extracellular matrix molecules and their receptors...

Annu. Rev. Neurosci. 1991.14:531-70Copyright © 1991 by Annual Reviews Inc. All rights reserved

EXTRACELLULAR MATRIXMOLECULES AND THEIRRECEPTORS" Functions in NeuralDevelopment

Louis F. Reichardt

Program in Neuroscience, Department of Physiology and Howard HughesMedical Institute, University of California School of Medicine,San Francisco, California 94143-0724

Kevin J. Tomaselli

Athena Neurosciences, South San Francisco, California 94080

KEY WORDS:integrins, proteoglyeans, axonal growth and guidance, neuronalmigration, neuronal cytoskeleton

INTRODUCTION

The development of neurons and virtually all other cell types in the organ-ism depends upon interactions with molecules in their environment. Stud-ies of individual cell types have revealed tremendous diversity in the mol-ecules that regulate the development of cells in the nervous system. Theseinclude chemotropic and trophic factors [e.g. nerve growth-factor (NGF)and brain derived neurotrophic factor (BDNF)], cell adhesion molecules[e.g. the neural cell adhesion molecule (NCAM) and N-cadherin], andmolecules secreted into the extracellular matrix (ECM) [e.g. laminin (LN)and fibronectin (FN)]. Each class of molecule has now been shown influence major steps in the development of the nervous system, includingneuronal survival, determination, and migration; axonal growth and guid-ance; synapse tbrmation; and glial differentiation. As molecules in the

5310147~?06X/91/0301 ~0531 $02.00

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532 REICHARDT & TOMASELLI

ECM influence all of these events and can be used to illustrate many ofthe principles derived from studies of the other classes of molecules, thisreview focuses upon constituents of the ECM and their receptors. The roleof the ECM in neural development has recently been reviewed in this series(Sanes 1989). This review focuses on cellular and molecular themes notemphasized in the previous one and includes examples of recent work onnonneural systems that illustrate probable future directions for researchin the nervous system.

Recent reviews on the composition and function of the ECM and itsreceptors include those of Hynes (1990), Hemler (1990), Kishimoto et (1989), Plow & Ginsberg (1989), Burgeson (1988), Buck & Horowitz(1987), McDonald (1988), Ruoslahti (1988, 1989), Fessler & Fessler (1989),and Erickson & Bourdon (1989). Reviews focusing on aspects of ECMfunction in neural development include those by Lander (1989), Sanes(1989), and Edgar (1989). Because of space limitations, only representativeexamples and references are cited in this review.

COMPOSITION AND FUNCTION OF THE

EXTRACELLULAR MATRIX

The ECM was originally defined morphologically as acellular material withstructure visible in the electron microscope (e.g. fibrils or basal lamina).It is now defined more broadly to include secreted molecules that areimmobilized outside cells, even if they lack organization that is detectablein the microscope. Major constituents identified initially in the ECMincluded collagens, noncollagenous glycoproteins, and proteoglycans.Recent work has revealed a surprising diversity in these molecules. Thenumber of distinct collagens is now greater than 12; the number of adhesiveglycoproteins is constantly growing. Multiple genes and differential splicingfurther add to diversity in both classes of ECM glycoproteins (cf. Ehrig etal, 1990, Hunter et al 1989a, Hynes 1985). Determination of the primarysequences of the core proteins for several proteoglycans has shown thatthis class of molecules is best considered as a group of diverse glycoproteinswith functions mediated both by their protein cores and by their carbo-hydrate side chains (cf. Ruoslahti 1989, Saunders et al 1989, Clement et al1989). Recent work also indicates that several cell-cell adhesion moleculesinteract with or have forms that are immobilized in the ECM (e.g. NCAM,myelin-associated glycoprotein) (Sanes et al 1986, Fahrig et al 1987).Finally, the ECM has also been shown to bind to and regulate the activityand stability of several growth factors, most notably basic-fibroblastgrowth factor (FGF) and transforming growth factor (TGF)-fl (cf. Rifkin& Moscatelli 1989, McCaffrey et al 1989).


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EXTRACELLULAR MATRIX 533

DOMAINS IN EXTRACELLULAR MATRIX

PROTEINS

The collagens and other ECM glycoproteins are molecular giants, in somecases spanning distances of several hundred nanometers. Primary sequenceanalysis has revealed that they are chaemeric proteins, assembled by exonshuffling. Multiple functional domains have been identified in individualECM glycoproteins such as FN or LN (Figure 1). These include bindingsites for cells, other ECM glycoproteins, glycosaminoglycans, and gly-colipids (e.g. Mann et al 1989). Some ECM glycoproteins and pro-teoglycans have also been shown to bind growth factors and proteases (cf.Rifkin & Moscatelli 1989, Silverstein et al 1986). With most of the ECMglycoproteins, it has been possible to localize specific functions, such ascell binding, to specific domains, sometimes as short as 3-20 amino acids(cf. Pierschbacher & Ruoslahti 1984, Wayner et al 1989, Guan & Hynes1990, Tashiro et al 1989). One of these short peptide sequences, RGD,functions as a cell attachment site in several different ECM glycoproteins,including FN (see Table 1). The demonstration of a specific function fora particular structure in one protein has raised the possibility that thisstructure functions similarly wherever it is found. By this criterion, manyof the domain structures in those ECM proteins that have been sequencedare potentially adhesive. Most prominent among these domains is thefibronectin type III repeat, one of which contains the seqt~ence RGDS asthe major cell attachment site in fibronectin (Pierschbacher & Ruoslahti1984). Multiple copies of this motif are found in FN, tenascin, and throm-bospondin (Figure 1). Currently, two of the 18 such domains in FN, oneof seven in tenascin, and one of six in thrombospondin have been shownto include cell attachment sites (Wayner et al 1989, Guan & Hynes 1990,Spring et al 1989, Lawler et al 1988). It seems likely that more of thesedomains will prove to have adhesive functions when tested with appro-priate cells. FN type III domains are differentially spliced in both FN andtenascin (cf. Hynes 1985, Spring et al 1989). In the case of FN, one of thedifferentially spliced type III domains has been shown to contain a cellattachment site recognized by both neural crest cells and sensory neurons(of. Dufour et al 1988, Humphries et al 1986, 1988).

A second major domain implicated in cell adhesion, the immunoglobulindomain, has recently been identified in the major HeS-proteoglycan, per-calin, which contains multiple copies of this structural motif (Noonan etal 1988). Similar domains have been found in several Ca2+-independentneural cell adhesion molecules, including NCAM, L1, contactin, neuro-glian, and fasciclin II (cf. Harrelson & Goodman 1988). Intriguingly, mostof these cell adhesion molecules also contain several FN type III repeat


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Heporin ,I Gelatin E!IIB ~ EIIIA {I Collagen ~u / Cell / Cell~ I I

~~ ~,,~ ~ Hen SS

NH2~’’ ’’

,’~COOHy ;Matrix ~sembly RGD Heparin

Fibron~tin

V

ITI IV V VI

I

4------

COOH

G I~G5

Laminin

~CI~LecfinEGF

HABR

NH2

Versican


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motifs. Even an ECM receptor subunit, integrin f14, contains three FNtype III repeats in its cytoplasmic domain (Suzuki & Naitoh 1990). There-fore neither the immunoglobulin nor FN type III domains distinguishcell membrane-associated adhesion molecules from adhesive glycoproteinsfound in the ECM.

Individual proteoglycans have recently been found to contain severalother domains that suggest adhesive or mitogenic functions, includingputative hyaluronic acid binding domains, lectin binding domains, leucine-rich repeats, and epidermal growth factor (EGF) repeats (cf. Ruoslahti1989). For example, the very large (2389 amino acids) chondroitin sulfateproteoglycan named versican, identified originally as a secretory productof fibroblasts, is identical to glial hyaluronic add-binding protein, whichhas been isolated from brain white matter (Perides et al 1989, Zimmerman

Figure 1 Examples of extracellular matrix glyeoproteins and proteoglycans. Schematicstructures for the adhesive glycoproteins tenascin, fibronectin, and laminin and for theproteoglyean versican are presented. Fibronectin is a dimer, but only one subunit is depicted.The sulfhydryl residues near its carboxyl terminus mediate its dimerization. The positionsof FN type I [ D ], FN type |1 [C)], and FN type II1 [1~] repeats are indicated in fibronectin.FN type III repeats are also found in tenascin, where their positions are marked by thesame symbol [I-q]. Shaded FN type 111 repeats in fibronectin and tenascin are encoded bydifferentially spliced exons and are thus missing from some forms of these glycoproteins.The positions of domains homologous to those in the EGF-precursor, the EGF repeats [~],are indicated in tenascin, laminin, and versican. The positions of domains homologous tofunctional domains in lectins][~], complement regulatory proteins [O], and hyaluronic

acid-binding proteins [~ are indicated in versican. Note that many of these domainsare also found in receptors, some of which are illustrated in Figure 2. In tenascin, thepositions of the RGD sequence, the major cell-binding domain, and the region implicatedin the anti-adhesive activity of this protein are shown. Similarly, in fibronectin, the positionsof the RGD sequence and domains mediating interactions with fibrin, heparin, gelatin,collagen, and cells are indicated. Both the N-terminal region and the major cell attachmentsite containing the RGD sequence are required for assembly of fibronectin into the extra-cellular matrix. The regions of fibronectin recognized by the integrins ~/~,, c¢3/~,, and ~4/~,are indicated by arrows. Note that the integrin ~4/~, recognizes a sequence located in adifferentially spliced exon that is not present in all forms of fibronectin. In laminin, homo-logous domains present in the A, BI, and B2 chains are indicated by roman numerals. Loopsindicate the locations of putative globular domains, many of which have been visualized inthe electron microscope. Domains I and II in the long arm of laminin’s cruciform structurerepresent regions in which the three subunits associate in a triple coiled rod. The majorproteolytic fragments of laminin E3, E8, E 1, and E I-4 described in the text contain approxi-mately the following domains: E3 (G4 and G5), E8 (G1, G2, G3, and most of I), E1 (partof II, III, IV, IIIa, IVa, IIIb, IVb), El-4 (part of II, 1II, IIIa, IIIb, IV, IV’, IVa, IVb, and VI). As indicated, the binding domains of the integrins ~ ~, ~3/3 ~, and ~6/3~ have beenroughly mapped by using these fragments. The site utilized by the integrin ~2/31 has not yetbeen defined. The wavy lines in versican indicate the positions of several chondroitin sulfateside chains. All of these glycoproteins contain N-linked carbohydrate, but this is not shown.


