Cytoskeleton interactions involved in the assembly and function of glycoprotein-80 adhesion complexes in dictyostelium

Adhesion complexes typically assemble from clustered receptors that link to the cytoskeleton via cytoplasmic adapter proteins. However, it is unclear how phospholipid-anchored adhesion molecules, such as the Dictyostelium receptor gp80, interact with the cytoskeleton. gp80 has been found to form adhesion complexes from raftlike membrane domains, which can be isolated as a Triton X-100-insoluble floating fraction (TIFF). We report here that the actin-binding protein ponticulin mediates TIFF-cytoskeleton interactions. Analysis of gp80-null cells revealed that these interactions were minimal in the absence of gp80. During development, gp80 was required to enhance these interactions as its adhesion complexes assembled. Whereas ponticulin and gp80 could partition independently into TIFF, gp80 was shown to recruit ponticulin to cell-cell contacts and to increase its partitioning into TIFF. However, these proteins did not co-immunoprecipitate. Furthermore, sterol sequestration abrogated the association of ponticulin with TIFF without affecting gp80, suggesting that sterols may mediate the interactions between ponticulin and gp80. In ponticulin-null cells, large gp80 adhesion complexes assembled in the absence of ponticulin despite the lack of cytoskeleton association. We propose that such nascent gp80 adhesion complexes produce expanded raftlike domains that recruit ponticulin and thereby establish stable cytoskeleton interactions to complete the assembly process.     

Assembly of glycoprotein-80 adhesion complexes in Dictyostelium. Receptor compartmentalization and oligomerization in membrane rafts

The phospholipid-anchored membrane glycoprotein (gp)-80 mediates cell-cell adhesion through a homophilic trans-interaction mechanism during Dictyostelium development and is enriched in a Triton X-100-insoluble floating fraction. To elucidate how gp80 adhesion complexes assemble in the plasma membrane, gp80-gp80 and gp80-raft interactions were investigated. A low density raft-like membrane fraction was isolated using a detergent-free method. It was enriched in sterols, the phospholipid-anchored proteins gp80, gp138, and ponticulin, as well as DdCD36 and actin, corresponding to components found in the Triton X-100-insoluble floating fraction. Chemical cross-linking revealed that gp80 oligomers were enriched in the raft-like membrane fraction, implicating stable oligomer-raft interactions. However, gp80 oligomers resisted sterol sequestration and were partially dissociated with Triton X-100, suggesting that compartmentalization in rafts was not solely responsible for their formation. The trans-dimer known to mediate adhesion was identified, but cis-oligomerization predominated and displayed greater accumulation during development. In fact, oligomerization was dependent on the level of gp80 expression and occurred among isolated gp80 extracellular domains, indicating that it was mediated by direct gp80-gp80 interactions. Rafts existed in gp80-null cells and such pre-existent membrane domains may provide optimal microenvironments for gp80 cis-oligomerization and the assembly of adhesion complexes.     

The membrane glycoprotein gp150 is encoded by the lagC gene and mediates cell-cell adhesion by heterophilic binding during Dictyostelium development

gp150 is a membrane glycoprotein which has been implicated in cell-cell adhesion in the postaggregation stages of Dictyostelium development. An analysis of its tryptic peptides by mass spectrometry has identified gp150 as the product of the lagC gene, which was previously shown to play a role in morphogenesis and cell-type specification. Antibodies raised against the GST-LagC fusion protein specifically recognized gp150 in wild-type cells and showed that it is missing in lagC-null cells. Immunolocalization studies have confirmed its enrichment in cell-cell contact regions. In mutant cells that lack the aggregation stage-specific cell adhesion molecule gp80, gp150 is expressed precociously. Moreover, these cells acquire EDTA-resistant cell-cell binding during aggregation, suggesting a role for gp150 in this process. Cells in which the genes encoding gp80 and gp150 are both inactivated do not acquire EDTA-resistant cell adhesion during aggregation. Strains transformed with an actin 15::lagC construct express gp150 precociously, but do not show EDTA-resistant adhesion during early development. However, vegetative cells expressing gp150 can be recruited into aggregates of 16-h lagC-null cells. These results, together with those obtained with the cell-to-substratum binding assay, indicate that gp150 mediates cell-cell adhesion via heterophilic interactions with another component that accumulates during the aggregation stage.     

