Bl chain.

BULXK

of

JW,

a motor

neuron-selective

PW.

DAMSKY

CH.

recognition of different cellof laminin (Pl, E3, EB) and evidence a6p functions as a major receptor for

1990, 110:214>2155. The a6pl is a receptor for the E8 fragment of laminin. Cell adhesion to the Pl fragment, a cryptic fragment exposed upon proteolysis, involves both pl integrins and the avp3 vitronectin receptor. 12.

SHAW

l

pendent toskeletal

IN.

ME~~ER JM. MERCNUO AM: The activation adhesion of macrophages to laminin involves anchoring and phosphorylation of the a6pl

decyin-

tegrin. J Cell Biol 1990, 110:2167-2174. Macrophages require activation of protein kinase C to adhere to laminin. Adhesion is mediated by the a6pl integrin and invokes phosphorylation of the cytoplasmic domain of a6 and anchoring of a6pl to the cytoskeleton.

13.

KRAMER RH,

l

express

MCDONALD

a novel

integrin

W

Vu

MP: Human

receptor

for

melanoma J Biol

laminin.

cells Cbem

1989, 264:15642-15649. Melanoma cells express a novel a integrin subunit (100 kD heavy chain and 3OkD light chain) that associates with pl and binds to lamininsepharose. 14.

ADAMS SP, MERUE JP,

MO~DENAN

binding li-agments that abpl but not fragment E8. J Cell Biol

Purifica-

comparison Uxm 1989,

SONNENHERG 4 INDEFLS CJT. Awt~1u.13’ M, TIMPI. R: lntegrin

l

KIRCHHOFER D. L+NGU[NO LR, RLIOSIAHTI E. PIENHHACHER MD: a201 integrins from different ceU types show different binding specilicities. J Biol Chem 1990, 265:615-518.

Laminin: Cell-type-dependent demonstrated.

ligand

specificity

for

the a2pl

integrin

is clearly

VAN KLIPPEVEL?’ T, LWGUINO LR, GMUT JO, SUZUKI S, Ruosvwn E: An alternative cytoplasmic domain of the integrin 83 subunit. Proc Nat1 Acad Sci USA 1989, 86:54155418. Differences in the cytoplasmic domain sequence of the f33 integrin subunit exist between human platelets and placental tissue. This difference may arise form alternative mRNA splicing. 15. 0

16. 0

CUMENT B, SEGUI-REAL B, SAVAGNER P. KLEINMAN HK, YAMADA Y: Hepatocyte attachment of laminin is mediated through multiple receptors. / Cell Biol 1990, 110:185-192. Non-integrin proteins that bind to laminin, as well as to functionally active synthetic peptides derived from laminin are described. 17. 0

WERE 2. TREM~U PM, BEHRENLXSEN 0, CROWLEY E, D~nis~v CH: Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol 1989, 109:877-889. ligation of the integrin fibronectin receptor induces expression of genes involved in matrix degradation. One of the first reports to demonstrate that integrins can function in signal transduction. 18. 0

INGBER DE, PRUSIY D, FRANGIONI JV, CRAGOE EJ JR, IICHENE C, SCHWAR’IZ MA Control of intracellular pH and growth by fibronectin in capillary endothelial cells. J Cell Biol 1990, 110:180~1811. Substratebound fibronectin alters intracellular pH in endothelial cells. Although not shown directly, it is likely that this effect is an example of signal transduction by an integrin. BUSBY JL Protein kinase C is involved in laminin stimulation 19. 0 of neurite outgrowth. Neuron 1989, 3~287-297. Evidence is presented to support the hypothesis that laminin-binding to receptors on neuronal cells activates protein kinase C. 20. @

NURCOMBE V, AUMAIUEY M, TIMPL R, EDGAR D: The highaEnity binding of laminin to cells. Assignation of a major cell-binding site to the long arm of laminin and a latent

multiple

forms,

multiple

receptors

Mercurio

cell-binding site to its short arms. Eur J Biocbem 1989, 1809-14. HT-1080 cell adhesion to laminin occurs through binding to the long arm of laminin (E8 fragment). A second potential binding site located in the short arms is masked and only accessible after proteotysis. 21. e

Woo HJ, SHAW LM, MESSIER JM, MERCURIO AM: The major nonintegrin laminin biding protein of macrophages is identical to carbohydrate binding protein 35 (Mac-2). J Biol C3em 199Q, 265:7097-7099 Purification and microsequence analysis of the major ceU surface laminin-binding protein demonstrate that it is identical to CBP 35, a weU characterized soluble or Sqpe, galactose-specific lectin. 22. a

LEE RT, IC~KAWA Y, AMEN HJ, LEE YC: Biding characteristics of galactoside-binding lectin (galaptin) from human spleen. J Biol C3em 199Q 26517-7871. A detailed biochemical Study of the binding specificities of S-type &tins such as CBP 35. Very useful for assessing the potential function of lectins in laminin adhesion. 23. 0

ZHOU Q, CUMMINGS sue binds selectively

RD: The S-type lectin from calf heart tisto the carbohydrate chains of laminin. Arch Biodwn B@bys 1990, 281:in press. S-type &tins bind with high affinity to the poly-N-ace+lactosamine chains of laminin. Together with [ 211, a strong case is made for the possible involvement of lectins in laminin interactions. 24. 0

DFAN JW III, CHANDRA~EKARAN S, TANZER ML Biological role for the carbohydrate moieties of laminin. J Biol Cbem 1990, 265:1255+12562. Spreading of 816 melanoma cells and neurite outgrowth of PC12 ceUs were impaired on un&co.sylated laminin substrate. This paper provides direct evidence for the importance of laminin oligosaccharirdes in cellular interactions. 25. 0

MECHAM RP, HINEK A, GRIFFIN GL, SENIOR RM, COTTA L% The elastin receptor shows structural and functional similarities to the 67 kDa tumor cell laminin receptor. J Biol &em 1989, 26416652-16657, Evidence that the much studied 67 kD laminin-binding protein may acNdb be a galactose lectin is provided.

849