Adhesion properties of a neuronal epitope recognized by the monoclonal antibody HNK-1

Brain Res'earch, 3~7 ( !98t~ 2~J 25

20

BRE 11484

Adhesion Properties of a Neuronal Epitope Recognized by the Monoclonal Antibody HNK- 1 RICHARD J. RIOPELLE 1, RONALD C. McGARRY2 and JOHN C. RODER 2 t Department of Medicine (Neurology) and 2Department of Microbiology and Immunology, Queen's University, Kingston, Ont. K7L 3N6 (Canada)

(Accepted July 2nd, 1985) Key words: neuronal adhesion protein - - neurite-promoting substrate-attached molecule - - myelin-associated glycoprotein

A carbohydrate epitope on adhesion proteins of the developing nervous system, and on myelin-associated glycoprotein, is recognized by the monoclonal antibody HNK-1. The HNK-1 epitope bearing proteins and the monoclonal antibody alter, in a dose-dependent manner, the interaction between neurons and neurite-promoting substrate-attached materials released from cultured neural cells.

INTRODUCTION The interaction of neurons with immobilized molecular species of the extracellular milieu plays a major role in axonal guidance during development of the nervous system. A number of cells of nervous system origin contribute to the extracellular environment of neurons 5. It has been demonstrated recently that neurons also produce immobilized materials24, and thus, like other cell types, neurons both respond to and produce constituents of the extracellular matrix. To date, there have been few studies devoted to characterization of neurite-promoting substrate-attached materials (NP-SAMs) produced by cells in culture and released into the medium of growth. However, Lander et al. ~5 have determined that many conditioned media (CMs) contain the well-characterized non-collagenous nectin glycoprotein laminin, and that this molecular species accounts for much of the neurite-promoting activity in CM. Little information is available on neuronal receptors or adhesion molecules for NP-SAMs. However, there is a growing literature devoted to characterization of a family of neuronal cell surface glycoproteins that subserve various aspects of cell-cell adhesion. Edelman et al. v have characterized an avian glyco-

protein species that is microheterogeneous and undergoes developmental changes. This molecule is known as N - C A M and is related to a similar adhesive protein of murine nervous tissue known as BSP-29. Recently, Schachner et al. 27 have described two glycoproteins of developing murine brain recognized by monoclonal antibodies and denoted L1 and L2. The L1 antigen subserves neuron-glia interaction and is functionally similar to neuron-glia C A M (Ng-CAM) described by Grumet et al.~0. N g - C A M and N - C A M are also related antigenically 10. Kruse et al.t4 have made the observation that L1, BSP-2 and N - C A M are antigenically related as they share epitopes recognized by the L2 antibody as well as by HNK-1, a murine monoclonal antibody reacting with granular lymphocytes of human peripheral blood I and the ubiquitous central and peripheral nervous system glycoprotein, myelin-associated glycoprotein (MAG) 17. The HNK-1 epitope is found on tetanus toxin-positive neurons of the central and peripheral nervous systems, as well as on galactocerebroside-Cpositive oligodendrocytes and on some glial fibrillary acidic protein-positive astrocytes (McGarry et al., in press). The HNK-1 epitope is a carbohydratel4 (McGarry et al., in press) and on neurons in vitro is protein-associated and synthesized de novo (McGar-

Correspondence: R.J. Riopelle, Room 101 La Salle Building, 146 Stuart Street, Kingston, Ont., Canada K7L 3N6.

0006-8993/86/$03.50© 1986 Elsevier Science Publishers B.V. (Biomedical Division)

21 ry et al., in press). It has been shown previously that the neuronal HNK-1 epitope interaction with the monoclonal antibody promotes a rapid regenerative neurite response ~8. In the present studies we demonstrate that the HNK-1 epitope is also an adhesion domain that mediates the interaction of avian neurons with NP-SAMs derived from cultures of neural tissue24,25. MATERIALS AND METHODS

