Cell–cell adhesion plays crucial roles in cell differentiation and morphogenesis during development of Dictyostelium discoideum. The heterophilic adhesion protein TgrC1 (Tgr is transmembrane, IPT, IG, E-set, repeat protein) is expressed during cell aggregation, and disruption of the tgrC1 gene results in the arrest of development at the loose aggregate stage. We have used far-Western blotting coupled with MS to identify TgrB1 as the heterophilic binding partner of TgrC1. Co-immunoprecipitation and pull-down studies showed that TgrB1 and TgrC1 are capable of binding with each other in solution. TgrB1 and TgrC1 are encoded by a pair of adjacent genes which share a common promoter. Both TgrB1 and TgrC1 are type I transmembrane proteins, which contain three extracellular IPT/TIG (immunoglobulin, plexin, transcription factor-like/transcription factor immunoglobulin) domains. Antibodies raised against TgrB1 inhibit cell reassociation at the post-aggregation stage of development and block fruiting body formation. Ectopic expression of TgrB1 and TgrC1 driven by the actin15 promoter leads to heterotypic cell aggregation of vegetative cells. Using recombinant proteins that cover different portions of TgrB1 and TgrC1 in binding assays, we have mapped the cell-binding regions in these two proteins to Lys537–Ala783 in TgrB1 and Ile336–Val360 in TgrC1, corresponding to their respective TIG3 and TIG2 domain.
- cell adhesion molecule
- Dictyostelium discoideum
- heterophilic interaction
- IPT/TIG domain
- mass spectrometry
How adhesive interactions between cells maintain multicellularity during morphogenesis and produce specific tissue structures and body patterns remains one of the most challenging problems in cell and developmental biology [1–3]. The social amoeba Dictyostelium discoideum has been widely used as a model organism in the study of cell–cell adhesion during development [4,5]. Dictyostelium feeds on bacteria in soil and reproduces by binary fission. When nutrients become exhausted, cells embark on a developmental cascade giving rise to aggregates of ~105 cells. Then the cell mound undergoes orchestrated cell differentiation and morphogenesis culminating in the formation of a fruiting body, where 75–80% of cells form viable spores within a sorus which is supported on the top of a stalk structure. Specific cell adhesion molecules are expressed at different developmental stages and they are critical to the integrity of cell mounds. Cell adhesion molecules also behave as morphoregulators, which generate signals required for subsequent cell differentiation [6–8].
Several adhesion systems are involved in the regulation of morphogenesis during development of Dictyostelium cells. The adhesion molecule DdCAD-1, which mediates the Ca2+-dependent cell–cell contacts, is encoded by the cadA gene and expressed soon after the initiation of development [9–12]. DdCAD-1 is involved in the formation of weak initial contacts and the recruitment of cells into cell streams during aggregation. Coinciding with the chemotactic migration stage is the expression of two Ca2+/Mg2+-independent cell adhesion systems, which are mediated by csA (also known as gp80) [13–16] and TgrC1 (also known as LagC, LagC1 and gp150; Tgr is transmembrane, IPT, IG, E-set, repeat protein) [17–19]. Both csA and TgrC1 contribute to the formation of stable cell–cell adhesion in the aggregation and post-aggregation phases of development.
Genetic manipulations have shown that cell adhesion proteins are critical to cell aggregation and pattern formation during Dictyostelium development [6–8]. Knockout of the gene encoding DdCAD-1 gives rise to abnormalities in cell sorting and cell-type proportioning, suggesting a role for DdCAD-1 in cell differentiation and morphogenesis in addition to cell aggregation [10,20]. Overexpression of csA in cells results in larger slugs and bigger fruiting bodies. The average slug size correlates with the level of DdCAD-1 and csA expression, indicating a role for adhesion molecules in regulating the stability and size of aggregates [21,22]. Expression of TgrC1 begins at the mid-aggregation stage and accumulates rapidly in the peripheral cells of the early mound structure [18,19,23]. Disruption of the tgrC1 gene leads to developmental arrest at the loose aggregate stage. Synthesis of components of the extracellular matrix and post-aggregation stage proteins is impaired and cell differentiation is blocked [18,19]. Genetic analysis has shown that the signalling pathway initiated by TgrC1 involves the comC and tgrD1 genes, with tgrC1 acting as the terminal node of this signalling network .