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& Ruoslahti 1989). In addition to a functional hyaluronate-bindingdomain, versican contains two EGF repeats, a lectin-like domain, and acomplement-regulatory protein-like repeat (Figure 1). All of these domainsare also found on one or more transmembrane cell surface glycoproteinsthat function as receptors (cf. Ruoslahti 1989). Thus, structural analysesdetect few motifs that distinguish cell-surface-associated and ECM-associ-ated adhesive glycoproteins.

Not all domains in ECM constituents are believed to function in celladhesion. In particular, domains homologous to the EGF precursor,named EGF repeats, are found in LN, entactin, thrombospondin, andtenascin, thereby raising the possibility that ECM molecules may have¯ interactions with cells similar to those mediated by EGF and its receptor(cf. Engel 1989).

STRUCTURE AND FUNCTION OF ECM

RECEPTORS: INTEGRINS

Progress has been so rapid in identifying receptors for the ECM that thefield is very fluid. Many receptor classes identified on nonneural cells havenot yet been found in the nervous system, although it seems likely thatthey will eventually be found there. At this point, neurons appear to usethe same general classes of receptors as nonneural cells, so studies on thelatter are useful in understanding the functions of these receptors in thenervous system. In this section, we first consider the major superfamily ofECM receptors, the integrins (listed in Table 1), and then consider otherreceptors likely to be important in mediating interactions with the ECM.

Integrin receptors are noncovalently associated heterodimeric glyco-protein complexes expressed on the surfaces of neurons and nonneuralcells that appear to be the primary cellular receptors for many ECMconstituents, including LN, FN, thrombospondin, and several collagens(see Table 1; cf. Hemler 1990). With assays of attachment or neuriteoutgrowth in vitro, antibodies to the integrins, notably the CSAT antibodythat binds to the chicken integrin fll subunit, appear sufficient to disruptvirtually all interactions of neurons with both purified ECM glycoproteinsand complex extracellular matrices (cf. Bozyczko & Horwitz 1986, Cohenet al 1986, Tomaselli et al 1986). Thus there is direct evidence that func-tional integrins are required for binding of neurons to the ECM. Injectionsinto developing avian embryos of antibodies to the integrin fll subunithave dramatic inhibitory effects on migration of several cell types, includ-ing cranial neural crest cells and myoblasts populating limb masses (Bron-ner-Fraser 1986, Jaffredo et al 1988). Thus this class of receptors also hasimportant functions in vivo.


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Table 1 Summary of integrin heterodimers and their ligands

~ I Ca/MgClass Mr Subunit M~ domain sites Cleaved Ligands References

fl~ l15K

fiE 90K

95K

f14 205K

fls 90K

tip 95K

cq 200K + 3 - col, LN (El) a

~2 150K + 3 - col, LNa b, c

~ 150K - 32 + LN (E8), 4, col~ c, d

~4 140K - 3 +/_3 FN (CS-t), VCAM-1 e

~5 150K - 4 + FN (RGD) f

~6 150K - 3 + LN (E8) g

~7 130K ?2 72 + LN h¢¢w 150K - 4 + FN4, VN~’4 i

0~LV^-t 170K + 3 -- I-CAM-l, I-CAM-2 j

~ua~-~ 180K + 3 - C3bi’~, FG k~,~ so 150K + 3 - ?

oq~, 136K - 4 + FN4, VN*, vWF4, FG~ 1~v~ 150K -- 4 + VN4, TS4, vWF4, FG~ m, n

~6 150K - 3 + ? o

~Xv~ 150K - 4 + VN4, FN’~ n

~,~ 140K -- 3 +/-- 3 ?

Six integrin classes, distinguished by distinct but homologous/~ subunits, are shown. Each/~ subunit can associatenoncovalently with a subgroup of homologous c~ subunits, lntegrin nomenclature is described in Hemler (1990). Thenumerical designations of ~-6 correspond to the human T cell VLA antigen c~ subunit nomenclature. The indicatedsubunit Mrs were determined by SDS-PAGE in nonredueing conditions. Note, some a/fl heterodimers recognize morethan one ligand. In some cases a ligand is recognized only by a subset of the cells expressing a heterodimer. Additionalligands may yet be discovered for those receptor heterodimers listed. Additional ~ and ,~ subunits and additionalassociations between listed ~ and fl subunits are also being discovered. The reference list below is selective and does notalways list original reports because of limited space.

Abbreviations: col, collagen; FG, fibrinogen; FN, fibronectin; ICAM, intercellular adhesion molecule; LN, laminin;TN, tenascin; TS, thrombospondin; VCAM, vascular cell adhesion molecule; VN, vitronectin; vWF, von Willebrandfactor; (RGD), RGDS site in FN; (CS-1), CS-1 site in FN; (El) and (E8), elastase fragments

~ Indicates ligands recognized in some, but not all cells.~ The sequence was not completed at the time of this review.~ Cleavage of ~ is variable.4 The RGD sequence is on this ligand and appears to be used by this receptor."Turner et al (1989), Hall et al (1990), Ignatius et al (1990), Tamkun et al (1986).b Takada & Hemler (1989), Elices & Hemler (1989), Languino et al (1989).~ Wayner & Carter (1987), Staatz et al (1989).~ Gehlsen et al (1989), Tsuji et al (1990).~ Guan & Hynes (1990), Holzmann et al (1989), Elites et al (1990), Takada et al (1989),fPytela et al (1985a).gHall et al (1990), Tamura et al (1990).~ K ramer et al (1989).i Vogel et al (1990), Suzuki et al (1985).~ Marlin & Springer (1987), Staunton et al (1989).~ Wright et al (1987).~ l?ytda et al (1986), Fitzgerald et al (1987).~ Pytela et al (1985b), Lawler et al (1988)."Cheresh et al 1989), Suzuki et al (1990).° Kajiji et al (1989), Suzuki & Naitoh (1990), Tamura et al (1990).P Holzmann & Weissman (1989).


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As illustrated in Table 1, the integrins are a large receptor family (see alsoHemler 1990). As shown in Figure 2, integrin receptors are transmembraneheterodimers composed of one ~ chain noncovalently associated with a flsubunit. Multiple genes encode families of both ~ and fl subunits. Ligandsfor almost all of the individual heterodimers have now been identified withfunction-blocking monoclonal antibodies and affinity chromatography on

CytoDlasmlcMetal Binding ii Domains

S~es r s-s~ II ’NH ~ | IIIIII IIIIII IIIIII Itllll I ~ CO~H (~ Integrin

H-CAM

C02H Syndecan

Extracellular Matrix ReceptorsFigure 2 Extracellular matrix receptors. Examples of three classes of integral membraneglycoprotein receptors are illustrated in this figure. An integrin similar to the fibronectinreceptor ctsfll is shown above. The ~ subunit contains four putative metal-binding sites, a siteat which it is cleaved into large and small fragments during maturation, a transmembranedomain, and a cytoplasmic domain (Argraves et al 1987). Some integrin ~ subunits containI domains inserted at approximately the position of the first metal-binding site, and lackprotease cleavage sites. The fl subunit contains four repeats of a cysteine-rich motif, atransmembrane, and a cytoplasmic domain. H-CAM, also named the Hermes antigen orCD44, is illustrated in the middle with positions of a hyaluronic acid-binding domain[1~il]. The positions of several carbohydrate chains are indicated by thin lines. Thestructure of syndecan, a proteoglycan that mediates binding of several cell types to severalcollagens and fibronectin is illustrated at the bottom. Positions of glycosaminoglycan chainsare indicated by thin lines.


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EXTRACELLU LAR MATRIX 539

purified ECM glyeoproteins (see Table 1). At present, there are six (andprobably more) distinct, but homologous,/3 subunits (/3L-/35,/3e), each which can associate with one or more of at least 12 a subunits. By con-vention (Hynes 1987), integrins have been classified into subfamiliesaccording to the /3 subunit that is shared among several different het-erodimers. Originally, groups of ct subunits were thought to associate withone particular/3 subunit. It has recently become clear, however, that some~ subunits can interact with more than one/3 subunit (cf. Cheresh et al1989b, Kajiji et al 1989, Hemler et al 1989, Holzmann & Weissman 1989).Nevertheless, it is still useful to refer to integrin/3-class subfamilies.

