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n% Function (Edelman, G. M. and Mountcastle, V. B., eds), pp. 13 Merzenich, M. M. et al. (1984) J. Comp. Neurol. 224, 591-605 7-50, MIT Press 10 Stone, G. O. (1986) in Parallel Distributed Processing 14 Finkel, L. H. and Edelman, G. M. (1985) Proc. NatlAcad. 5ci. USA 82, 1291-1295 (Rumelhart, D. E. and McClelland, J. L., eds) pp. 444--459, 15 Churchland, P. S. (1986) Neurophilosophy MIT Press MIT Press 11 Maunsell, J. H. R. and Van Essen, D. C. (1983) J. Neurosci. 3, 16 Marr, D. (1982) Vision W. H. Freeman 17 Reeke, G. N., Jr and Edelman, G. M. (1984) Ann. New York 2563-2586 Acad. 5ci. 426, 181-201 12 Trends Neurosci.(1987) [special issue] 10, 263-302

Neuronal laminin receptors David Edgar DavidEdgaris at the Departmentof Anatomyand Cell Biology, PO Box 147, Universityof LiverpoolL693BX, UK.

The ability of the extracellular matrix protein/aminin to regulate the survival and differentiation of neurons has led to the search for a neuronal laminin receptor. This article reviews the evidence for the existence of laminin receptors, and then goes on to discuss the difficulties in making thejump from a pharmacologicaldemonstration of the receptor to its molecular identification.

induced neurite outgrowth. Thus it was determined that the site on the laminin molecule responsible for its interactions with neural cells is likely to be at or near the end of the long arm of this cruciform molecule (see Fig. 1), since domain-specific inhibitory antibodies all recognize epitopes located in this g e n e r a l a r e a 3'7.

It is important to realize that such blocking antibodies do not necessarily recognize the neuritepromoting site of laminin per se, but may equally well rely on the presence of epitopes either spatially or functionally associated with the site, with blockade occurring indirectly by steric hindrance or allosteric inhibition, respectively. Indeed, variants of laminin from a variety of sources have been described, many of which can stimulate neurite outgrowth although they lack the epitopes recognized by blocking antibodies 8, presumably as a result of differing subunit composition (see Ref. 9). Hence it is likely that most (if not all) inhibition of the neurite-promoting effects of laminin is due to indirect blockade of the neurite-promoting site of the molecule. The corollary of these observations is that this site is a weak antigen, possibly due to conservation between species (see Ref. 9) and immunological tolerance (long-arm epitopes are found on the exterior of the basement membrane 1° where they can presumably be contacted by cells, including those of the immune system). Despite these reservations, similar antibody inhibition experiments with a variety of nonneuronal cells have subsequently indicated that many cell types can also interact with either the same or closely associated sites on the long arm of laminin 1~'~2. These observations are of significance because if both neurons and non-neuronal cells can interact with the same sites on laminin, then any 'neuronal' laminin receptor will not necessarily be unique to neurons, but may also be found on nonneuronal cells. Using the complementary approach with antibodies directed against cell membrane components, it has been shown that anti-integrin antibodies block neurite outgrowth on laminin 4,13-~s. Integrins, as Indirect analysis of laminin-neuron interactions: their name suggests, constitute a widely distributed antibody inhibition Early studies to analyse the molecular basis of family of integral membrane protein dimers, comlaminin-neuron interactions used antibodies di- prising non-covalently linked e~- and ~-subunits (see rected against either laminin or components of the insert to Fig. 1). These proteins are involved in many neuronal membrane in attempts to block laminin- cell-cell and cell-matrix interactions 16, as a