Materials Trypsin and DNase were obtained from Worthington Biochemical; Hank's balanced salt solution, Ham's F12 medium, Dulbecco's modified Eagle's medium (DMEM), gentamycin and fetal calf serum (FCS) from Gibco; poly-D-lysine, bovine serum albumin (BSA) (Fraction V), cytosine arabinoside, insulin, progesterone, transferrin, selenium and putrescine from Sigma; anti-thy 1.1 from Cedarlane; antifibronectin, anti-laminin, laminin and fibronectin from Bethesda Research Labs. Fertilized eggs were obtained from H and W Hatcheries, Belleville, Ont. The MeWo cell line was obtained from Dr. Jorgen Fogh, Sloan-Kettering Labs., Rye, NY. N-CAM and rabbit anti-N-CAM were gifts of Dr. Urs Rutishauser. Antibody 299, a monoclonal IgM recognizing a cell surface epitope on small cell lung carcinoma cell line H69, was a gift of Dr. John Minna. HNK-1 was produced as ascites fluid. Culture plasticware was purchased from Falcon. M A G was prepared from human brain by the method of Quarles and Pasnak 22. Nerve growth factor was prepared by the method of Mobley et a1.19.

Methods Cell preparation. Sensory (dorsal root ganglion, D R G ) neurons were obtained from 7 - 8 day embryo chick as previously described 23.24. Ganglia were incubated with 0.01% trypsin in 0.05 mg/ml DNase in Ca2+-Mg2+-free buffer for 12 min at 37 °C. The reaction was stopped by removal of the incubation solution and by addition of supplemented H a m ' s F12 medium containing 5% FCS (SF12) 23. Following trituration with a 5-ml pipette, the cell suspension was centrifuged (750 g x 10 min, 4 °C) through a 100% concentrated FCS gradient, suspended in 4 ml SF12, and plated on 100-mm Falcon culture dishes for 3 - 4 h at

37 °C, 5% CO 2. The non-adherent neuron-enriched cell population was harvested, washed and resuspended in defined medium 4. Spinal cord neurons were prepared as described previously 23. Briefly, 1-mm3 pieces of 7 - 8 day chick embryo spinal cord (SC) were incubated in 0.1% trypsin in 0.05 mg/ml DNase for 25 min at 37 °C, 5% CO2. The trypsin solution was removed and the tissue triturated in SF12 + 5% FCS to a single cell suspension. The suspension was centrifuged (750 g × 10 min at 4 °C) through a 100% FCS gradient and the cells then resuspended in 4 ml SF12 + 5% FCS and seeded onto 100-mm Falcon culture plates for 3 - 4 h at 37 °C, 5% CO 2. The non-adherent, neuron-enriched cell population was harvested, washed and resuspended in defined medium if cells were to be used in assay, or in defined medium + 5% FCS if cells were to be used to prepare CM. Conditioned medium (CM) preparation. As described previously 24, to prepare SC neuron CM, cells were seeded densely in defined medium + 5% FCS for 24 h (37 °C, 5% CO2), following which time medium was removed and replaced by serum-free defined medium (with gentamycin) containing 10-5 M cytosine arabinoside. CM was collected after 7 days and filtered through 0.2-/~m millipore filters. MeWo cultures were maintained in D M E M + 5% FCS and gentamycin. CM was collected after 5 days and filtered through 0.2-~m millipore filters. Controis for both neuron and tumor cell CM was medium handled in the same way as CM but not exposed to cells. Assay preparation. Poly-D-lysine-coated (0.1 mg/ml, 24 h) microcul~re wells were treated for 16-20 h with 100% CM or control medium at 37 °C, 5% CO 2. After extensive washing, wells were seeded with D R G cells or SC cells at 2000-3000 per well in defined medium. Wells with D R G cells had nerve growth factor 90 pg/ml added at the same time. The appropriate proteins or antibodies to be tested were dialyzed against 0.1 M phosphate buffer (24 h, 4 °C) and aliquoted in defined medium; the appropriate solutions were added to the assay at 1/100 the final volume. The microculture wells were incubated at 37 °C, 5% CO 2 for 15-20 h (DRG) or 40 h (SC), and neuronal performance was then scored using a Leitz Diavert microscope with phase optics. Neuron performance was defined as process formation in excess

of 1.5 cell diameters. One representative diameter of

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each well at 200× magnification was scored, and for each additive, triplicate wells were analyzed. I ) R G neuron processes were easily measured after 15-20 h, while at least 40 h were required to analyze process formation of SC neurons. RESULTS