Integrins are expressed by neurons and glial cells in both the central andperipheral nervous systems (cf. Bozyczko & Horwitz 1986, Cohen et al1987, Tomaselli et al 1986, Hall et al 1987, Pesheva et al 1988, Letourneau& Shattuck 1989, Tawil et al 1990). In particular,/31-class integrins havebeen shown to mediate neuronal attachment and process outgrowth inresponse to several ECM proteins, including LN, FN, and several colla-gens. Of the eight known/3L integrins (Table 1), five (~1, 0~, 0~5, ~6, ~vN) been identified in either primary neurons or neuronal cell lines (Tomaselli etal 1988a,b, 1989, and unpublished, Turner et al 1989, Ignatius & Reichardt1988, Ignatius et al 1990, Toyota et al 1990, Vogel et al 1990). Strongindirect evidence for the expression of~4 by peripheral neurons comes fromstudies of their interactions with the alternatively spliced IIICS domain infibronectin (Humphries et al 1988, Guan & Hynes 1990)./33 integrins havealso been described on retinal neurons (K. M. Neugebauer and L. F.Reichardt, unpublished). Members of the/32 family, ~he lymphocyte inte-grins, have yet to be identified on neural cells./32 integrins are unlikely toplay a prominent role in the nervous system, however, since mutations inthe/32 subunit have no obvious effects on the development or function ofthe nervous system (cf. Kishimoto et al 1989). Although which of therecently discovered/3 subunits (/34,/3s,/3p) are expressed in the nervoussystem is not yet clear, those found on epithelial cells, f14 and fls, seemlikely to be present there. A major challenge for the future is to catalogthe integrin a and fl subunits expressed in different neural cells and toidentify the functions of individual receptors in mediating the interactionsof these different cells with ECM-bound and other ligands.

The integfins have several important features to consider in under-standing their demonstrated and potential functions in the nervous system.First, as shown in Table 1, their ligands include not only virtually alldescribed ECM constituents, but also cell surface molecules. In the/3~family, 0~4/31 has been shown to interact directly with FN and with anintegral membrane glycoprotein ligand, VCAM-1, which contains severalextracellular immunoglobulin-like domains (Elices et al 1990). Based upon


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REICI-IARDT & TOMASELLI

their distributions, it has been suggested that other members of this family,notably a2fll and a3fll, may also have cell surface ligands. Such ligandsseem likely to be particularly important in the central nervous system,where many ECM constituents that are present in the peripheral nervoussystem are largely absent. Secondly, both the ~ and fl subunits have beenshown to be important in determining ligand binding properties of integrinheterodimers (cf. Cheresh et al 1989). As a consequence, tremendouspotential functional diversity is encoded by the integrin family. Since manysubunits have only been discovered in the past year, additional subunitswill very likely be discovered in coming years, adding further to diversityof these receptors. Third, the integrins include both heterodimers thatrecognize many ligands, e.g. ~3fl, which binds LN, FN, and collagen, andheterodimers that bind only one ligand, e.g. ~6fl and ~sf~, which arespecific for LN and FN, respectively. Thus, some heterodimers are likelyto play very general roles whereas others play much more specific roles inregulating development of neurons and other cells in the nervous system.Finally, cell-specific factors have recently been shown to modify the ligand-binding specificities of two integrins. Both ~2f~ and ~3f~ heterodimersexhibit different ligand-binding specificities in different cell types (Lan-guino et al 1989, Elices & Hemler 1989, Gehlsen et al 1989). The ~2flreceptor mediates binding to collagen wherever it is expressed but mediatesbinding to LN in only a subset of cells. The ability of the ~3f~ receptor tobind collagen is also regulated by cell-specific factors. The molecular basesfor these surprising results are not yet understood.

Structurally, both integrin ~ and f subunits are transmembrane glyco-proteins. Primary sequence analysis of the five sequenced f subunits indi-cates that each has a large N-terminal extracellular domain, containing afourfold repeat of a 40-amino-acid cysteine-rich segment, a single trans-membrane domain, and, except for fin, a short cytoplasmic domain of ca.40 amino acids (Figure 2; cf. Tamkun et al 1986, Fitzgerald et al 1987,Suzuki et al 1990). The integrin f4 subunit has a very large cytoplasmicdomain containing more than 1000 amino acids (Suzuki & Naitoh 1990).The f-subunit cytoplasmic domains contain potential phosphorylationsites for tyrosine and scrinc/thrconinc kinases. Differential splicing hasbeen shown to result in f3 and f4 subunits with two different cytoplasmicdomains (van Kuppevelt et al 1989, Suzuki & Naitoh 1990). It is not yetclear whether these are both functionally significant. As is discussed below,the cytoplasmic domains appear to mediate interactions with cytoskeletalelements and are required for the adhesive functions of integrins on cells(Solowska et al 1989, Hayashi et al 1990).

Primary sequence analysis of the 11 sequenced ~ subunits indicates thateach also has a large N-terminal extracellular domain, a single trans-


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membrane domain, and a short cytoplasmic domain (see Figure 2; cf.Hemler 1990). The N-terminal half of each ~ sequence encodes sevenrepeats of an approximately 50-amino-acid domain, three or four of which,depending on the subunit, contain putative divalent cation binding sites.Cross linking with small peptide ligands has shown that the ligand-bindingsite is located in the vicinity of the first three divalent cation-binding sitesin the vitronectin receptor, ~vNfl3 (Smith & Cheresh 1990). Insertions ca. 200 amino acids are present in the leukocyte family of integrins (aLVA.l,

~Mao-l, ~gp150) and at least two members of the fl~ family (~ and ~2) (cf.Kishimoto et al 1989, Takada & Hemler 1989, Ignatius et al 1990). These"interactive" or I domains are inserted between the second and thirdrepeats within a putative ligand binding site defined by inferred homologyto ~VNfl3. These I domains are similar to I domains in several collagen-binding ECM proteins, such as von Willebrand’s factor, thereby suggestingthe possibility that they mediate interactions of the 0¢~ fl I and ~2fl i receptorswith collagen. Differential splicing has been shown to insert an exon in theputative ligand-binding region of the Drosophila integrin ~ subunit, PS2(Brown et al 1989). To date, there is no evidence for differential splicingof vertebrate integrin ~ subunits.

In the electron microscope, integrins appear as long, double rods extend-ing ca. 20 nm from the membrane with globular domains at their extra-cellular termini that appear to mediate subunit association (Carrell et al1985, Nermut et al 1988). Association of the two subunits is required forsurface expression and ligand binding (cf. Bodary et al 1989). Cross-linkingstudies with small peptide ligands have shown that segments of bothsubunits form the ligand binding site in the integrins "ilbfl3 and 0~¥Nfl3

(D’Souza et al 1988, Smith & Cheresh 1988, 1990). Subunit association some integrins requires low concentrations of a divalent cation (Fitzgerald& Phillips 1985). The ligand-binding activity of all integrins requires muchhigher, millimolar concentrations of Mg2+ or Ca2+ (cf. Marlin & Springer1987, Gailit & Ruoslahti 1988). The divalent cation dependence of integrinfunction has been exploited recently t6 isolate several individual integrinheterodimers by affinity chromatography on ECM ligands.

Integrin functions have been shown to be regulated on several levels.Both FN and LN receptors are dynamically regulated on neurons. Ratsensory neurons have been shown to lose functional FN receptors betweenEl6 and P4 (Kawasaki et al 1986). Chick ciliary ganglion neurons losefunctional LN receptors between E8 and El4 (Tomaselli & Reichardt1988). Chick retinal neurons have been shown to lose functional FN andLN receptors between E6 and El2 (Cohen et al 1986, Hall et al 1987). Allof these decreases in functional receptors correlate with the establishmentof functional contacts with the targets of these diverse neuronal popu-


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lations. Ablation of the optic tectum reduces the loss of functional LNreceptors on retinal ganglion cells, thus suggesting that target contact isresponsible for loss of receptor function (Cohen et al 1989). Althoughthese changes seem likely partly to reflect changes in gene expression,unambiguous evidence for loss of individual subunits has not yet beenobtained (see e.g. Hall et al 1987). In addition to the postulated loss integrin subunits, evidence suggests that the affinity of LN receptors onretinal ganglion cells is reduced upon target contact (Cohen et al 1989).Whatever the detailed mechanisms, the dynamic regulation of integrinreceptor function in several distinct classes of neurons seems likely to beimportant in regulating changes in neuronal behavior in vivo.

Growth factors and oncogenes have been shown to regulate integrinsubunit expression in nonneural systems. At least one integrin subunit, e 1,is strongly induced by NGF on PC12 pheochromocytoma cells (Rossinoet al 1990). The cell adhesion molecules L1 and NCAM are also inducedby NGF (cf. Prentice et al 1987). If similar inductions of integrins andCAMs occur on primary neurons, they are likely to facilitate collateralsprouting that is induced, at least partly, by increases in NGF levels indenervated fields (cf. Diamond et al 1987). Since axons of severed per-ipheral and central neurons have been shown to be capable of regeneratingon ECM substrates (cf. Davis et al 1987), it seems very likely that theyre-express functional integrin receptors. Understanding the factors thatregulate expression of these receptors is potentially useful for developingconditions that promote maximal nerve regeneration.