The survival and development of neurons are influenced not only by soluble molecules such as neurotransmitters, hormones and trophic factors, but also by cell adhesion molecules anchored either on cell membranes or in the extracellular matrix ~. Current interest in laminin (Fig. 1) stems from the observation that it is unique among defined extracellular matrix molecules in being able to stimulate the rapid growth or regeneration of neuronal processes2; for example, neurite outgrowth can be initiated within minutes of exposure of neurons to tissue culture substrata containing laminin, the rate of neurite growth being about 1 ~m min -~ (Ref. 3). Furthermore, an analogous role in vivo seems likely because laminin immunoreactivity appears only transiently in the developing optic nerve, coinciding both with axonal growth in the optic tract and with the period in which retinal ganglion cells have been shown to respond to laminin 4. Perhaps even more strikingly, laminin and appropriate target-derived neurotrophic factors can act synergistically to promote neuronal survival during early development both in vitro 3 and in vivo 5. Subsequently, it appears that laminin helps to maintain the adrenergic phenotype: both a rapid activation and an enhanced expression of the enzyme tyrosine hydroxylase have been demonstrated in chromaffin cells after culture on laminin substrata 6. The intracellular mechanisms responsible for these various activities of laminin remain unknown, although the potency and specificity of its effects on neurons and other cells point to the existence of specific receptors, and these are currently being characterized by three distinct but complementary approaches.

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consequence of which the anti-integrin antibodies reported to block neurite outgrowth on laminin 4'13-~ are not specific, in the sense that they also block the attachment and spreading of many cell types on a variety of extracellular matrix molecules ~7'~8. The reason for this is that these antibodies recognize epitopes present on the l~-subunit common to many integrins (for discussion see Ref. 19). Given that the structural domains of laminin that interact with cellular receptors have polypeptide sequences that apparently are not shared by other extracellular matrix molecules 8,2°, then it is to be expected that the cellular receptors for laminin are also unique. Thus, as with the domain-specific antilaminin antibodies, the epitopes of the anti-13subunit antibodies so far used to block neurites are unlikely to be the actual molecular structures directly involved in laminin binding, consistent with the notion that the specificity of individual integrin molecules is conferred by their c~-subunits (see Ref. 16). It is therefore not surprising that such anti-~subunit antibodies have not yet led to the unambiguous demonstration of a neuronal integrin specific for laminin 14'~5,~9. Nevertheless, the results of the antibody inhibition experiments may be taken together as an indication that a site located on the long arm of laminin interacts with integrin(s) or integrin-associated molecules on the membranes of neurons and other cells. Direct analysis of laminin-neuron interactions: ligand binding studies Many of the ambiguities inherent in indirect attempts to characterize laminin-cell interactions by antibody inhibition can be circumvented by assaying directly the ability of laminin to interact with its responsive cells. Dissection of the laminin molecule by proteolytic fragmentation has thus confirmed that a site near the end of the laminin long arm mimics the effects of the whole molecule on neural 3 and other cells11,12. In addition, however, the use of proteolytic fragments has shown also that the short arms of laminin contain at least two other cell binding sites3'21. In contrast to the long-arm site, those of the short arms appear to be latent within the native molecule in that their activities can only be demonstrated after release from intact laminin by proteolysis (see Refs 3, 21, 22), and because antibodies against epitopes found on the long arm completely block the effects of native laminin on neurons and most other cell types 3'11. One of these cryptic sites is found only in complete short-arm fragments (e.g. fragment 1-4, see Fig. 1), and while few non-neuronal cells respond to this site (even after activation - see Refl 21), it does display weak neurite outgrowth-promoting activity 3. In contrast, the second short-arm site is exposed only after more extensive proteolysis to remove the globular domains from these arms22. Although this site acts as a potent substrate for many non-neuronal cells11'2~, it is unable to affect the behaviour of neurons 3. ~NS, Vol. 12, No. ~ 1989

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Fig. 1. Cartoon of laminin as isolated from the mouse Engelbreth-Holm-Swarm sarcoma tumour, and visualized by electron microscopy. The molecule is composed of three polypeptide chains that are covalently linked together to form a cross-shaped molecule of gO0 kDa 3z. The locations of those fragments of laminin shown to interact with neurons and other cells are indicated: 1, internal short-arm fragment that binds only non-neuronal cells; 1-4, complete short-arm fragment that has weak neurite-promoting activity; 8, long-arm fragment that contains the major site interacting with both neurons and non-neuronal cells. Scale bar represents 20 nm. Insert: cartoon of an integrin receptor, drawn approximately to the same scale as the laminin molecule 38. The integrins comprise two polypeptide chains, non-covalently linked to form a complex of about 300 kDa. Hatched area represents the cell membrane. Taken together, therefore, the experiments to examine the effects of laminin fragments on cell behaviour point towards the existence of three structurally distinct cell binding sites on the laminin molecule, and thus implicate three distinct cellular receptors for laminin and its fragments. However, the determined changes in cell behaviour (e.g. neurite outgrowth) are open to the complication that they cannot be expected simply to reflect the initiating molecular interactions between laminin and its cellular receptor: a change in cell behaviour is the end point of a complex, generally undefined series of cell biological processes, any one of which may be modified or distorted by events unrelated to the laminin-receptor interaction. In order to gain more definitive information on the molecular properties of laminin-receptor interactions it is therefore necessary to look at the binding of laminin directly. Maximally, some 105 high-affinity (KD 10-9M) binding sites specific for laminin have been demonstrated on the membranes of its responsive cells by ligand-binding assays, the numbers of binding sites 249