Whereas both DdCAD-1 and csA are homophilic binding cell adhesion proteins [9,14,25], TgrC1 mediates cell–cell adhesion via heterophilic interaction with a cell-surface receptor [18,26]. In order to elucidate the mechanism of TgrC1-mediated cell adhesion and its role in signal transduction, we have undertaken the task of identifying the heterophilic partner of TgrC1. In the present study, we demonstrate that TgrC1 binds to TgrB1 to form an adhesion complex during Dictyostelium development. Both TgrB1 and TgrC1 contain multiple IPT/TIG (immunoglobulin, plexin, transcription factor-like/transcription factor immunoglobulin) domains, which adopt an immunoglobulin-like fold and have been found in proteins involved in transcriptional regulation and signal transduction [27–29]. Using recombinant proteins in binding assays, we have demonstrated that the IPT/TIG domains in these two cell adhesion molecules participate directly in protein–protein interactions to mediate cell–cell adhesion.
Cell strains and culture conditions
The wild-type strain NC4, the axenic strain AX4 and two mutant strains, GT10 (csaA−)  and AK127 (tgrC1−) , were used. Cells were cultured on SM plates in association with Klebsiella aerogenes or grown axenically in HL5 medium at 22°C according to standard protocols . Transfectants were cultured in liquid medium containing G418 (10 μg/ml). For development, cells were collected at the mid-exponential growth phase and washed free of bacteria or medium. Cells were suspended in 17 mM sodium phosphate buffer (pH 6.4), and then spread at densities of between 5×105 and 2.5×106 cells/cm2 on non-nutrient 2% agar plates for development.
Plasmids and cell transfection
Two plasmids for ectopic expression of TgrB1 and TgrC1 driven by the actin15 promoter were constructed for cell transfection. Both plasmids contained the G418-resistance cassette for selection. AX4 cells at the mid-exponential growth phase were collected, washed twice with ice-cold H-50 buffer [20 mM Hepes, 50 mM KCl, 10 mM NaCl, 1 mM MgSO4, 5 mM NaHCO3 and 1 mM NaH2PO4 (pH 7.0)] and then suspended in H-50 buffer at 1×108 cells/ml. The cell suspension (100 μl) was transferred to an ice-cold 0.1-cm-pathlength electroporation cuvette containing 10 μg of DNA in 10 μl of H-50 buffer. Electroporation was carried out twice at 0.85 kV and 25 μF, with 5 s between pulses. After 5 min of incubation on ice, cells were suspended in 12 ml of HL5 medium and transferred into a 12-well Petri dish. G418 was added at 1 μg/ml in HL5 medium on the next day and gradually increased to a final concentration of 10 μg/ml .
Expression of recombinant proteins
DNA fragments encoding different portions of the extracellular domain of TgrB1 and TgrC1 were generated by PCR amplification and subcloned into pET22b+ or pGEX vectors between BamHI and XhoI sites. GST (glutathione transferase)–TgrC1-FP9 (72 bp) was constructed by annealing of two primers (see Supplementary Table S1 at http://www.biochemj.org/bj/452/bj4520259add.htm) and then fused to the GST cDNA. The His6-tagged proteins were expressed in BL21(DE3) Escherichia coli cells and subjected to a denaturation/renaturation process before purification on an Ni-NTA (Ni2+-nitrilotriacetic acid) agarose column . GST-fusion proteins expressed in JM101 E. coli cells were purified by glutathione–Sepharose affinity chromatography .
Preparation of rabbit antibodies against TgrB1
A mixture of equal amounts (20 mg each) of three His6-tagged TgrB1 extracellular domain fragments containing the N-terminal, middle and C-terminal regions (see Supplementary Figure S1 at http://www.biochemj.org/bj/452/bj4520259add.htm) emulsified in complete Freund's adjuvant was injected into rabbits for antibody production. The use of animals complied fully with the appropriate ethical guidelines. Boosts were carried out in incomplete Freund's adjuvant given after 3, 6 and 9 weeks. Immunoreactivity of the antiserum was initially tested by dot blot analysis using both native and denatured proteins.