FUNCTIONS OF OTHER ECM RECEPTORFAMILIES

Precedents from nonneural systems and preliminary studies on neuralcells suggest that nonintegrin receptor systems may also be important inmediating interactions of cells in the nervous system with several ECMconstituents. Laminin is an intriguing example of a glycoprotein for whichthere is now evidence for interactions mediated by both integrins and otherreceptors. Several adhesive sites have been identified on the classic lamininisoform that contain the A, B1, and B2 chains (summarized in Kleinman& Weeks 1989, Beck et al 1990). These include RGD-containing and.IKVAV-containing sites in the A chain, both of which appear capableof promoting neurite outgrowth by PC12 cells (Tashiro et al 1989).Antibodies to the IKVAV peptide inhibit PC12 interactions with intactLN, thereby indicating that this site is functional. Similar experimentsneed to be done by using antibodies to the RGD-containing peptide todetermine whether the RGD site is also functional in intact LN. Inter-


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actions of PC12 cells with LN are mediated by two integrins, ~lfl~ and

~3fl~, either or both of which may recognize these sites in LN (Tomaselliet al 1990). Recent work has identified proteolytic fragments of LN thatare utilized by individual integrin heterodimers. The ~3 fit and ~6 fl~ dimershave been shown to bind sites in the long arm of LN present in thefragment E8 (Gehlsen et al 1989, Hall et al 1990). The ~ fl~ receptor hasbeen shown to interact with a site near the center of LN’s cruciformstructure, present in the fragment El (Tomaselli et al 1990, Hall et al 1990).Based on their locations in the structure of LN, the RGD and IKVAVsites are candidates to interact with the integrins ~flx and ~3fll, respec-tively.

In addition to the integrins, a set of antigenically related proteins withMrs of 32K, 45K, and 67K bind LN with high affinity and have been foundon many cells and tissues, including chick brain (see Kleinman & Weeks1989, Douville et al 1988). The 67K protein is reported to utilize a sequenceYIGSR located in an EGF repeat in the B 1 chain of LN (Graf et al 1987).The 32K protein is identical to the galactose-specific lectin CBP 35(Woo et al 1990). Although antibodies to the 67K protein do not in-hibit LN binding by neuronal cell lines (Kleinman et al 1988), they arereported to inhibit the binding of some other cell types (Graf et al 1987).Evidence that a cell surface enzyme, galactosyltransferase, functions as aLN receptor has recently been obtained (Runyon et al 1986). Antibodiesto or modifiers of this enzyme, and modifications of carbohydrate sub-strates on LN for this enzyme, partially inhibit both initiation and exten-sion of neurites by a neuronal cell line on LN without significantly affectingcell attachment (Begovac & Shur 1990). Similar results have been obtainedin assays of avian ncural crest cell migration on LN (Runyon et al 1986).These intriguing observations suggest roles for both a neuronal cell surf~eenzyme and LN-linked oligosaccharides in regulating growth conemotility. Finally, gangliosides and sulfatides are present on neural cellsand have been shown to bind LN (cf. Roberts et al 1985). Exogenous braingangliosides GD~A and GT~B can inhibit neurite outgrowth on LN byneuroblastoma cells (Laitinen et al 1987). Still uncertain, though, whether these gangliosides compete with receptors or have less directeffects.

The receptors that mediate neuronal interactions with the two mostprominent ECM glycoproteins in the CNS--tenascin and thrombo-spondin--have not yet been unambiguously identified. A neuronal proteo-glycan has been shown to interact with tenascin and is consequently onecandidate to be a tenascin receptor (Hoffman et al 1988). Though associ-ated with the neuronal surface, this proteoglycan is not an intrinsic mem-brane constituent, and thus would presumably function indirectly in


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attaching neurons to tenascin. A second candidate, described in nonneuralcells, is an apparently novel integrin, purified by affinity chromatography ontenascin (Bourdon & Ruoslahti 1989). This receptor’s function is inhibitedby RGD peptides, though, and it is not certain that cell interactions withtenascin are mediated by an RGD site (cf. Spring et al 1989). Althoughavian tenascin contains an RGD site, this site is not conserved in all species.Moreover, mapping both adhesive and anti-adhesive sites to individualdomains of tenascin has been possible by using fusion proteins and anti-bodies (Spring et al 1989). The most prominent adhesive site in tenascinhas been mapped to a FN type III repeat distant from the repeat containingthe RGD sequence; the site on tenascin that inhibits cell flattening andmotility has been mapped to EGF repeats, again far from the RGD-containing site. As discussed by Spring et al (1989), convincing identi-fication of any receptor, integrin or otherwise, that mediates either of thesetwo activities is still lacking. Identifying the receptor that mediates theanti-adhesive activity of tenascin will be particularly interesting, becausethis is a novel activity for an ECM macromolccule. In the case of thrombo-spondin, both the integrin ~vNfl3 and an 88 kDa intrinsic membraneglycoprotein named GPIV have been shown to function as receptors innonneural cells (Lawler et al 1988, Tandon et al 1989). Though neural cellsclearly interact with thrombospondin (O’Shea et al 1990, Boyne et al 1989),the question of which, if either, of these receptors mediates the observedinteractions remains obscure.

Although their neural functions can only be addressed speculatively,several other ECM receptor classes seem likely to be important in thenervous system. Prominent among these are hyaluronic acid receptors.Hyaluronic acid is located in pathways utilized by many cells, includingneural crest cells, for migration. Ligands that bind cell surface-anchoredhyaluronate dramatically inhibit migration of neural crest cells (Perris Johansson 1990). The Hermes antigen (CD44) is a transmembrane recep-tor that functions in lymphocyte binding and has a domain with stronghomology to several hyaluronic acid-binding proteins (Goldstein et al1989). This receptor has been shown to interact with both ECM and cellsurface-associated ligands (cf. St. John et al 1990). Whether these areexclusively hyaluronic acid is not yet clear. This protein has recently beenlocalized on both CNS glia and Schwann cells (Picker et a11989). Althoughit appears not to be expressed by adult neurons, its distribution in thedeveloping nervous system merits attention.

Proteoglycans function as both ECM constituents and receptors (cf.Ruoslahti 1989). Individual proteoglycans have important functions in celladhesion, proliferation, and differentiation. Specific examples documentthat heparan-sulfate proteoglycans can function as receptors, binding cells


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ExTRACELLULAR MATRIX 545

to ECM glycoproteins, including several collagens, FN, and thrombo-spondin (cf. LeBaron et al 1989, Saunders et al 1989). In addition, pro-teoglycans have been shown to modulate the binding of other receptors toECM proteins. As one example, a chondroitin sulfate proteoglycan withweak affinity for FN has been shown to inhibit binding of a purifiedintegrin to FN, probably by steric hindrance (Hautanen et al 1989). Pro-teoglycans are widely distributed in the nervous system. Despite appreci-ation of their presence and probable importance, they have received com-paratively little attention. With the exception of versican, which of thecomparatively well-characterized nonneuronal proteoglycans are alsofound in the nervous system is not yet clear. Recently, new, more powerfulmethods for characterizing proteoglycans have been applied to analyzeproteoglycans in the brain. The results suggest that there are very largenumbers of distinct proteoglycans in the mammalian brain (Herndon Lander 1990). Perhaps the most interesting proteoglycan that has beenidentified in the brain is a chondroitin sulfate proteoglycan defined bythe monoclonal antibody Cat-301 (cf. Zaremba et al 1989). This is extracellular proteoglycan, present on subsets of neurons in the brain,whose expression is regulated by synaptic activity. Although no functionis known for this proteoglycan, its expression pattern suggests that it maybe involved in synaptogenesis.

Finally, gangliosides and other glycolipids are differentially expressedmacromolecules that are prominent neuronal surface constituents. Theseinteract directly with many ECM glycoproteins, including FN, LN, andthrombospondin (cf. Roberts et al 1985). Thus they may function directlyas receptors. In addition, gangliosides have been shown to modulate thefunctions of integrins (cf. Stallcup 1988, Santoro 1989). The integrin het-erodimer--~VN fl 3--has been purfied in tight association with a ganglioside(Cheresh et al 1987). Gangliosides have been shown to be essential forfunctional reconstitution of this integrin. Hence, gangliosides may act onneurons in part by interacting with integrins.

ECM REGULATION OF CELL MIGRATION AND

AXONAL GUIDANCE

The importance of extrinsic cues in guiding migrating cells and motilegrowth cones is now well established in both invertebrate and vertebratemodel systems (cf. Harrelson & Goodman 1988, Dodd & Jessell 1988). several systems, cues localized on cells and within the extracellular matrixappear to provide the major guides for cell movements. In the developinggrasshopper limb, for example, growth cones of pioneer neurons follow


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546 REICHARDT & TOMASELL!

trajectories restricted on one side by a basal lamina and on the other byepithelial cells (cf. Bentley & Caudy 1983). Developing limbs can isolated, opened, stripped of mesoderm, and cultured as a fiat explant(Lefcort & Bentley 1987). In these conditions, designed to eliminate theeffects of electric fields, gradients of diffusible molecules, and tissues extrin-sic to the limb, growth cones of pioneer neurons still follow normal tra-jectories. Thus the limb epithelium and its associated ECM appear toprovide adequate guidance cues. Retraction of growth cones as a conse-quence of removal of the basal lamina by proteolysis implies that thereare adhesive interactions between the growth cones and basal lamina(Condic & Bentley 1989a). In such preparations, the polarity of initialoutgrowth and selective interactions with limb segment boundaries andneuronal cells still occur normally, thereby indicating that the intact basallamina is not required in this system for normal guidance (Condic Bentley 1989b). Oriented axon outgrowth has also been examined inexplants of embryonic chick and quail retinae (cf. Halfter & Deiss 1984). these cultures, also designed to eliminate guidance by diffusible molecules,already existing cues are followed by axons in developmentally olderportions of the retina, but these cues cannot be established in the devel-oping retinal rim. When tested on the isolated ECM, growth cones growat normal rates but are not oriented normally, thus implying that necessaryguidance cues are not contained in the retinal ECM (Halfter et al 1987).To summarize, both of these examples indicate that ECM molecules invivo can provide permissive substrates for growth cone motility. Neithersuggests the presence of information in the ECM that can steer them.