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As previously reported 24, neurons seeded in numbers that minimize cell-cell interaction show increased adherence and more rapid neurite formation if the poly-D-lysine substrate of growth has been pre-

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treated with CM from highly enriched chick embryo neuronal cultures. Similar observations have been made with CM from a h u m a n neural crest cell line (MeWo) 2s. In the present experiments, poly-D-lysine-treated microculture wells were treated for 16-20 h with 100% CM or medium similarly handled without exposure to cells. To the treated and extensively washed wells, D R G or SC cells highly enriched for neurons by preplating 23 were added at 2000-3000 per well in defined medium. Because of the rates at

(anti-thy 1.1), did not inhibit the n e u r o n - N P - S A M interaction (Fig. 1 and Table 1). These data were interpreted to suggest that HNK-1 blocks an epitope on neurons that is responsible for process formation on

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6

7

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110,

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bearing D R G cells on MeWo-derived N P - S A M (Fig. 1A), and a similar inhibition of neurite-bearing SC cells on SC n e u r o n NP-SAM (Fig. 1B). A n u m b e r of control immunoglobulins, including those antibodies recognizing the defined non-collagenous glycoproteins, laminin and fibronectin, and an antibody which recognizes a surface glycoprotein on neuronal cells

4

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ble neurite formation by SC neurons. In these conditions, between 140 and 160 process-bearing cells per

tives - - monoclonal and polyclonal antibodies, M A G , N - C A M and BSA. Fig. 1 demonstrates a dose-dependent inhibition by HNK-1 of neurite-

3

120

~ ~60

Using this culture paradigm, n e u r o n - e n r i c h e d suspensions were co-incubated with the various addi-

2

Concentration

which neuronal processes are extended in vitro, assays employing D R G neurons could be read at 15-20 h, while 40 h were required to detect easily measura-

microculture well diameter at 200× magnification were counted in those wells treated with CM. Where control medium was used, the response was 25 +_ 4% of the response in conditioned wells.

I

% Conclntrotion ( ,,ug/ml )

Fig. 1. Concentration-dependence of inhibition of neuronal performance on NP-SAMs by various additives. Fig. 1A illustrates the performance of DRG neurons on NP-SAMs from the MeWo cell line25and Fig. 1B illustrates the performance of SC neurons on NP-SAMs from spinal cord neuron-enriched cultures24. NP-SAMs were harvested from CMs prepared as described in Materials and Methods and used at 100% to coat poly-D-lysine-derivatized(0.1 mg/ml, 24 h, 37 °C) and washed microculture wells. Neuron-enriched cell suspensions23 were seeded at 2000-3000 per well in defined medium4. Nerve growth factor (90 pg/ml) was added to wells containing DRG neurons. The various proteins and antibodies indicated were added in triplicate over the concentration range indicated. Plates were incubated at 37 °C, 5% CO2for 15-17 h (DRG) or 40 h (SC), and the wells were then scored at 200× magnification by counting neurite-bearing cells in a representative well diameter. Results are expressed as means (_+S.D.) of percent maximal response (140-160 neurons per diameter in the absence of additives). The performance in control wells was 25% (+_4) of maximum. A HNK-1; O MAG; • myeloma IgM; ® IgM monoclonal antibody 299; [] BSA.

23 TABLE I Modulation of neurite formation on substrate-attached material

Substrate-anached materials from spinal cord neuron-enriched cultures24 and MeWo cultures25 were harvested by passaging conditioned medium (CM) prepared as described at 100% on poly-D-lysine-coated(0.1 mg/ml, 37 °C, 24 h) and washed microculture wells. Control wells were treated with medium handled in a similar manner but never exposed to the cells. Freshly dissociated, neuron-enriched, 7-8 day dorsal root ganglion (DRG) cells23were seeded at 2000-8000 per well in defined medium4 containing nerve growth factor 90 pg/ml. Various solutions were added in triplicate at the concentrations (ug/ml) or dilutions indicated in parentheses at the time the cells were aliquoted. Plates were incubated at 37 °C, 5% CO2 for 15-17 h and then scored on a Leitz Diavert microscope as described in Materials and Methods. Results are expressed as mean (+ S.D.) percent inhibition of neurite extension, where maximum performance was the number of cells with neurites in wells with no additives (140-160/well diameter), and where 100% inhibition was chosen arbitrarily as neurite extension in wells treated with control medium (25 + 4% of maximum). Addition