Molecular mechanisms of cell-cell interaction in Dictyostelium discoideum

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in response to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr80,000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell-cell adhesion. One of them has a Mr of 150,000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell-cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.     

Mutations affecting a surface glycoprotein, gp80, of Dictyostelium discoideum.

We isolated two independent mutations in Dictyostelium discoideum that result in the absence of the antigenic determinant recognized by monoclonal antibody E28D8. This antibody reacts with a post-translational modification on the surface glycoprotein gp80 and several other proteins. Both of the mutations occur in the same locus, modB, which was mapped to linkage group VI. The modB mutations result in sufficient alteration of gp80 that it is absent or unrecognizable by two-dimensional gel electrophoresis. Strains carrying modB mutations exhibit "contact sites A"-mediated cell-cell adhesion although more weakly than do wild-type strains and develop to fruiting bodies carrying viable spores. Although gp80 has been implicated in the mechanism of cell-cell adhesion in D. discoideum, it is clear from the behavior of these mutant strains that the determinant on gp80 recognized by E28D8 is not necessary for either morphogenesis or reduced EDTA-resistant adhesion.     

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.     

Adhesion-blocking antibodies prepared against gp150 react with gp80 of Dictyostelium

A membrane glycoprotein of 150000 D, gp150, has been implicated in the mechanism of cell-cell adhesion which arises during development of Dictyostelium discoideum. This conclusion was founded on the observation that monovalent Fab′ fragments prepared from an antiserum raised against partially purified gp150 are able to block cell-cell adhesion. We show that this serum contains antibodies to a distinct membrane glycoprotein, gp80, previously implicated in cell-cell adhesion. Reaction of Fab′ to this surface molecule can account for the adhesion-blocking activity in the antiserum to gp150. Moreover, binding of gp80 neutralized Fab′ to gp150 does not block adhesion. If gp150 carries other determinants which bind adhesion-blocking Fab′, these determinants must also be present on gp80. Thus, it is not clear that gp150 is directly involved in cell-cell adhesion of Dictyostelium.     

Phagocytosis in Dictyostelium discoideum is inhibited by antibodies directed primarily against common carbohydrate epitopes of a major cell-surface plasma membrane glycoprotein

Using a water-soluble, reversible biotinylating reagent, we retrieved three surface-exposed proteins from a complex mixture of crude membrane proteins. The compound, sulfosuccinimidyl 2-(biotinamido)ethyl-1-3'-dithiopropionate (sulfo-NHS-SS-biotin), which has a cleavable disulfide bond, was used to label Dictyostelium discoideum amebae. Cells were lysed and a crude membrane preparation was isolated and solubilized with Triton X-100. Biotinylated molecules were bound to immobilized streptavidin and then eluted from the affinity matrix with dithiothreitol. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that out of the original complex mixture of detergent-solubilized membrane proteins, three major species at 130, 100, and 77 kDa were specifically bound and eluted with thiol reagents. These three proteins were glycoproteins (gp) since they bound concanavalin A. As demonstrated by one-dimensional peptide mapping, the retrieved gp130 and gp100 also were present in specialized plasma membrane subdomains called contact regions which are regions of cell-cell cohesion isolated from aggregated, developed amebae. This finding provides preliminary evidence that the two proteins may be involved in cell-cell interactions during both the vegetative and aggregation stages of the D. discoideum life cycle. The retrieved gp130 species has a relative mobility on SDS-gels similar to that of gp126, a surface-exposed glycoprotein. gp126 has been suggested to play roles both as a phagocytosis receptor and as a cohesion molecule (C.M. Chadwick, J.E. Ellison, and D.R. Garrod, (1984) Nature (London) 307, 646). To test if the retrieved gp130 was the same as gp126, a polyclonal antiserum was raised against gel-purified, endoglycosidase F-treated gp130. The immune serum recognized epitopes, apparently carbohydrates, present on many D. discoideum membrane proteins. Univalent IgG fragments from this antiserum inhibited phagocytosis, suggesting that anti-carbohydrate activity was responsible for the functional inhibition of phagocytosis.     