TABLE I. Neuronal laminin receptor candidates Molecular mass (kDa)

Sourceand properties

Proteins identified by antibodiesthat block neurite outgrowth

110, 125, 135,145 a 120, 140, 180a

220a

From chick neural retina, proteins metabolically labelled before immunoprecipitation by antibodies against chick integrin I~-subunits. Slight changes in protein pattern occur during development 15 From rat pheochromocytoma (PC12) cells, both metabolically and surface labelled. 120 kDa protein recognized by antibodies against integrin l~l-subunit, so other proteins co-precipitated. Some binding to laminin and type IV collagen demonstrated after incorporation into liposomes14,19 From PC12 cells, metabolically labelled. Immunoprecipitation with antibodies that block fibre outgrowth on laminin and type 1/111 collagen. Immunoreactivity demonstrated on neurons and astrocytes35

Proteins that bind laminin

120a

110, 180a

120, 2 0 0 ~ 67, 120-140 b

From rodent and chick tissue, not specific for nervous system. Detected after electroblotting by binding labelled laminin under isotonic salt concentrations. Binding sensitive to detergents, so protein would not be seen on affinity chromatography. Binding dependent on divalent cations, with KD apparently less than 1 nmoP 6 From neuroblastoma-glioma cells, both proteins bind laminin on affinity chromatography (low pH elution), the 180 kDa protein is specific to the nervous system and also binds laminin under hypotonic conditions after electroblotting. High concentrations of antisera (30%) against either protein block fibre outgrowth from neuroblastoma cells28 From PC12 cells and rat neuroblastoma. Binding to laminin on affinity chromatography is dependent on divalent cations. 120 kDa protein identified as I~l-subunit of integrin 29 From chick brain. Proteins bind laminin on both on affinity chromatography (low pH elution), and under hypotonic conditions after electroblotting. High affinity for laminin demonstrated 23

aDeterminedby SDS-polyacrylamidegel electrophoresisundernon-reducingconditions. bDeterminedunder reducingconditions. being characteristic of the cell type 11'21-24. Significantly, it could also be shown that the long-arm fragment of laminin competitively blocks the binding of intact laminin to cells 22, whereas it does not interfere with the binding of short-arm fragments 1~'22. This result supports and extends those previously mentioned by indicating that the long arm of laminin does indeed contain the major cellular binding site of the native molecule. In contrast, although the short-arm fragments bind independently to distinct cellular receptors, these sites do not appear to be used by native laminin in its neuronal interactions. The significance (if any) of these latent cell binding sites for the interactions of laminin with neurons remains to be established. The results of the ligand binding assays do not, of course, prove that the high-affinity binding sites for laminin molecules in solution are the physiological receptors mediating cellular responses to laminin anchored in an extracellular matrix. That this may well be the case, however, is supported by the observation that the presence of the binding sites correlates well with the ability of a variety of cell types to adhere and spread on laminin substrata ~. In particular, it has been shown that the number of high-affinity laminin receptors on embryonic retinal ganglion cells decreases during maturation, this loss being reflected by the decline in laminin-stimulated 250

neurite outgrowth from these neurons 24. The information derived from such ligand binding studies may therefore provide useful criteria for the assessment of putative receptor molecules, in terms of estimating how many receptors can be expected from a given cellular source, their affinity for laminin, and details of the specificity to be expected in their molecular interactions with laminin 11'22. Thus the long-arm laminin receptor common to both neurons and other cells is unlikely to be the 35/70 kDa cellmembrane-associated protein previously identified and cloned from various (non-neural) sources 25'26, that has been shown to bind to a site in the short arms of laminin 27. Isolation of receptor candidates