HNK-1 (IgM) (10) Anti-thy 1.1 (IgG) (10) Antibody 299 (IgM) (20) Myeloma (IgM) (20) Anti-fibronectin (1/200) Anti-laminin (1/200) MAG (10) N-CAM (5) BSA (20)

Percent inhibition of DRG neurons on substrates Spinal cord neuron CM

Neural crest cell CM

59 + 12" NT 4+6 0 0 0 63 _+10" 74 + 11" 0

88 + 20* 4+7 15 + 13 0 0 0 89 + 7* 77 + 9* 0

* P<0.01; NT, not tested.

thus far uncharacterized molecular species in NP-

domain involved in the interaction of neurons with

SAM, Similar but less striking inhibition of neuronal performance on N P - S A M was seen where the neurons were pretreated with HNK-1 and washed before seeding onto the NP-SAMs (data not shown). These differences would be predicted since, in the preincubation experiments, most of the HNK-1 is b o u n d ,

materials released by cells to the extracellular matrix of growth.

while free HNK-1 is limiting and therefore unavailable as more epitope appears on the n e u r o n a l cell surface during process extension. Since the HNK-1 epitope is also present on N - C A M TM and on M A G 14,17, these proteins should compete with neurons for N P - S A M interactions. Fig. 1 illustrates the dose-dependent inhibition of neuron performance provided by h u m a n M A G , while Table I also demonstrates significant inhibition of D R G n e u r o n - N P - S A M interactions by N - C A M at 5 #g/ml. A n o t h e r glycoprotein, BSA, at high concentrations did not inhibit the n e u r o n - N P - S A M interaction. DISCUSSION The present studies provide evidence to suggest that the HNK-1 epitope on n e u r o n s is an adhesion

The molecular species of the extracellular matrix consists of collagens, non-collagenous glycoproteins and glycosaminoglycans complexed to protein and known as proteoglycans. To date, the NP-SAMs from neuron-enriched cultures 24 or the neural crest tumour cell line 25 have not been characterized. Sodium dodecyl sulfate polyacrylamide gels reveal a restricted n u m b e r of proteins released by cultured SC neurons, some of which interact with poly-D-lysine (Dow and Riopelle, unpublished observations). NPSAMs have been detected in CMs of cultures of a n u m b e r of n o n - n e u r o n a l cell types3. Only two of these factors have been partially characterized2, ~6. Recently, however, L a n d e r et al.15 have found that much of the NP-SAMs activity of CMs from a number of cultured cells can be accounted for by laminin complexed within NP-SAMs, but that anti-laminin does not inhibit the NP-SAM activity in vitro. Observations made in the present studies are consistent with these latter findings. Antibodies to laminin and fibronectin at concentrations that inhibited n e u r o n performance on the respective substrates by 90%

24

and 60% (data not shown) had no influence on the SC neuron or neural crest cell line NP-SAMs. Preliminary data suggest that the HNK-1 epitope is also involved in neuronal interactions with NP-SAMs from other cell types grown in vitro (Riopelle, unpublished observations). Studies are currently in progress to determine whether the epitope recognized by HNK-1 is involved in neuron adhesion to laminin and fibronectin. A number of molecular species that mediate neuronal adhesion have been described. Edelman and his colleagues have characterized a microheterogeneous neuronal glycoprotein N-CAM which is involved in the process of fasciculation of neuronal processes 7 and in neuromuscular interaction 1~, and which is restricted to nervous system from the time of neural induction 7. Antibody to N-CAM blocks neuronal fasciculation and has been shown to recognize an NH2-terminal peptide sequence of the protein. NgCAM, a protein antigenically related to N-CAM, has been shown to mediate neuron-gila interactions 10. Goridis and his colleagues 9 have described a family of adhesive proteins known as BSP-2 which share the properties of N-CAM. Schachner et al. z7 have described two antigens (L1 and L2) in developing mouse cerebellum that are involved in adhesive interactions. The L1 antigen mediates the migration of developing cerebellar granule cells along Bergmann glia in explants in vitro. This antigen would appear to have similar functional properties to Ng-CAM described by Grumet et al. 10. The adhesion domain recognized by HNK-1 appears to differ from that recognized by anti-N-CAM. While anti-N-CAM recognizes a polypeptide sequence 7, HNK-1 recognizes a carbohydrate epitope 14 (McGarry et al., in press). Furthermore, preliminary observations suggest that anti-N-CAM does not interfere in the neuron-NP-SAM interaction (Riopelle et al., unpublished). On the other hand, the present studies indicate that chick N-CAM inhibits the neuron-NP-SAM interaction, as does human MAG. MAG has previously been shown to carry the HNK-1 epitope 17 and has been implicated in adhe-