A mechanism by which ECM fibrils can guide cell migration is providedby recent studies on neural crest cells (Newgreen 1989). Fibrils of ECMare easily aligned by a variety of forces. On a major substrate for crest cellmigration, the medial face of the somites, fibrils of matrix are alignedparallel to the direction of neural crest cell migration. In vitro, crest cellsmigrate preferentially along the axis of alignment of the matrix fibrils, thussuggesting that this directional cue is utitized in vivo. Thus, structural cuesmay promote preferential migration in one direction (Boocock 1989).

Substantial progress in identifying individual ECM glycoproteins impli-cated in cell migration has come from studies on the developing neuralcrest and cerebellum. FN, LN, tenascin, thrombospondin, and hyaluronicacid are each found in pathways followed by neural crest cells (cf. Perris& Bronner-Fraser 1989, Epperlein et al 1988). Several reagents that inhibitinteractions of neural crest cells with ECM constituents in vitro dra-matically impair the migration of chick cranial neural crest cells. Theseinclude RGDS-containing peptides, modeled as competitors for a cell-binding site in FN; the INO antibody, which prevents cell interactions


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with an isoform of LN; tenascin antibodies; and an antibody that blocksthe functions of fl~ subunit-containing integrins (Boucaut et al 1984, Bron-ner-Fraser & Lalier 1988, Bronner-Fraser 1986, 1988). These results sug-gest that integrins are important receptors on neural crest cells and thatan RGD-containing ECM glycoprotein (e.g. FN, thrombospondin, orvitronectin), a LN isoform, and tenascin are essential substrates for thesecells. Since neural crest cells can migrate efficiently on substrates coatedwith individual ligands in vitro, such as FN or LN, it is not clear whyinterfering with the function of a single ligand in vivo has such dramaticeffects. In analogous in vitro systems in which cells or growth cones moveon substrates with multiple ligands, blocking the function of a singlemolecule usually has only a partial inhibitory effect (cf. Bixby et al 1988).One possibility is that some of the peptides and antibodies used in the invivo studies do not inhibit migration directly, but instead inhibit steps inneural crest cell differentiation prior to migration. This can now beaddressed more directly by visualizing individual cells during migrationvia confocal microscopy.

The ability to study cerebellar granule cell migration in tissue slices invitro has provided an opportunity to assess the role of ECM proteins inthe migration of central neurons. During postnatal development, cerebellargranule ceils proliferate in the external granule cell layer and then migratethrough the molecular layer to reach their final positions in the internalgranule cell layer. Several ECM components and CAMs have been ident-ified in the developing cerebellar cortex, including tenascin (cytotactin),thrombospondin, chondroitin sulfate proteoglycan, hyaluronic acid, andL1 (Hoffman et al 1988, Chuong et al 1987, O’Shea et al 1990, Ripellinoet al 1989). Perturbation experiments implicate several of these moleculesin granule cell development. Antibodies to the CAM, L1, strongly inhibitgranule cell migration (Chuong et al 1987). Cells accumulate in the pre-migratory zone of the external granule cell layer. Since these cells maynot have initiated migration, these antibodies may inhibit differentiationofpremigratory granule cells instead of interfering directly with migration.Tenascin surrounds granule cells in the external granule layer and is alsoexpressed in the molecular layer, primarily on the surfaces of Bergrnannglia (Chuong et al 1987). In the presence of tenascin antibodies, granulecells accumulate in the molecular layer, thus indicating that they start,but do not complete, migration. Both observations suggest that tenascinfunctions as an important substrate for granule cell migration. Throm-bospondin is associated with granule cells in the premigratory zone of theexternal granule cell layer and in the molecular layer (O’Shea et al 1990).In contrast to tenascin, thrombospondin is not associated with the surfacesof the Bergmann glia, but is instead concentrated at the leading edges


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of the migrating granule cells. Thrombospondin antibodies prevent themigration of granule cells into the molecular layer. Although throm-bospondin may stimulate migration as a substrate, its ability to bind andactivate plasminogen and tissue plasminogen activator (Silverstein et al1986) suggests that it may function instead by localizing and activatingproteases released by the granule cells (Verrall & Seeds 1989, O’Shea etal 1990). Consistent with this idea, inhibitors of the tissue plasminogenactivator/plasminogen system can also inhibit cerebellar granule cellmigration from the external granule cell layer into the molecular layer(Moonen et al 1982). To summarize, L1, tenascin, and thrombospondinhave been shown to promote cell attachment in vitro and may thereforealso act as substrates in vivo. Differences in their distributions, activities,and effects of antibody injection suggest, though, that each also has a veryspecific, nonredundant function.

The characterization of genes that guide circumferential migrations ofmesodermal cells and pioneer axons in C. elegans has provided convincingevidence for a role for an ECM constituent in providing directional infor-mation (Hedgecock et al 1990). In C. elegans, pioneer axons migratebetween the epidermis and basal lamina. Mesodermal cells migrate on theopposite side of the basal lamina. Three genes unc-5, unc-6, and unc-40--are required for guiding both classes of cells along the dorsoventralbut not anterior-posterior axis. Unc-6, which affects migrations in bothdorsal and ventral directions, has been shown to be a homologue of theLN B2 chain (cited in Hedgecock et al 1990). Considering the importanceof LN in regulating epithelial cell development (cf. Klein et al 1988), seems likely that some isoforms of LN are still present in unc-6 mutants.The genetic analyses of migration-deficient mutants in C. elegans hasprovided the most specific role for an ECM molecule in cell guidance thathas been identified thus far. That guidance and motility are not affectedalong the anterior-posterior axis is particularly striking. There is persuasivegenetic evidence for interaction of two other gene products--unc-5 andunc-40--with unc-6 (Hedgecock et al 1990). Uric-5 has been cloned andhas properties consistent with its being a receptor, including a predictedexternal lg domain and a putative transmembrane domain (cited in Hedge-cock et al 1990). Existence of opposing adhesive gradients have beenproposed as a model to explain guidance in C. eleyans. One gradient couldpotentially be formed by a LN isoform containing the unc-6 product. Invertebrate systems, though, neurons in vitro do not seem able to followgradients of LN (McKenna & Raper 1988).

Many ECM molecules stimulate neuronal process outgrowth in vitro.These include LN, FN, several collagens, thrombospondin, and tenascin(cf. Manthorpe et al 1983, Rogers et al 1983, O’Shea et al 1990, Wehrle


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Chiquet 1990). Some of these glycoproteins are also capable of guidingaxons in vitro (cf. Gunderson 1987). Essentially all of these molecules arelocalized in vertebrate embryos where they could potentially serve aspermissive substrates or provide guidance cues for growth cones (reviewedin Sanes 1989). LN isoforms, for example, have been detected in both thevertebrate peripheral and central nervous systems in locations that wouldsuggest a role in axonal growth: in developing sensory and autonomicganglia and spinal nerve roots (Rogers et al 1986), in the pathway trigeminal nerve fibers (Riggott & Moody 1987), in developing muscleprior to innervation (Sanes & Chiu 1983), in the spinal ventral longitudinalpathway (Letourneau et al 1988), and in the optic nerve (cf. Cohen et 1987). In almost all cases, LN expression appears to be transient, occurringat high levels during periods of axonal growth and, at least, in the centralnervous system, diminishing later in development. Two notable exceptionsare the continued expression of LN isoforms in the basal lamina of peri-pheral nerve (Chiu et al 1986) and the optic nerves of goldfish (Hopkinset al 1985, Liesi 1985), two favorable sites in the adult for either continuedaxonal growth or nerve regeneration. In contrast to studies on cellmigration, surprisingly few attempts have been made to use antibodies orother reagents to interfere with axonal growth. In one of these, the INOantibody, which prevents cell interactions with an isoform of LN, wasshown to slow, but not prevent reinnervation of the iris by sympatheticneurons (Sandrock & Matthew 1987).

Although experimental evidence is largely missing, it seems likely thatECM constituents function as permissive substrates for axonal growth inmany areas of the developing nervous system. The ability of many ECMconstituents to guide growth cones in vitro has suggested that they mightalso guide growth cone movements in vivo (cf. Gunderson 1987). principle, observed heterogeneities in distribution of ECM proteins orchanges in their concentrations could direct axonal growth. Recent resultsindicate that heterogeneity in both the number and function of ECMglycoproteins has been underestimated. Most strikingly, there are manymore genes for ECM glycoproteins than was previously appreciated. Thecollagens, for example, have proliferated in both number of new collagensand number of genes encoding new isoforms of existing collagens (Bur-geson 1988). Some of these have quite restricted distributions (cf. Hostikkaet al 1990). The recently described heterogeneity in LN is of particularinterest to neurobiologists, because LN has many dramatic effects onneuronal differentiation. In addition to its effects on neurite outgrowth,LN also modulates the activities of neurotrophic factors, regulatesexpression of transmitter enzymes, and has many other striking effects onthe differentiation of neurons, not mimicked by other ECM glycoproteins


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(of. Edgar et al 1984, Kalcheim et al 1987, Acheson et al 1986). Althoughit has been appreciated for several years that laminins from differentsources have different structures and antigenic properties (cf. Lander et al1985, Edgar et al 1988), only in the past year, with the discovery of newgenes encoding LN homologues, has a definite advance been made inunderstanding the causes of this heterogeneity.