Cell-cell adhesion in Dictyostelium discoideum

Three separate mechanisms of cell-cell adhesion have been shown to appear at different stages of development in Dictyostelium discoideum. During the first few hours of development, the cells synthesize and accumulate a glycoprotein of 24,000 daltons (gp24) that is positioned in the membrane. The time of appearance of gp24 correlates exactly with the time of appearance of cell-cell adhesion in two strains in which temporal control varies by several hours. Antibodies specific to gp24 are able to block cell-cell adhesion during the first few hours of development but not during later development. By 8 hr of development, another glycoprotein, gp80, that is not recognized by antibodies to gp24 accumulates on the surface of cells. This membrane protein mediates an independent adhesion mechanism during the aggregation stage that is resistant to 10 mM EDTA. Antibodies specific to gp80 can block EDTA-resistant adhesion during this stage. During subsequent development, gp80 is removed from the cell surface and replaced by another adhesion mechanism that is insensitive to antibodies to either gp24 or gp80. A lambda gt11 expression vector carrying a Dictyostelium cDNA insert was isolated that directs the synthesis of a fusion protein recognized by antibodies specific to gp24. This cDNA was used to probe a genomic library. A clone carrying a 1.4-kb insert of genomic DNA was recognized by the cDNA and shown to hybridize to a 0.7-kb mRNA that accumulates early in development. This unusually small RNA could code for the small protein, gp24. Southern analysis of restriction fragments generated by various enzymes on Dictyostelium DNA with both the cDNA and genomic clones indicated the presence of two tandem copies of the gene. This may account for the failure to recover mutations resulting in the lack of gp24. Mutations have been recovered that result in the lack of accumulation of gp80, and cells carrying these mutations have been shown to be missing the second adhesion mechanism. These mutant strains are able to complete development because the other adhesion mechanisms are not impaired. Sequential addition of adhesion mechanisms provides a means for the formation of multicellular organisms from previously solitary cells.     

Cell-cell adhesion molecules in Dictyostelium

Multicellularity in the cellular slime mold Dictyostelium discoideum is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive adhesion sites are expressed very early in the development cycle and a surface glycoprotein of 24,000 Da is known to be responsible for these sites. The EDTA-resistant contact sites begin to accumulate on the cell surface at the aggregation stage of development. Several glycoproteins have been implicated in the EDTA-resistant type of cell-cell binding and the best characterized one has an Mr of 80,000 (gp80). gp80 mediates cell-cell binding via homophilic interaction and its cell binding site has been mapped to an octapeptide sequence. The mechanism by which gp80 mediates cell-cell adhesion will be discussed.     

Filopodia are enriched in a cell cohesion molecule of Mr 80,000 and participate in cell-cell contact formation in Dictyostelium discoideum

During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodial surfaces had a twofold higher gold density. Both TEM and scanning electron microscopy showed that contact areas were enriched in filopodial structures. Filopodia often appeared to adhere to either smooth surfaces or similar filopodial structures of an adjacent cell. These observations suggest that the formation of stable cell-cell contacts involves at least four sequential steps in which filopodia and gp80 probably play an important role in the initial stages of recognition and cohesion among cells.     

Regulation of spatiotemporal expression of cell-cell adhesion molecules during development of Dictyostelium discoideum

The social amoeba Dictyostelium discoideum is a simple but powerful model organism for the study of cell-cell adhesion molecules and their role in morphogenesis during development. Three adhesive systems have been characterized and studied in detail. The spatiotemporal expression of these adhesion proteins is stringently regulated, often coinciding with major shifts in the morphological complexity of development. At the onset of development, amoeboid cells express the Ca(2+) -dependent cell-cell adhesion molecule DdCAD-1, which initiates weak homophilic interactions between cells and assists in the recruitment of individuals into cell streams. DdCAD-1 is unique because it is synthesized as a soluble protein in the cytoplasm. It is targeted for presentation on the cell surface by an unconventional protein transport mechanism via the contractile vacuole. Concomitant with the aggregation stage is the expression of the contact sites A glycoprotein csA/gp80 and TgrC1, both of which mediate Ca(2+) /Mg(2+) -independent cell-cell adhesion. Whereas csA/gp80 is a homophilic binding protein, TgrC1 binds to a heterophilic receptor on the cell. During cell aggregation, csA/gp80 associates preferentially with lipid rafts, which facilitate the rapid assembly of adhesion complexes. TgrC1 is synthesized at low levels during aggregation and rapid accumulation occurs initially in the peripheral cells of loose mounds. The extracellular portion of TgrC1 is shed and becomes part of the extracellular matrix. Additionally, analyses of knockout mutants have revealed important biological roles played by these adhesion proteins, including size regulation, cell sorting and cell-type proportioning.     