sive interactions of axons and glia -'~. Krusc ct al. ~a have made the observation that BSP-2/N-CAM also carries the HNK-1 epitope. Both the L2 monoclonal antibody and HNK-1 recognize epitopes shared bctween BSP-2/N-CAM, L1 and MAG 14. All of these data suggest that there is a family of glycoproteins of the developing nervous system which act as integral membrane receptors to promote various forms of neuronal adhesion with extracellular ligands - - neuron-neuron (BSP-2/N-CAM), neuron-gila (Ng-CAM/L1) and neuron-substrate (HNK-1). That the HNK-I epitope can be found on L1, BSP-2/N-CAM and MAG 14 and, as shown in the present studies, that HNK-1, MAG and N-CAM interfere with neuron-SP-SAM interactions, are compelling evidence to suggest that antigenically related glycoproteins possess heterogeneous adhesion domains. Changes in concentration of N-CAM 7 and alterations of carbohydrate composition of N-CAM 13 and BSP-226 significantly affect binding rates of these adhesion molecules in an indirect manner. The presence on the same glycoprotein of multiple domains subserving a variety of adhesive functions represents an additional mechanism to modulate neuronal interactions. For example, heterogeneity of expression of the HNK-1 epitope on N-CAM molecules within a population of neurons could provide biases that might explain some forms of selective fasciculation of individual axons. Heterogeneity of expression of adhesion domains within a family of glycoproteins, and differences in kinetics of adhesive interactions provide molecular mechanisms that begin to address two ideas emerging from a host of recent observations: first, exquisite neuronal connectivity during early development is the result of temporal and spatial opportunity 2°, competition12 and recognition processes occurring at the level of axonal guidance2°; secondly, the recognition process is a relative or bias phenomenon based upon modulations that occur as a result of past and present cell interactionsO.S.20.

Note added in proof

Reference 'McGarry et al., in press' should be added to list of References as: McGarry, R.C., Riopelle, R.J., Frail, D.E., Edwards, A.M., Braun, P.E. and Roder, J.C., The characterization and cellular distribution of a familyof antigens related to myelin associated glycoproteinin the developing nervous system, J. Neuroimmunol., in press.

25 REFERENCES 1 Abo, T. and Balch, C.M., Characterization of HNK-1 (Leu-7) human lymphocytes. II. Distinguishing phenotypic and functional properties of natural killer cells from activated NK-like cells, J. Immunol., 129 (1982) 1758-1761. 2 Barbin, G., Manthorpe, M. and Varon, S., Purification of the chick eye ciliary neuronotrophic factor, J. Neurochem., 43 (1984) 1468-1478. 3 Berg, D.K., New neuronal growth factors, Annu. Rev. Neurosci., 7 (1984) 149-170. 4 Bottenstein, J.E., Skaper, S.D., Varon, S.S. and Sato, G.H., Selective survival of neurons from chick embryo sensory ganglionic dissociates utilizing serum-free supplemented medium, Exp. CellRes., 125 (1980)183-190. 5 Carbonetto, S., The extracellular matrix of the nervous system, Trends Neurosci., 7 (1984) 382-387. 6 Edelman, G.M., Cell adhesion and morphogenesis: the regulator hypothesis, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1460-1464. 7 Edelman, G.M., Hoffman, S., Chuong, C.-M., Thiery, J.-P., Brackenbury, R., Gallin, W.J., Grumet, M., Greenberg, M., Hemperly, J.J., Cohen, C. and Cunningham, B.A., Structure and modulation of neural cell adhesion molecules in early and late embryogenesis, Cold Spring Harbor Symp. Quant. Biol., 48 (1983) 515-526. 8 Goodman, C.S., Bastiani, M.J., Raper, J.A. and Thomas, J.B., Cell recognition during neuronal development in grasshopper and Drosophila. In W.M. Cowan (Ed.), Molecular Bases of Neural Development, NRP Press, New York, in press. 9 Goridis, C., Deagostini-Bazin, H., Hirn, M., Hirsch, M.-R., Rougon, G., Sadoul, R., Langley, O., Gombos, G. and Finne, J., Neural surface antigens during nervous system development, Cold Spring Harbor Symp. Quant. Biol., 48 (1983) 527-537. 10 Grumet, M., Hoffman, S. and Edelman, G.M., Two antigenically related neuronal CAM's of different specificities mediate neuron-neuron and neuron-glia adhesion, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 267-271. 11 Grumet, M., Rutishauser, U. and Edelman, G.M., Neural cell adhesion molecule is on embryonic muscle cells and mediates adhesion to nerve cells in vitro, Nature (London), 295 (1982) 693-695. 12 Hamburger, V. and Oppenheim, R.W., Naturally occurring neuronal death in vertebrates, Neurosci. Comment., 1 (1982) 39-55. 13 Hoffman, S. and Edelman, G.M., Kinetics of homophilic binding by E and A forms of the neural cell adhesion molecule, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 5762-5766. 14 Kruse, J., Mailhammer, R., Wernecke, H., Faissner, A., Sommer, I., Goridis, C. and Schachner, M., Neural cell ad-

hesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1, Nature (London), 311 (1984) 153-155. 15 Lander, A.D., Fujii, D.K. and Reichardt, L.F., Laminin is associated with the 'neurite outgrowth-promoting factors' found in conditioned media, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 2183-2187. 16 Mathew, W.D. and Patterson, P.H., The production of a monoclonal antibody that blocks the action of a neurite outgrowth-promoting factor, Cold Spring Harbor Symp. Quant. Biol., 48 (1983) 625-631. 17 McGarry, R.C., Helfand, S.L., Quarles, R.H. and Roder, J.C., Recognition of a myelin-associated glycoprotein by the monoclonal antibody HNK-1, Nature (London), 306 (1983) 376-378. 18 McGarry, R.C., Riopelle, R.J. and Roder, J.C., Accelerated regenerative neurite formation by a neuronal surface epitope reactive with the monoclonal antibody, Leu 7, Neurosci. Lett., 56 (1985) 95-100. 19 Mobley, W.C., Schenker, A. and Shooter, E.M., Characterization and isolation of proteolytically modified Nerve Growth Factor, Biochemistry, 15 (1976) 5543-5552. 20 Purves, D. and Lichtman, J.W., Specific connections between nerve cells, Annu. Rev. Physiol., 45 (1983) 553-565. 21 Quarles, R.H., Everly, J.L. and Brady, R.O., Evidence for the close association of a glycoprotein with myelin in rat brain, J. Neurochern., 21 (1973) 1171-1191. 22 Quarles, R.H. and Pasnak, C.F., A rapid procedure for selectively isolating the major glycoprotein from purified rat brain myelin, Biochern. J., 163 (1977) 635-637. 23 Riopelle, R.J. and Cameron, D.A., Neurite growth-promoting factors of embryonic chick - - ontogeny, regional distribution and characteristics, J. Neurobiol., 12 (1981) 175-186. 24 Riopelle, R.J. and Cameron, D.A., Neurite-promoting factors from embryonic neurons, Dev. Brain Res., 15 (1984) 265-274. 25 Riopelle, R.J., Haliotis, T. and Roder, J.C., Nerve growth factor receptors of human tumours of neural crest origin: characterization of binding site heterogeneity and alteration by theophylline, CancerRes., 43 (1983) 5184-5189. 26 Sadoul, R., Hirn, M., Deagostini-Bazin, H., Rougon, G. and Goridis, C., Adult and embryonic mouse neural cell adhesion molecules have different binding properties, Nature (London), 304 (1983) 347-349. 27 Schachner, M., Faissner, A., Kruse, J., Lindner, J., Meier, D.H., Rathjen, F.G. and Wernecke, H., Cell-type specificity and developmental expression of neural cell-surface components involved in cell interactions and of structurally related molecules, Cold Spring Harbor Symp. Quant. Biol., 48 (1983) 557-568.