Inhibition of cell reassociation and development by anti-TgrB1 antibodies
GT10 (csaA−) cells were developed for 14 h and dissociated in 20 mM EDTA in 17 mM sodium phosphate buffer (pH 6.4). Cells were resuspended at 2.5×107 cells/ml in 5 mM EDTA and 15 μl of either TgrB1 antiserum or pre-immune serum was added to 150 μl of cells. All samples were incubated for 20 min at 4°C on a platform shaker. To avoid divalent IgG-induced cell–cell association, cell samples of 30 μl each were diluted to 200 μl with 0.2 mg/ml goat anti-rabbit IgG Fab and incubated for another 20 min at 22°C on a platform shaker . Cell samples were briefly vortex-mixed and cells were allowed to reassociate on a platform shaker rotating at 180 rev./min at 22°C. The number of single cells was counted at different time points and the percentage of cell reassociation was calculated.
The effects of anti-TgrB1 antibodies on development were examined by collecting AX4 cells at 12 h of development and then mixing 0.9 ml of cells (2.5×107 cells/ml) with 100 μl of antiserum in the presence of 5 mM EDTA. Samples were incubated for 20 min at 4°C on a platform shaker. Then cells were pelleted, washed twice and resuspended in 1.2 ml of goat anti-rabbit IgG Fab (0.2 mg/ml) for another 20 min at 22°C. Next, 1×107 cells were taken from each sample and plated on to 2% plain agar in 12-well plates. Cells were allowed to develop for 24 h and the number of fruiting bodies in each well was counted. Fruiting bodies were collected and treated with 0.25 ml of 0.2% SDS. The numbers of spores collected from each well was counted using a haemocytometer.
Isolation of plasma membrane and ConA (concanavalin A)-binding glycoproteins
Plasma membrane was isolated by the aqueous two-phase polymer systems [35–37]. Plasma membranes derived from 3×109 14-h cells were solubilized in 10 ml of 0.5% Triton X-100 in PBS and centrifuged (10000 g for 15 min at 4°C) to remove insoluble material. The supernatant was loaded on to a 0.5 ml ConA-conjugated Sepharose 4B (Sigma–Aldrich) column and washed twice with PBS. Glycoproteins bound on the column were eluted with 0.3 M α-methylmannoside. Both plasma membrane and ConA-binding proteins were subjected to SDS/PAGE and far-Western blot analysis.
Far-Western blot analysis
Cell lysates were prepared from AX4 cells at different stages of development by rocking cells (3×108 cells/ml) in a lysis buffer [20 mM Tris/HCl (pH 8.0), 120 mM NaCl, 0.1 mM PMSF, 2 mM EDTA, 1 mM sodium orthovanadate, 0.5% Nonidet P40 and 1:100 dilution of a protease inhibitor cocktail (Sigma)] for 30 min at 4°C. After centrifugation at 10000 g for 15 min to remove the particulate, the supernatant was collected and prepared for SDS/PAGE under reducing conditions. Proteins were transferred on to nitrocellulose membranes and far-Western blot analysis was carried out by incubating the protein blots with the recombinant protein probe at 0.5–2.0 μg/ml for 1 h in PBS containing 4% non-fat dried skimmed milk. In peptide competition studies, a peptide corresponding to the TgrC1 heterophilic binding site (Lys338–Leu358) was mixed with the probe before adding to the nitrocellulose filter. After the removal of excess probe and extensive washing with PBS containing 0.1% Tween 20, the blots were probed with antibodies directed against either the protein probe or its tag, followed by treatment with horseradish peroxidase-conjugated secondary antibodies and the ECL (enhanced chemiluminescence) detection kit (GE Healthcare).