Merosin is an A-chain homologue that associates with the B1 and B2chains to form a novel isoform of LN (Ehrig et al 1990). Merosin-con-taining LN generally appears late in development and is the major LN inadult skeletal muscle, cardiac muscle, and peripheral nerve (Leivo Engvall 1988, Sanes et al 1990). Specific functions relevant for cell differ-entiation appear to be differentially encoded by different A chain genes.For example, expression of the classic A chain appears necessary fornormal development of kidney epithelial cells in organ cultures of embry-onic kidney anlage (Klein et al 1989, Sorokin et al 1990). Consistent withthe possibility that A chain genes differentially encode sites relevant foraxon growth, recent work has demonstrated that sensory neurons utilizedifferent combinations of integrins to extend neurites on merosin-con-taining and A-chain containing isoforms of LN (K. Tomaselli et al 1991,unpublished).

S-laminin was originally identified as a synapse-specific component ofmuscle basal lamina (cf. Sanes & Chiu 1983). Recently shown by sequ-encing to be a homologue of the LN B1 chain (Hunter et al 1989a), it hasnot yet been shown to associate with other LN subunits. S-laminin has amuch more restricted distribution than LN in vivo (Hunter et al 1989a).Only a subset of neurons, at this point only motoneurons from the ciliaryganglion, appear to interact with it (Hunter et al 1989b). It is associatedwith sites at which axons cease elongation, suggesting that it effects onneuronal differentiation arc quite different from those of other LN iso-forms (Covault et al 1987). Recent work has implicated a peptide sequenceLRE as the attachment site for motoneurons in S-laminin fusion proteins(Hunter et al 1989b). Since S-laminin is likely to associate with A and chain homologues, the function of this sequence needs to be confirmed byusing native isoforms of LN containing this subunit.

The discoveries of merosin and S-laminin make it possible in theory toassemble at least four different LN isoforms. In addition, there is evidencesuggesting additional diversity in LN isoforms, not attributable to differentgenes. The INO antibody was isolated as an inhibitor of the activity ofLN-containing neurite outgrowth factors (Matthew & Patterson 1983). clearly binds an epitope associated with LN-heparan sulfate complexes(Chiu et al 1986). It also inhibits axonal extension on sections of theperipheral nerve where merosin, B1, and B2 are the major visible LN


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subunits (Sandrock & Matthew 1987b, Sanes et al 1990). Even thoughthis suggests that INO recognizes LN isoforms containing merosin, itsdistribution in vivo is quite distinct from that of merosin or any otheridentified LN subunit (Chiu et al 1986, Sanes et al 1990). This suggeststhat the structure and possibly biological activities of LN can be modifiedby associating with other molecules, such as proteoglycans.

FN, which promotes axonal outgrowth by peripheral neurons, illustrateshow differential splicing may contribute to diversification of the ECM(Hynes 1990). Embryonic chick sensory and sympathetic neurons extendneurites on two non-overlapping fragments of FN, a 75 kDa fragmentcontaining the RGDS sequence and a 33 kDa fragment containing analternatively spliced FN type III repeat (cf. Humphries et al 1988, Rogerset a11987). Spinal cord neurons interact poorly with the RGDS-containingfragment, but interact efficiently with the 33 kDa fragment (Rogers et al1987). These differences in behavior almost certainly reflect differences inintegrin subunit expression by the different populations of neurons. Theintegrin ~4/~1 interacts with the alternatively spliced CS-1 domain in the37 kDa fragment (cf. Guan & Hynes 1990) and is thus probably presenton both central and peripheral neurons. The integrins ~5/~ and ~3/~1 bothinteract with the RGDS-containing fragment of FN (cf. Hemler 1990) andare consequently not likely to be expressed by spinal cord neurons. Asmentioned previously, expression of specific domains in other ECM glyco-proteins, including tenascin, is also regulated by differential splicing. Inprinciple, this could generate tremendous diversity in the temporal andspatial patterns of ECM protein expression during development of thenervous system.

Tenascin is a particularly intriguing ECM glycoprotein because it isexpressed at high levels in the nervous system, has already been implicatedby perturbation experiments as a mediator of neural crest and granule cellmigration, and has an anti-adhesive as well as adhesive domain (Spring etal 1989; for review, see Erickson & Bourdon 1989). The anti-adhesivedomain actually prevents responsive cells from attaching strongly to per-missive substrates such as FN (cf. Lotz et al 1989). Tenascin’s anti-adhesiveactivity in vitro suggests that it might function in vivo to inhibit thegrowth of responsive axons, similar to intrinsic membrane proteins isolatedrecently from myelin and posterior somite, two tissues avoided by axonsin vivo (Caroni & Schwab 1988, Davies et al 1990). In particular, sincetenascin is found in boundaries of vibrissae-related barrel fields in thesomatosensory cortex of mice (Steindler et al 1989), it has been suggestedthat its anti-adhesive activity may function in vivo to separate the fieldsof different afferent inputs. Recent work indicates that there is a family oftenascins and tenascin-like glycoproteins. First, there are several isoforms


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of tenascin derived from one gene by differential splicing (cf. Spring et al1989). These differ in the number of FN type III repeats included in thetranslation product. Still more diversity is provided by a second geneencoding an isoform of tenascin that was discovered recently by virtue ofits proximity to the gene encoding complement factor IV (Morel et al1989). Further heterogeneity in tenascin-like proteins has been discoveredby characterization of the Jl family of proteins (cf. Pesheva et al 1989).ECM proteins recognized by J1 antibodies include the major isoforms oftenascin and, in addition, two smaller proteins, Jl-160 and Jl-180. Whenexamined in the EM, these smaller forms are dimeric and trimeric kinkedarm rods, respectively, whereas tenascin is a hexamer of kinked arm rods(Pesheva et al 1989). Jl-160 and Jl-180 thus have striking structuralsimilarities with tenascin. In contrast to tenascin, which is primarily syn-thesized by astroglia and fibroblasts (ffrench-Constant et al 1986, Gat-chalian et al 1989), the Jl-160 and Jl-180 are synthesized by oli-godendrocytes (Pesheva et al 1989). Like tenascin, Jl-160 and Jl-180 arenonpermissive substrates for attachment of some cells, including cerebellarneurons and astroglia. Similar to tenascin, J1 isoforms also have anti-adhesive activities on mixed substrates (cf. Pesheva et al 1989, Lotz et al1989). To summarize, tenascin and its relatives are expressed at high levelsin the nervous system. In different parts of the nervous system, evidencesuggests that tenascin may have quite different functions, e.g. promotingmigration of cerebellar granule cells in the cerebellum but limiting theextent of afferent branching in the barrel fields of the somatosensory cortex(Chuong et al 1987, Steindler et al 1989). The recent discoveries of newgenes, differential splicing, and related glycoproteins increase its potentialfunctions and the interest in identifying TN receptors.

To summarize this section, there is comparatively strong evidence thatECM constituents function as permissive substratcs for cell and growthcone migration. Except for studies in C. elegans, there is little evidencethat ECM constituents provide directional cues. Recent discoveries of newgenes encoding ECM glycoprotein homologues and differential splicing oftranscripts encoding these molecules indicate that the ECM in the devel-oping nervous system may well be much more heterogeneous spatiallythan was previously appreciated. Thus there is at least the potential forthe ECM to provide some of the guidance cues required for establishingmigratory routes and axon tracts in the nervous system. At this point,though, ECM proteins have not yet been shown to distinguish between¯ individual fiber pathways, as has been shown for some cell adhesionmolecules, e.g. fasciclins I, II, and III in the insect nervous system (Har-relson & Goodman 1988). On balance, these results suggest that much ofthe information specifying direction and pathway choice is likely to be


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provided by other molecules, such as cell adhesion molecules and chemo-tropic factors (cf. Harrelson & Goodman 1988, Dodd & Jessell 1988).

ECM REGULATION OF SYNAPSE FORMATION

Studies on regeneration of amphibian neuromuscular junctions have dem-onstrated a critical role for the basal lamina in inducing formation of bothpre- and postsynaptic specializations (see Nitkin et al 1983, Sanes & Chiu1983). The basal lamina at the neuromuscular synapse has been shown tobe molecularly distinguishable from non-synaptic basal lamina. ECMglycoproteins present only in synaptic basal lamina include two LN sub-units (A and S) and two collagen IV chains [e3(IV) ~4(I V)] (Sanes etal 1990); agrin, an apparently novel ECM glycoprotein (Nitkin et al 1987);and, in Torpedo, a large proteoglycan named TAP-1 (Carlson & Wight1987). Studies of these molecules imply that synaptic basal lamina acquiresits unique molecular constituents from both neurons and myotubes. Thus,S-laminin is a biosynthetic product of myotubes (Sanes & Chiu 1983).Although its function in the synapse is unknown, its effects on peripheralmotoneurons described above suggest that it may function to induce oneor more steps in differentiation of the presynaptic terminals of moto-neurons. The glycoprotein agrin was identified initially as a factor thatinduces clustering of acetylcholine receptors on skeletal myotubes (cf.Nitkin et al 1983). Purified agrin has been shown to induce concentrationsof both ECM-associated and membrane-associated synaptic molecules onskeletal myotubes. Specifically it dusters acetylcholine receptors, acetyl-choline esterase, and butyrylcholine esterase (Nitkin et al 1987, Wallace1986). Agrin antigen is specifically localized at neuromuscular junctionsin vivo (cf. Reist et al 1987), making it a strong candidate to induceclustering of the same molecules in vivo. Agrin is transported antero-gradely by motoneurons (Magill-Solc & McMahan 1988). Agrin can alsobe detected in uninnervated muscle masses (Fallon & Gelfman 1989).Thus, it seems likely that it is synthesized by both neurons and skeletalmyotubes. TAP-1 is a large synaptic vesicle-associated proteoglycan thatappears to be synthesized by motoneurons in Torpedo (Carlson & Wight1987). An isoform of this proteoglycan is associated with the synapticbasal lamina in the Torpedo electric organ. Since it also has properties ofan integral membrane protein, it is a good candidate to function as areceptor that mediates binding of the nerve terminal plasma membrane tothe synaptic basal lamina.