Table I shows that several membrane proteins from neurons and related cells have been identified, based either on their recognition by antibodies capable of blocking neurite outgrowth, or alternatively on the demonstration that the proteins bind to laminin after their extraction from the cell membrane. Unfortunately, the characterization of the candidate molecules is presently limited mainly to estimates of their apparent molecular weights. Thus it is not clear to what extent the various proteins of similar size, but isolated using different sources and techniques, are related. In addition to the difficulties arising from the use of inhibitory antibodies to identify putative receptor molecules (see above), the demonstration that an isolated protein can bind laminin obviously does not guarantee that the protein acts as the physiological laminin receptor; one of the major problems with the affinity chromatography technique has been the lack of precise knowledge of the peptide sequences of laminin involved in the binding of the native molecule to its neuronal receptors 8 (or indeed the sequences of any other antagonists of lamininreceptor binding). It has therefore not been possible specifically to elute laminin affinity columns as, for example, has proved invaluable for the successful elution of fibronectin-specific integrins with ArgGly-Asp-containing peptides (see Ref. 16). Consequently, the only methods available for elution of putative neuronal laminin receptors have relied on changes in the ionic conditions 23,28,29. The specificity of affinity columns eluted by manipulating ionic concentrations might not be expected to be very high. However, the adhesion of TINS, Vol. 12, No. 7, 1989

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integrins to extracellular matrix molecules has been shown to be dependent on divalent cations. In particular, the efficiency of integrin binding to fibronectin is markedly enhanced by manganese 3°. Based on this observation it was demonstrated that distinct integrin subunits extracted from glioblastoma cells will also bind well to laminin affinity columns in the presence of manganese 31. Furthermore, it appeared that the integrin binding involved long-arm rather than short-arm structures of laminin, indicating that integrins may well be a common laminin receptor shared between neuronal and non-neuronal cells. Indeed, support for this possibility has recently been provided by showing that the same technique can be used to isolate integrins from neuroblastoma and pheochromocytoma cells 29, fibre outgrowth from which is known to be dependent on magnesium, and presumably also on similar divalent cations like manganese 32. It remains to be shown, however, that the cation requirements of fibre outgrowth are due to a cation-dependent interaction of laminin with the integrins present in the membranes of these cells.

Future directions Direct evidence for the involvement of an identified receptor molecule in the neuronal response to laminin is still lacking. Although none of the techniques employed individually can be considered as providing definitive evidence for the identification of a neuronal laminin receptor, results from both the antibody approach 4'13-15 and affinity chromatography 23'29 are consistent with the involvement of integrins in the neuronal response to laminin. In the near future it is to be expected that both techniques will be refined to the point where the likelihood that the molecules they each identify are indeed laminin receptors will be markedly improved. Thus, recent reports that antibodies specific for integrin o~-subunits are also able to inhibit cell 33 and blood platelet 34 attachment to laminin substrates will doubtless provoke similar inhibition experiments with neuronal cells. If such antibodies can indeed block neurite outgrowth, then the way is open for their use in isolating a neuronal laminin-specific integrin. Similarly, the use of laminin fragments containing the neurite-promoting site 3,12, possibly together with synthetic oligopeptides derived from the sequences involved 8, should improve the effectiveness of affinity chromatography by both reducing the chances of non-specific interactions and increasing the specificity of elution. While subsequent estimates of the amounts of these receptor candidates and their affinity for laminin binding will help substantiate their role as receptors, all these experiments will still not provide definitive proof. It will only be certain that a neuronal laminin receptor has been identified when it can be shown that transfection of cells with the cloned gene(s) for the candidate results in the expression of functional receptors with the appearance of cellular responses to laminin.