Cell lysates were prepared as described above, except that the concentration of Nonidet P40 was decreased to 0.3%. In His6-mediated pull-down assays, 1.2 ml of AX4 cell lysate was incubated with 20 μg of His6–TgrB1 or His6–TgrC1 for 45 min at 22°C. Then, 15 μl of Ni-NTA resin (Invitrogen) was added and the mixture was incubated for 45 min at 22°C to retrieve the His6-tagged proteins. The resin was pelleted, washed and prepared for SDS/PAGE, followed by immunoblot analysis. In GST-mediated pull-down assays, 25 μg of GST-tagged recombinant protein (TgrC1-FP9, TgrB1-TIG1, TgrB1-TIG2 and TgrB1-TIG3) was incubated with 15 μl of glutathione–Sepharose 4B beads for 30 min at room temperature. Then 1.2 ml of cell lysate or His6–TgrC1-TIG2 recombinant protein (0.5 mg/ml) was incubated with 15 μl of probe-conjugated beads for 45 min at 22°C. After incubation, the beads were pelleted, washed and prepared for SDS/PAGE, followed by immunoblot analysis.
To assess the binding of GST–TgrC1 to His6–TgrB1 fragments in solution, 5 μg of GST–TgrC1 was incubated with 5 μg of His6–TgrB1 for 1 h at 4°C in 0.5 ml of the pull-down buffer [20 mM Tris/HCl (pH 7.4), 1 mM EDTA, 100 mM NaCl and 0.1% Triton X-100]. The GST–protein complexes were retrieved using 10 μl of packed glutathione–Sepharose beads. The bound proteins were subjected to SDS/PAGE, followed by immunoblot analysis.
AX4 cells transfected with the tgrB1::tgrB1-myc plasmid had developed for 14 h before collection for cell lysate preparation. Cell lysates were prepared in the same way as those used in the pull-down assays (described above). Sepharose beads conjugated with the anti-Myc-tag monoclonal antibody 9B11 (20 μl) (Cell Signaling Technology) were added to 2 ml aliquots of cell lysate and the mixture was incubated at 4°C for 45 min. After three washes, the beads, together with the bound protein complexes, were prepared for SDS/PAGE and immunoblot analysis.
CBB (Coomassie Brilliant Blue)-stained bands were excised from gels and diced, destained with 100 μl of 50 mM ammonium bicarbonate and treated with 100 μl of 50% acetonitrile/50 mM ammonium bicarbonate. To break the disulfide bridges, the gel pieces were reduced with 100 μl of 10 mM DTT (dithiothreitol) for 30 min at 55°C, followed by alkylation with 110 mM iodoacetamide for 15 min in the dark at 22°C. Then the gel was treated with 100 μl of 50% acetonitrile/50 mM ammonium bicarbonate and digested with 130 ng of trypsin (MS grade; Roche) in 30 μl of ammonium bicarbonate at 37°C for 8 h. Peptides were extracted in turn with 20 μl of 25 mM ammonium bicarbonate, 100% acetonitrile and 5% formic acid. The extracted peptides were freeze-dried and subjected to LC-MS/MS (liquid chromatography–tandem MS).
Antibody-induced capping of cell-surface receptors
AX4 cells were collected at 16 h of development and resuspended in 20 mM EDTA and 17 mM phosphate buffer (pH 6.4), at 5×107 cells/ml. To induce clustering of TgrB1 or TgrC1 by antibody cross-linking, cell samples (200 μl) were incubated with antibodies directed against either TgrB1 or TgrC1 (1:100 dilution) for 30 min, followed by Alexa Fluor® 568-conjugated goat anti-rabbit secondary antibody (1:50 dilution) for 30 min at 22°C. Cells were fixed in 10% formaldehyde in 20 mM sodium phosphate buffer (pH 6.4) at room temperature for 15 min and then mounted for fluorescence microscopy.
To examine the binding of His6–TgrB1 to TgrC1 on the cell surface, cell samples (200 μl) were incubated with 30 μg of His6–TgrB1, followed by the anti-His6-tag monoclonal antibody (1:1000 dilution) and then Alexa Fluor® 568-conjugated goat anti-mouse secondary antibody (1:50 dilution), each for 30 min at 22°C. Cells were fixed with 4% paraformaldehyde, washed extensively and immunostained with anti-TgrC1 antibodies and Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibody before mounting for microscopy.