Although synapse-specific constituents of the ECM have been identified,very little is known about the intracellular signaling mechanisms involvedin induction of pre- and postsynaptic differentiation. Extracellular Caa+


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~4 REICHARDT & TOMASELLI

is required for the formation of agrin-induced aggregates of acetylcholinereceptor (Wallace 1988). Inhibitors of energy metabolism and activatorsof kinase C both inhibit clustering of this receptor. Future studies extend-ing these promising initial results should be informative.

At this point, it is possible to propose how molecular differences betweensynaptic and nonsynaptic basal lamina are established. In part, this mustreflect the contributions to the synaptic basal lamina of constituents syn-thesized by motoneurons, such as TAP-1 and agrin and, in turn, mustreflect localization in the synaptic basal lamina of muscle-specific products,such as S-laminin. Mechanisms by which ECM constituents synthesizedby skeletal myotubes become localized to synaptic basal lamina are notwell understood. Preferential transcription of genes encoding synapticmolecules in myotube nuclei located near the synapse is one potentialmechanism. The genes encoding the acetylcholine receptor appear to beregulated in this fashion (cf. Goldman & Staple 1989).

ECM REGULATION OF CELL PROLIFERATIONAND DIFFERENTIATION

At early stages of development of the CNS, shortly after neural tubeclosure, the neuroepithelium is a single cell layer in thickness. At this time,germinal neuroepithelial cells in many areas of the nervous system contactat their basal surfaces an ECM that is rich in fibronectin, laminin, thrombo-spondin, and entactin (e.g. Tuckett & Morris-Kay 1986, O’Shea & Dixit1988). At later stages, in some parts of the CNS that have undergoneconsiderable cell proliferation (e.g. the retina) contact between germinalcells and overlying basement membrane is maintained. Recent studies onretinal neurogenesis in frogs provide evidence for a role of ECM moleculesin controlling the extent of proliferation of retinal germinal neuroepithelialcells. When slices of tadpole retinae are maintained in culture with thesurrounding basement membrane intact, neuroepithelial cells in the pro-liferative zone continue to divide for up to 3 weeks. Removal of thebasement membrane by collagenase treatment, however, leads to a sig-nificant decline in germinal cell proliferation, and this deficit can be par-tially ameliorated with a basement membrane extract that is rich inlaminin, collagen, and heparan sulfate proteoglycan (Reh & Radke 1988).Therefore, at least in some regions of the CNS, cell proliferation apparentlymay be stimulated by the presence of proteins within a basal lamina. Inregions of the CNS where proliferating cells are not in contact with thebasement membrane (e.g. the cerebral cortex), other ECM molecules,including FN, thrombospondin, and tenascin, are often in the proliferativezone (cf. Chun & Schatz 1988, O’Shea & Dixit 1988).


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Although the mechanisms by which ECM constituents regulate pro-liferation of neuroepithelial cells are not well understood, studies on non-neural systems provide evidence for two roles of the ECM. First, the ECMlocalizes growth factors. FGF, for example, binds heparan sulfate onproteoglycans in the ECM (cf. Saksela et al 1988). This has been shownto protect FGF from proteolysis. As a result, the ECM serves as a reservoirofFGF (cf. Rifkin & Moscatelli 1989). Since FGF is both a potent mitogenand a differentiation factor for embryonic neuroepithelial cells (cf. Bartlettet al 1988), these observations are very relevant for understanding earlydevelopment of the nervous system. Other growth factors, including amitogen for Schwann cells, also bind to heparin (e.g. Ratner et al 1988).The activity of TGF-fl is regulated in two distinct ways by proteoglycans.First, heparin activates it by dissociating it from an inactive complex withanother protein (McCaffrey et al 1989). Second, a cell surface proteoglycannamed decorin seems to function as a low-affinity receptor, binding andconcentrating TGF-fl on the cell surface, where it can then be bound by thehigh-affinity TGF-fl receptor (Boyd & Massague 1989, Ruoslahti 1989). addition to indirect effects mediated by growth factors, the ECM containsmajor constituents, such as LN, tenascin, and thrombospondin, that candirectly promote cell proliferation, in vitro, LN has been shown to promoteproliferation of several cell types, including fibroblasts and Schwann cells(cf. Panayatou et al 1989, McGarvey et al 1984). Thrombospondin andtenascin also promote the proliferation of specific cell types, includingsmooth muscle cells and fibroblasts, respectively (see Engel 1989). All threeproteins contain domains with homology to EGF. In the case of solubleLN, mitogenic activity has been localized within a fragment containingmultiple EGF repeats that does not contain the major cell attachment orneurite outgrowth-promoting sites (Panayatou et al 1989). A cell linelacking EGF receptors does not respond to soluble LN, thus making itattractive to imagine that the EGF receptor mediates the mitogenicresponse. Soluble LN does not compete with EGF for its receptor,however, so the mechanisms of LN’s action are unclear. Whatever themechanisms, the effectiveness of soluble LN suggests that the mitogenicaction of LN is due to direct modulation ofintracellular second messengersrather than effects on cell adhesion.

ECM REGULATION OF CELL SURVIVAL AND

DIFFERENTIATION

The effects of the ECM on neuronal survival and differentiation also seemlikely to reflect modulations of cytoplasmic second messenger systems. Inparticular, LN has striking effects on the survival of early embryonic


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neurons and on the responsiveness of older neurons to neurotrophicfactors. Early embryonic sympathetic neurons require an LN substratebut not atrophic factor to survive in vitro (Ernsberger et al 1989). Forinitial survival in vitro, embryonic sensory neuron precursors have a simi-lar requirement for an ECM-coated substrate, but not atrophic factor(Ernsberger & Rohrer 1988). At later stages, both sympathetic and sensoryneurons become dependent on trophic factors. Some sympathetic neuronsremain dependent on a LN substrate (cf. Edgar et al 1984). Those capableof surviving without LN can be maintained on LN by much lower con-centrations of NGF than would otherwise be effective. As LN is a promi-nent constituent of embryonic sympathetic and sensory ganglia at thesestages of development, the ECM is likely to be critical in regulating pro-liferation and differentiation of these cells in vivo. Consistent with thisview, embryonic sensory neuron precursors, when separated from theneural tube in vivo, can be rescued from cell death by the trophic factorBDNF only in combination with LN (Kalcheim et al 1987).

The effects in the nervous system of ECM molecules are not limited toneurons. Studies on Schwann cells, the myelin-forming cells of the per-ipheral nervous system, indicate that constituents of the basal lamina arerequired for myelination. Agents that disrupt Schwann cell basal laminaproduction in vitro--e.g, inhibitors of collagen or proteoglycan biosyn-thesis-prevent myelination of axons in vitro (Carey et al 1987, Eldridge etal 1987). Basal lamina-deficient Schwann cells can be induced to myelinateaxons by the addition of LN. Eldridge et al (1989) have proposed that promotes assembly of a normal basal lamina that in turn enables Schwanncells to myelinate the available axons. Schwann cells thus provide aninteresting example of an autocrine role for the ECM in inducingexpression of a highly differentiated phenotype. The mechanisms by whichthe ECM promotes Schwann cell differentiation are not understood. Bothadhesive interactions that polarize the Schwann cells and modulation ofsecond messenger systems may be important.

ECM components have been shown to regulate differentiation of neuralprecursor cells into neurons and to regulate the expression of transmitterenzymes in neural cells. Thus, LN has been shown to induce amphibianretinal pigment epithelial cells to express a neuronal phenotype (Reh et al1987). A substrate-attached factor from conditioned medium, which isprobably an isoform of LN, dramatically enhances the ability of NGF toconvert neonatal adrenal chromaffin cells to sympathetic neurons (Doupeet al 1985). The same factor is absolutely required for NGF-inducedconversion of adult adrenal chromaffin cells. ECM components have alsobeen shown to induce expression of an adrenergic phenotype by neuralcrest cells and adrenal chromaffin cells (Maxwell & Forbes 1987, Acheson


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et al 1986). When used as a substrate for adrenal chromaffin cell growth,LN increases the amount of tyrosine hydroxylase over a period of days,but is also able to increase the activity of tyrosine hydroxylase within afew hours (Acheson et al 1986). The latter result suggests that LN acts tyrosine hydroxylase by regulating the activity of one of the many proteinkinases known to modulate its activity (cf. McTigue et al 1985).

MECHANISMS OF ACTION OF ECM

CONSTITUENTS AND THEIR RECEPTORS

ECM constituents obviously influence many aspects of cell behavior duringdevelopment of the nervous system. Knowledge of the mechanisms bywhich ECM components exert their effects is still sketchy; however, in thecase of integrin-mediated interactions with the ECM, a picture has begunto emerge. Upon binding of ligand, integrins appear to transduce signalsacross the plasma membrane in two ways: by interacting directly withcytoskeletal proteins, and by regulating the levels of second messengers.Some integrin-mediated actions may, in principle, reflect simply ~effects ofinteractions with the cytoskeleton. The functions of macromolecules suchas FN in modulating migration of neural crest cells, for example, seempotentially understandable in this conceptual framework. Other effects ofECM constituents, though, such as effects upon celt survival, mitosis, ortrans-determination, are difficult to explain as simple consequences ofadhesion. Instead, they seem much more easily explicable if ECM macro-molecules also influence the intracellular second messenger systems of thecells. Studies on integrins, particularly in lymphocytes and platelets,provide evidence that they can indeed transfer signals across the membranethat influence both the cytoskeleton and intracellular second messengersystems. There is also convincing evidenc~ that intracellular metabolismcan alter the ligand-binding functions of several integrin receptors.