Mediation of cell-cell adhesion by the altered contact site A glycoprotein expressed in modB mutants of Dictyostelium discoideum

In Dictyostelium discoideum, a surface glycoprotein with Mr 80,000 (gp80) has been found to mediate the EDTA-resistant contact sites A at the aggregation stage of development. To evaluate the role of the carbohydrate moiety in cell-cell adhesion, we have examined the accumulation and activity of an altered gp80 molecule in two glycosylation (modB) mutants. Both mutants synthesize an altered gp80 of lower molecular size. This modB-gp80 can be detected by the monoclonal antibody 80L5C4, which is capable of blocking cell-cell adhesion (C. -H. Siu, T. Y. Lam, and A. Choi, (1985) J. Biol. Chem. 260, 16,030-16,036). The mutant cells exhibit both EDTA-sensitive and EDTA-resistant types of cell-cell binding, though to a lesser extent than that of the parental strain, and the EDTA-resistant binding sites are blocked in the presence of 80L5C4 Fab. Mutant cells can also bind Covaspheres conjugated with gp80. These results suggest that the modB-gp80 protein still retains the domain essential for its cell binding activity and the carbohydrate moiety affected by the modB mutation is not directly involved in cell-cell adhesion.     

Inhibition of cell-cell binding at the aggregation stage of Dictyostelium discoideum development by monoclonal antibodies directed against an 80,000-dalton surface glycoprotein

Monoclonal antibodies were prepared against a putative cell-cell adhesion molecule, a surface glycoprotein with an apparent Mr of 80,000 (gp80), from Dictyostelium discoideum. Seven monoclonal antibodies directed against gp80 were characterized and found to fall into three distinct classes. Class I consisted of one monoclonal antibody, is monospecific for gp80, and probably recognizes the peptide portion of the molecule. This class was capable of blocking the EDTA-resistant contact sites effectively. Class II recognized the carbohydrate moiety of gp80 and cross-reacted with a large number of glycoproteins. These monoclonal antibodies partially inhibited cell reassociation. Class III recognized gp80 and one other glycoprotein of Mr 95,000. This class had no effect on cell-cell binding. The class I monoclonal antibody was most potent in inhibiting cell reassociation at the aggregation stage of development. Its effect decreased drastically as development progressed and became negligible by the culmination stage. These observations are consistent with a direct role of gp80 in cell-cell binding and suggest a transient function for gp80 at the aggregation stage.     

Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum

At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.     

Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum

A membrane glycoprotein of 24,000 Da (gp24) was purified from developed cells of Dictyostelium discoideum and shown to neutralize a crude antiserum (R695) that blocks EDTA-sensitive cell-cell adhesion during the early developmental stages of this organism. Purified gp24 was used to raise rabbit polyclonal antibodies and mouse monoclonal antibodies. Rabbit antiserum R851 was shown to be highly specific to gp24 by both Western analysis and immunoprecipitation. IgG of R851 is able to block adhesion of dissociated cells swirled in suspension. Adhesion of wild-type cells is blocked by R851 antibodies during the first 8 hr of development but not thereafter when other adhesion mechanisms come into play. The glycoprotein gp80 plays an essential role in the second adhesion system that appears during the aggregation stage of D. discoideum. By adding both anti-gp24 and anti-gp80 antibodies, adhesion of aggregation stage cells could be blocked. Late in development a third adhesion mechanism appears that is not blocked by either antibodies to gp24 or gp80 or both antibodies together. Western analysis and immunoprecipitation with monoclonal antibody mLJ11, specific for gp24, indicated that gp24 is absent in cells growing exponentially on bacteria but is rapidly synthesized and accumulated following the initiation of development. Synthesis of gp24 is maximal during the first 4 hr of development and then continues at a reduced rate throughout the remainder of development. The coordinate appearance of gp24 and EDTA-sensitive cell-cell adhesion as well as the ability of this glycoprotein to neutralize the adhesion blocking activity of R695 and R851 antibodies indicates that it plays a role in early cell-cell adhesion.     