Cell-to-substratum binding assay
The cell-to-substratum binding assay was carried out as previously described . Petri dishes were coated with solubilized nitrocellulose (5 cm×5 cm nitrocellulose membrane in 12 ml of methanol) and then 50 μl aliquots of different GST-tagged TgrC1 extracellular fragments were placed on to spots of 1-cm diameter on the plastic surface for 90 min at room temperature. Generally, ~30% of the input protein became adsorbed on to the plastic. After removal of the unbound protein, excess binding sites were blocked by incubation with 1% BSA for 60 min. Each spot was washed three times with 17 mM sodium phosphate buffer (pH 6.4). NC4 cells were collected at 16 h of development and suspended in17 mM sodium phosphate buffer (pH 6.4), containing 20 mM EDTA to prevent the formation of aggregates. Aliquots of cells [50 μl at (2–4)×105 cells/ml] were placed on the protein-coated areas. After incubation for 30 min at 22°C, the unbound cells were removed and each spot was washed gently three times with 20 mM EDTA. The bound cells were fixed using 4% paraformaldehyde and stained for microscopic scoring. All experiments were repeated three or more times.
Identification of TgrB1 as the heterophilic partner of TgrC1
In order to identify the heterophilic binding partner of TgrC1, the recombinant protein His6–TgrC1, which contained the entire extracellular portion of TgrC1 (Met20–Gly851), was purified from bacteria (Figure 1A) and used as a protein probe in far-Western blots. Blots containing plasma membrane proteins and ConA-binding proteins derived from NC4 cells collected at 16 h of development were probed with His6–TgrC1, followed by anti-His6-tag antibodies to detect the bound protein. The antibodies detected a 130-kDa band which was highly enriched in the ConA-binding protein fraction (Figure 1B). The ConA flow-through fraction was included as a negative control and no protein band was recognized by the probe. To establish the identity of the 130-kDa glycoprotein, the corresponding band in the CBB-stained gel of ConA-binding proteins was excised, reduced and alkylated with iodoacetimide, and then digested with trypsin (Figure 1C). The peptides were extracted for LC-MS/MS. TgrB1 [accession number gi|224975602(+5)] was identified as the TgrC1 receptor with 100% probability. The tgrB1 gene is located on chromosome 3 and is situated in the opposite orientation adjacent to the tgrC1 gene linked by a common promoter of 523 bp (Figure 1D). The mature transcript of tgrB1 encodes a polypeptide of 902 amino acids. Both TgrB1 and TgrC1 contain three IPT/TIG domains in their extracellular region (Figure 1D).
Binding of recombinant TgrB1 with endogenous TgrC1
In order to confirm the TgrB1–TgrC1 interaction, a His6-tagged TgrB1 fragment (Thr45–Asp783) which contained most of the extracellular portion of TgrB1, was prepared and subjected to a variety of binding assays. In far-Western blots, His6–TgrB1 bound specifically to the endogenous TgrC1 in 16 h AX4 cell lysates (Figure 2A). The probe detected a doublet band consistent with our previous observation that cells produce a TgrB1 proteolytic fragment of 145 kDa . As negative controls, His6–TgrB1 was used to probe blots containing 0 h lysates of AX4 cells, and 0 h and 16 h lysates of AK127 (tgrC1−).
To investigate the interactions between native TgrB1 and TgrC1, pull-down studies were carried out using either His6–TgrB1 or His6–TgrC1, which was incubated with cell lysates derived from AX4 cells at 14 h of development. The His6-tagged protein complexes were pulled down by the Ni-NTA resin. Immunoblot analysis showed that TgrB1 had pulled down TgrC1 and vice versa (Figure 2B). Additionally, co-immunoprecipitation studies were carried out using AX4 transfectants which expressed TgrB1–Myc ectopically. Cell lysates were prepared from transfectants at 14 h of development and incubated with Sepharose beads conjugated with an anti-Myc monoclonal antibody. The immunoprecipitates were subjected to SDS/PAGE and immunoblot analysis and the results showed that TgrC1 co-precipitated with TgrB1–Myc (Figure 2C), suggesting that TgrB1 and TgrC1 formed complexes in these cells.