An important effector of integrin-mediated signaling is likely to be achange in integrin structure, mediated by proteins inside and outside thecell. With regard to extracellular mediators, both binding of ligands andof divalent cations have been shown to alter in.tegrin conformation (Pariseet al 1987). Regulated conformation-dependent epitopes have been ident-ified on the platelet integrin ~Ibfl3 and on the lymphocyte integrins byusing monoclonal antibodies (Frelinger et al 1988, Dransfield & Hogg1989). The epitope on glycoprotein ~bfl3 is located in the ~ub subunitsequence at a site that is well separated in the linear sequence from theligand and cation-binding sites, a position suggesting that it is a reporterfor a significant structural change (Frelinger et al 1988).

Binding of antibodies and ligands to external domains of integrin recep-


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REICHARDT & TOMASELLI

tors has been shown to regulate several cytoplasmic events. Among these,binding to an ECM protein-coated substrate promotes the formation offocal contacts in which intcgrins are associated with the termini of F-actinfilaments and several other proteins, most notably talin, vinculin, andactinin (cf. Burridge et al 1988, Fath et al 1989). Each of these proteinshas been detected in growth cones (cf. Letourneau & Shattuck 1989).Purified integrins have been shown to bind talin, which can in turn bindvinculin (Horwitz et al 1986, Tapley et al 1989). Based on observed inter-actions of these proteins in vitro, it has been proposed that talin, vinculin,and ~-actinin link the integrins to the cytoskeleton (reviewed in Burridgeet al 1988). Analysis of early events in cell attachment has demonstratedthat binding to ligand induces integrin-talin coaggregafion at sites ofmembrane-ECM contact, which is followed by association of other cyto-skeletal proteins (Mueller et al 1989). Agents that disrupt adhesion, suchas RGD-containing peptides, .have been shown to dissociate these cyto-skeletal proteins from integrins. In addition to regulation by adhesion,colocalization can also be regulated by intracellular messengers and proteinphosphorylation (cf. Tapley et al 1989).

The cytoplasmic domain of the integrin fl~ subunit has been shown to beimportant for its colocalization with the cytoskeleton and for its function incell adhesion (Solowska et al 1989, Hayashi et al 1990). When expressedin heterologous cells, integrin fll subunits lacking the cytoplasmic domainassociate with ~ subunits to form receptors that can still bind ligands inbiochemical assays. Within the cell, though, they do not localize to focalcontacts and are not effective at promoting cell adhesion. This indicatesthat integrins must interact with the cytoskeleton to promote cell adhesion(cf. Lotz et al 1989). The cytoplasmic domains of several integrin subunitsare substrates in vivo for tyrosine or serine-threonine protein kinases (cf.Tapley et al 1989). Some evidence suggests that phosphorylation of thesedomains regulates associations of integrins with the cytoskeleton.

Regulation of integrin interactions with the cytoskeleton is importantfor controlling the motility of cells and growth cones. In stationary cells,ligand binding by integrins leads to their accumulation in stable focaladhesions. Integrin associations with adhesive zones are comparativelystable (Duband et al 1988). In motile cells, however, integrins have beenshown to diffuse rapidly in the membrane, thereby indicating that associ-ations with the ECM and cytoskeleton are transient. The importance ofactin dynamics in growth cone motility and guidance is well established(cf. Smith 1988, Mitchison & Kirschner 1988). Growth cone motility driven in part by the behavior of the actin-based cytoskeleton. The flowof actin depends on actin polymerization at the leading edge of cells,energy-dependent retrograde movement of actin filaments, and depoly-


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EXTRACELLULAR MATRIX J59

merization of these filaments in the cell interior. Integrin-mediated inter-actions with F-actin can in principle regulate any of these steps. Lateralinteractions have the potential to slow retrograde movements, generatingtraction against the substrate. Interactions at filament ends may regulatecapping, polymerization, or depolymerization. Receptors interacting withmoving filaments of actin will generate tension inside the growth cone,which can potentially regulate the shape and dynamics of all cytoskeletalelements in the growth cone. In addition, stretch-activated and stretch-inactivated K+ channels have recently been discovered in growth cones(cf. Sigurdson & Morris 1989). By regulating membrane potential, thesechannels may modulate voltage-dependent Ca2+ channels and cytoplasmicCa2+ levels, which in turn may affect several other second messengers. AsCa2+ has been shown to be an important modulator of growth conemotility (cf. McCobb et ai 1988), critical examination of the functions these channels should prove interesting.

Binding to integrin receptors of antibodies and ligands has been shownto regulate other cytoplasmic events, at least some of which are not relatedto cell shape or adhesion. Some anti-integrin monoclonal antibodies caninitiate signaling cascades; for example, binding of some monoclonal anti-bodies to the platelet integrin (~Ilb~3 induces platelet activation (Jennings eta11985). During platelet activation, tyrosine protein kinases phosphorylat¢proteins in three waves. The function of the platelet integrin glycoprotein~llbfl3 is required for the third wave of phosphorylation, which does notoccur in platelets lacking this integrin or in platelets activated in thepresence of RGDS peptides (Ferrell & Martin 1989). Similarly, in fibro-blasts, interactions of the fibronectin receptor with fragments of fibronectinor with antibodies capable of crosslinking this receptor induce expressionof genes encoding ECM-degrading proteases (Werb et al 1989). Theseeffects are not related to visible changes in cell shape or cytoskeletalorganization. Consistent with this evidence implicating integrins as modu-lators ofintracellular signaling, the functions ofintegrins have been shownto be required for differentiation of several cell types (e.g. myoblasts) substrates for which they are not essential for cell adhesion (cf. Menko Boettiger 1987).

Mechanisms by which integrins regulate signal cascades are not under-stood. In platelets, there is evidence that the integrin t~llbfl 3 can associatewith an additional membrane-associated protein, CD9, which itself isinvolved in platelet activation (Slupsky et al 1989). Similar associations other proteins with integrins may potentially be involved in transducingsignals in other cells. Intriguingly, the cytoplasmic domain of an individualintegrin subunit has typically proven to be highly conserved among species(cf. Marcantonio & Hynes 1988), but ~ and fl subunits within a species


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have quite different cytoplasmic domains (cf. Takada & Hemler 1989). seems possible, therefore, that these domains interact with additional,unknown proteins, and that different subunits have significant differencesin their interactions and signaling capabilities. This may provide at leasta partial explanation for the existence of multiple integrin receptors thatinteract with a single ECM glycoprotein (Table 1).

Intracellular metabolism can alter both the structure and function ofthe extracellular domains of the integrins. The ligand-binding functionsof several integrins, most strikingly the platelet integrin ~Ilbfl3 and thelymphocyte integrins ~LFA-~ r2 and ~tMac.~ f12, have been shown, to be regu-lated by ligand binding to other receptors, by physiological stimuli, andby agonists or activators of second messenger systems (cf. Frelinger et al1988, Dustin & Springer 1989). Appearance of a conformation-dependentepitope shared by each of the lymphocyte integrins (~LFA-I f12, ~Mac-I ~2, and

~50fl2) has been shown to require not only Mg2+ binding but also cellularmetabolism (Dransfield & Hogg 1989). Phosphorylation of cytoplasmicdomains of integrin subunits seems likely to be one mechanism by whichintegrin-mediated cell adhesion is regulated (TapIey et al 1989, Danilov Juliano 1989). This may be one mechanism by which trophic factors andchemotropic factors influence neuronal growth cone interactions with theECM.

Additional mechanisms for rapid regulation of integrin function havebeen described in lymphoid cells and may be useful paradigms for under-standing effects of substrates, growth factors, and chemotropic agents ongrowth cone behavior. Large fractions of the integrins aMa~.1 r2 and agp~0fl2are sequestered in intracellular vesicles in neutrophils (Bainton et al 1987).Physiological stimuli induce exocytosis of these granules, transferring themto the cell surface. Factors that induce aggregation of these receptors ortheir ligands have also been shown to enhance their activity (cf. Her-manowski-Vosatka et al 1988). In a series of ingenious experiments inwhich a membrane-impermeant, cleavable crosslinking agent was used,integrins have been shown to cycle rapidly between intracellular and sur-face compartments in fibroblasts, being inserted preferentially at leadingedges of motile cells (Bretscher 1989). By similar mechanisms, chemotropicagents and trophic factors could potentially regulate growth cone behaviorby regulating the cycling or by targeting the insertions of integrins andother adhesion molecules.

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Bainton, D. F., Miller, L. J., Kishimoto, T.K., Springer, R. A. 1987. Leukocyte adhe-sion receptors are stored in peroxidase-negative granules of human neutrophils.J. Exp. Med. 166:1641-53

Bartlett, P. F., Reid, H. H., Bailey, K. A.,Bernard, O. 1988. Immortalization ofmouse neural precursor cells by the c-myconcogene. Proc. Natl. Acad. Sci. USA 85:3255-59

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extracellular matrix molecules and their receptors...

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