Immunological analysis of a glycoprotein (contact sites A) involved in intercellular adhesion of dictyostelium discoideum

We have prepared antisera in rabbits to the “contact sites A” glycoprotein (gp80) purified from Dictyostelium discoideum. IgG isolated from these anti-sera reacts with a number of different proteins in D discoideum lysates, as analyzed by immune precipitation and by antibody staining of gel electropherograms transferred to nitrocellulose. Blocking experiments indicate that this cross-reactivity reflects the presence of common antigeneic determinants on gp80 and other cellular proteins, rather than the presence of extraneous antibodies in the antisera. The spectrum of reactive proteins is different a: different stages of development. In particular, gp80 itself is synthesized only for a restricted period during the cell aggregation phase. The protein persists throughout development and can be detected in spores. Anti-gp80 Fab fragments bind to the surface of developing D discoideum cells and specifically block their developmentally regulated adhesion. After absorption with vegetative cells, the IgG stains only gp80 and (to a lesser extent) one other band in lysates of aggregation-competent cells. The absorbed antibodies also can block adhesion. Several proteins that appear late in development also arc stained by the absorbed IgG.     

盘基网柄菌发育中的细胞粘附分子及其信号转导

在盘基网柄菌发育早期,DdCAD-1和csA调节了变形虫细胞间的粘着,调控该过程的机制类似于胚胎发育中上皮细胞层的闭合。完成网柄菌发育的一个必需分子是gpl50异嗜性粘附分子。盘基网柄菌β-连环蛋白同源物Aardvark(Aar)的缺乏使细胞间失去粘着连接,Aar也有信号转导功能,调控了前孢子细胞基因的表达。因此,细胞间的粘着是盘基网柄菌发育的一个重要组成部分,并与调控形态发生过程的信号转导有密切相互作用关系。     

Regulation of cell-cell adhesion during Dictyostelium development

During development of Dictyostelium, four adhesion systems have been identified and adherens junction-like structures have been discovered in the fruiting body. The temporal and spatial expression of cell adhesion molecules (CAMs) is under stringent developmental control, corresponding to major shifts in morphological complexity. Genetic manipulations, including over-expression and knockout mutations, of the adhesion genes, cadA (encoding DdCAD-1), csaA (gp80) and lagC (gp150), have shed light on new roles for cell adhesion molecules in aggregate size regulation, cell-type proportioning, cell differentiation and cell sorting. As cell-cell interactions remain highly dynamic within cell streams and aggregates, mechanisms must exist to facilitate the rapid assembly and disassembly of adhesion complexes. Studies on gp80 have led to a model for the rapid assembly of adhesion complexes via lipid rafts.     

Cell-cell adhesion and morphogenesis in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire EDTA-resistant cell-cell adhesion at the aggregation stage. The EDTA-resistant cell binding activity is associated with a cell surface glycoprotein of Mr 80,000 (gp80), which mediates cell-cell binding via homophilic interaction. Analysis of the structure of gp80 deduced from cDNA sequence reveals the presence of three internally homologous segments in the NH2-terminal domain, which also contains regions with homology to the neural cell adhesion molecule. Secondary structure predictions show an abundance of beta-structures and very few alpha-helices. This is confirmed by circular dichroism measurements. It is likely that the homologous segments are organized into globular structures, extended from the cell surface by a Pro-rich stalk domain. The cell binding activity of gp80 resides within the first globular repeat of the NH2-terminal domain and has been mapped to a 51 amino acid region between Val123 and Leu173. Synthetic oligopeptides corresponding to sequences within this region have been prepared and assayed for their ability to bind to cell surface gp80. Results lead to identification of the homophilic binding site to an octapeptide sequence within this region. Synthetic peptides containing this octapeptide sequence and univalent antibodies directed against this site block the formation of organized cell streams during aggregation. Although cell aggregates are eventually formed, most fail to undergo further development to give rise to slugs and fruiting bodies, indicating that cell-cell adhesion involving gp80 is an important step in normal morphogenesis.