To examine whether recombinant TgrB1 was capable of binding to cell-surface-associated TgrC1, post-aggregation-stage cells were incubated with His6-tagged TgrB1. The bound His6-tagged TgrB1 protein was clustered to form large patches or caps by treatment with a mouse anti-His6-tag antibody followed by Alexa Fluor® 568-conjugated goat anti-mouse secondary antibody. Cells were subsequently fixed and incubated with anti-TgrC1 antibody and Alexa Fluor® 488-conjugated secondary antibody. Fluorescence micrographs showed the co-localization of an abundance of TgrC1 in the cap structure (Figure 2D), indicating that His6–TgrB1 bound especially to TgrC1 on the cell surface.
Co-ordinated expression of TgrB1 and TgrC1
In order to raise anti-TgrB1 antibodies for further studies, three purified recombinant protein fragments that covered the entire extracellular region of TgrB1 (see Supplementary Figure S1A) were used to immunize rabbits. The rabbit antibodies recognized all three recombinant fragments (see Supplementary Figure S1B) and the endogenous TgrB1 in cell lysates (see Supplementary Figure S1C). The fact that tgrB1 and tgrC1 are two adjacent genes linked by a common promoter suggests that their expression might be stringently co-ordinated during development. Immunoblot analysis of cell lysates derived from different stages of development revealed that the temporal expression pattern of TgrB1 was remarkably similar to that of TgrC1 . Interestingly, in AK127 (tgrC1−) cells, the expression of TgrB1 was turned on earlier than AX4 cells and began to decline after peaking at 12 h, which corresponded to the mound stage. This protein expression pattern is consistent with tgrB1 mRNA levels expressed in the tgrC1− strain during development .
Effects of anti-TgrB1 antibodies on cell cohesion and development
Since both csA and TgrC1 mediate Ca2+/Mg2+-independent cell–cell adhesion in the post-aggregation stages of development, GT10 (csaA−) cells were used to assess the effects of anti-TgrB1 antibodies on cell–cell adhesion in order to avoid complications due to the expression of csA in wild-type cells. When post-aggregation-stage cells were dissociated and rotated in the presence of anti-TgrB1 antibodies, cell reassociation was inhibited by ~60%, whereas pre-immune serum had negligible effects (Figure 3A), consistent with the role of TgrB1 as a cell adhesion protein.
Development of tgrC1− cells is arrested at the loose aggregate stage . To assess the role of the adhesive interactions between TgrB1 and TgrC1 in cell differentiation, post-aggregation-stage cells were collected and treated with an anti-TgrB1 antibody before re-depositing on 2% KK-2 agar for development to resume. Although some of the cells were capable of completing development, the number of fruiting bodies was reduced by ~70%, and spore yield showed a corresponding reduction (Figure 3B), suggesting that TgrB1 and TgrC1 are involved in terminal cell differentiation.
TgrB1 mediates cell adhesion with TgrC1
To provide in vivo evidence that TgrB1 and TgrC1 are adhesive partners, two strains, AX4(actin15::tgrB1) and AX4(actin15::tgrC1), were constructed. These two strains expressed TgrB1 and TgrC1 respectively during vegetative growth. AX4(actin15::tgrB1) cells and AX4(actin15::tgrC1) cells were collected at mid-exponential growth from liquid medium and labelled with DiO (3,3′-dioctadecyloxacarbocyanine perchlorate) and DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) (both from Invitrogen) respectively. Cells were mixed at a 1:1 ratio and rotated on a platform shaken to allow aggregate formation. Mixtures of AX4(actin15::tgrB1) and AX4(actin15::tgrC1) cells formed chimaeric aggregates and showed 40% cell aggregation at 60 min, whereas mixtures of AX4 cells with either AX4(actin15::tgrB1) or AX4(actin15::tgrC1) showed only background levels of cell association over the entire 60 min period (Figure 4).