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Selected references 1 Jessel,T. M. (1988) Neuron 1, 3-13 2 Baron-Van Evercooren, A. et al. (1982) J. Neurosci. Res. 8, 179-194 3 Edgar, D., Timpl, R. and Thoenen, H. (1985) EMBO J. 3, 1463-1468 4 Cohen, J., Burne, J. F., McKinley, C. and Winter, J. (1987) Dev. Biol. 122,407--418 5 Kalcheim, C., Barde, Y-A., Thoenen, H. and Le Douarin, N. M. (1987) EMBO J. 6, 2871-2873 6 Acheson, A. L., Edgar, D., Timpl, R. and Thoenen, H. (1986) J. Cell BioL 102, 151-159 7 Engvall, E. etal. (1986) J. Cell Biol. 103, 2457-2465 8 Edgar, D., Timpl, R. and Thoenen, H. (1988) J. CellBiol. 106, 1299-1306 9 Hunter, D. D., Shah, V., Merlie, J. P. and Sanes,J. R. (1989) Nature 338, 229-234 10 Schittny, J. C., Timpl, R. and Engel, J. (1988) J. CellBiol. 107, 1599-1610 11 Aurnailley, M., Nurcombe, V., Edgar, D., Paulsson, M. and Timpl, R. (1987)J. Biol. Chem. 262, 11532-11538 12 Dillner, L., Dickerson, K., Manthorpe, M., Ruoslahti, E. and Engvall, E. (1988) Exp. Cell Res. 177, 186-198 13 Bozyczko, D. and Horwitz, A. (1986) J. Neurosci. 6, 1241-1251 14 Tomaselli, K. J., Damsky, C. H. and Reichardt, L. F. (1987) J. Cell Biol. 105, 2347-2358 15 Hall, D. K., Neugebauer, K. M. and Reichardt, L. F. (1987) J. Cell Biol. 104, 623-634 16 Hynes, R. O. (1987) Cell 48, 549-554 17 Greve, J. M. and Gottlieb, D. I. (1982) J. Cell Biochem. 18, 221-230 18 Neff, N. T. etal. (1982) J. CellBiol. 95, 654-666 19 Tomaselli, K. J., Damsky, C. H. and Reichardt, L. F. (1988) J. Cell Biol. 107, 1241-1252 20 Sasaki, M., Kleinman, H. K., Huber, H., Deutzmann, R. and Yamada, Y. (1988) J. Biol. Chem. 263, 16536-16544 21 Terranova,V. P., Rao, C. N., Kalebic, T., Margulies, I. M. and Liotta, L. A. (1983) Proc. NatlAcad. Sci. USA 80, 444-448 22 Nurcombe, V., Aumailey, M., Timpl, R. and Edgar, D. (1989) Eur. J. Biochem. 180, 9-14 23 Douville, P. J., Harvey,W. J. and Carbonetto, S. (1988) J. Biol. Chem. 263, 14964-14969 24 Nurcombe, V. and Edgar, D. (1988)J. PhysioL (London)400, 68P 25 Rao, N. C., Barsky, S. H., Terranova, V. P. and Liotta, L. A. (1983) Biochem. Biophys. Res. Commun. 111,804-808 26 Yow, H. et al. (1988) Proc. Natl Acad. Sci. USA 85, 6394-6398 27 Graf, J. etal. (1987) Cell48, 989-996 28 Kleinman, H. K. et al. (1988) Proc. Natl Acad. Sci. USA 85, 1282-1286 29 Ignatius, M. J. and Reichardt, L. F. (1988) Neuron 1,713-725 30 Gailit, J. and Ruoslahti, E. (1988) J. Biol. Chem. 263, 12927-12932 31 Gehlsen, K. R., Dillner, L., Engvall, E. and Ruoslahti, E. (1988) Science 241, 1228-1229 32 Turner, D. C., Flier, L. A. and Carbonetto, S. (1987) Dev. Biol. 121, 510-525 33 Wayner, E. A. and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884 34 Sonnenberg, A., Modderman, P. and Hogervorst, F. (1988) Nature 336, 487-489 35 Turner, D. C., Flier, L. A. and Carbonetto, S. (1987) J. Cell Biol. 105, 137a 36 Smalheiser, N. R. and Schwartz, N. B. (1987) Proc. NatlAcad. Sci. USA 84, 6457-6461 37 Martin, G. R. and Tirnpl, R. (1987) Annu. Rev. Cell. Biol. 3, 57-85 38 Nermut, M. V., Green, N. M., Eason, P., Yamada, S. S. and Yamada, K. M. (1988) EMBO J. 7, 4093-4098

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