Mapping the active binding site in TgrC1
To identify the active binding site in TgrC1, we performed cell-to-substratum binding and cell aggregation inhibition assays using GST-tagged TgrC1 extracellular fragments (Figures 5A and 5B). These recombinant proteins were attached on 1-cm-diameter spots marked out on nitrocellulose-coated plastic dishes. Wild-type cells were collected at 16 h of development and deposited on these coated spots. The number of cells attached was scored after 30 min of incubation and the results showed that FP3(Met20–Val360) functioned as efficiently as FP1(Met20–Gly851) which contained the entire TgrC1 extracellular region (Figure 5C). The inability of FP7(Met20–Val335) to elicit cell attachment suggested that the heterophilic binding site of TgrC1 resides between Ile336 and Val360, which lies within the second extracellular IPT/TIG (TIG2) domain.
When cell cohesion assays were carried out using GT10 (csaA−) cells at 16 h of development, similar results were obtained with cell reassociation inhibited by ~50% by both FP1 and FP3, whereas the other recombinant proteins had negligible effects (Figure 5D). The data thus confirmed that TgrC1 mediates cell–cell binding via sequences within its TIG2 domain.
In vitro analysis of the interaction between the cell-binding sites of TgrC1 and TgrB1
To investigate the binding specificity of the heterophilic binding site of TgrC1, the GST-fusion peptide FP9(Ile336–Val360) was synthesized for far-Western blot and pull-down assays. When GST–FP9 and GST were immobilized on nitrocellulose for far-Western analysis, His6-tagged TgrB1 recognized GST–FP9, but not GST (Figure 6A). Conversely, when GST–FP9 was used to probe immobilized His6–TgrB1, FP9 bound to TgrB1, but not BSA (Figure 6B), confirming that the binding between TgrC1 and TgrB1 is mediated by sequences within the TgrC1-TIG2 domain. In addition, far-Western blots of cell lysates were probed with GST–FP9 in the presence of a competing synthetic peptide, which spanned Lys338–Leu358 of TgrC1. Dose-dependent inhibition of the binding of GST–FP9 to the endogenous TgrB1 was observed (Figure 6C), confirming the specificity of the binding interaction. The same peptide also inhibited the binding of His6–TgrB1 to the endogenous TgrC1 in cell lysates.
To examine the interactions between GST–FP9 and TgrB1 in solution, pull-down studies were carried out using glutathione–agarose beads coupled with either GST–FP9 or GST. Cell lysates were prepared from AX4 cells at 16 h of development and then incubated with the beads. TgrB1 was pulled down from the lysate by the GST–FP9-coupled beads, but not the GST-coupled beads (Figure 6D).
Involvement of the TgrB1 IPT/TIG domain in TgrC1 binding
To map the TgrC1-binding site inTgrB1, far-Western blots were carried out using the three His6-tagged TgrB1 extracellular fragments N, M and C (see Supplementary Figure S1A) to probe cell lysates derived from AX4 cells at 0 and 18 h of development. The results showed that only fragment C recognized a band at 150 kDa in the 18-h cell lysate, corresponding to the position of TgrC1 (Figure 7A). Peptide competition experiments were performed to determine whether the fragment C was indeed bound to TgrC1. When the inhibitory peptide (TgrC1 Lys338–Leu358) was included in far-Western blots, the binding of fragment C to the 150-kDa band was abrogated (Figure 7B). The data therefore indicated that fragment C bound to TgrC1 via its interaction with the TgrC1-TIG2 domain.
Since fragment C (TgrB1 Lys537–Ala783) contains almost two entire IPT/TIG domains (TIG2, Pro603–Ser680 and TIG3, Ile704–Phe788), it is likely that the TgrB1-to-TgrC1 interaction might involve TIG-to-TIG binding. To test this hypothesis, GST-fusion proteins containing the different IPT/TIG domains of TgrB1 were prepared and used in pull-down assays. Both GST–B-TIG2 and GST–B-TIG3 pulled down the endogenous TgrC1 from cell lysates (Figure 7C, panel a). However, neither GST nor GST–B-TIG1 recognized TgrC1. In a separate pull-down experiment, all four types of conjugated glutathione beads were incubated with His6-tagged TgrC1-TIG2 (C-TIG2, Ile294–Tyr379). The immunoblot results showed that both GST–B-TIG2 and GST–B-TIG3 pulled down His6–C-TIG2 (Figure 7C, panel b). Consistently, the B-TIG3 domain was more effective in binding the C-TIG2 domain than the B-TIG2 domain.
The physical interactions between the TIG3 domain of TgrB1 and the TIG2 domain of TgrC1 suggested that these peptides should interfere with cell–cell adhesion. When GT10 (csaA−) cells were collected at 16 h of development, dissociated and allowed to reassociate in the presence of different TIG-domain-containing recombinant proteins, GST–B-TIG3 and His6–C-TIG2 exhibited the strongest inhibitory effects, both reducing cell reassociation by ~60% (Figure 7D). In control samples, neither GST nor GST–B-TIG1 interfered with cell reassociation, whereas GST–B-TIG2 exhibited an intermediate level of inhibitory effect, consistent with the results of the pull-down experiments.
In the present study, we have used both genetic and biochemical approaches to identify TgrB1 as the receptor of the heterophilic cell adhesion protein TgrC1, the first pair of heterophilic cell adhesion proteins identified in D. discoideum. TgrB1 is encoded by the gene tgrB1 which is situated adjacent to the gene tgrC1 on chromosome 3 [26,38]. These two genes are arranged in opposite directions sharing a common promoter. The promoter region contains sequences similar to those of several known cAMP-response elements [39,40]. However, tgrC1 transcription is not affected by low concentrations of cAMP pulses; it is stimulated by high concentrations of cAMP that arise in the post-aggregation stages . Microarray analysis and quantitative RT (real-time)-PCR studies show that transcription of tgrB1 and tgrC1 is initiated at low levels at aggregation stage and increases rapidly during mound formation [23,26].
The bimodal pattern of TgrB1 and TgrC1 accumulation in cells suggests that they may serve different functions at different stages during development. Although only low levels of TgrB1 and TgrC1 are detected during chemotactic migration, the study of chimaeras made up of strains showing polymorphism in TgrC1 has highlighted the role of this pair of cell adhesion proteins in selective cell recognition, which eventually results in kin discrimination . Altered residues due to single-nucleotide polymorphism may impair the TgrB1–TgrC1 interaction when mutations reside in the heterophilic binding sites of these cell adhesion proteins. Alternatively, mutations may lead to conformational changes that can affect the binding site. In order to distinguish biochemically between self and non-self cells, tgrB1 and tgrC1 encoded by the same genome must evolve together to preserve structural complementarity. The observation that cell sorting between two geographically isolated strains happens at the cell streaming stage indicates that both tgrB1 and tgrC1 have co-evolved in certain strains resulting in optimal interaction between TgrB1 and TrgC1 of the same strain, but becoming incompatible with a different strain. Cellular recognition and aggressive reactions against foreign cells occur in diverse invertebrates, which can be understood as the primitive formation of innate immune system in evolution . Therefore the cell sorting phenomenon and phagocytosis exhibited by D. discoideum are reminiscent of a primitive immune system involved in self-recognition.
During cell aggregation, both DdCAD-1 and csA/gp80 are expressed at high levels. Both tgrB1− and tgrC1− strains are capable of completing the aggregation stage [18,26], suggesting that the contribution of TgrB1 and TgrC1 to cell–cell adhesion is less significant than the other two adhesive systems. However, both knockout strains are arrested at the loose mound stage, although both csA/gp80 and DdCAD-1 are present in great abundance at this stage. It is therefore likely that TgrB1 and TgrC1 play a much more important role in cell–cell adhesion in the post-aggregation stages. Aggregates made up of the knockout cells eventually scatter, whereas some cells may re-aggregate to form small and granular mounds. Three-dimensional time-lapse microscopy reveals that tgrC1− cells exhibit random aberrant motions in aggregates as opposed to the organized rotational motion in parental cell mounds, suggesting a role for TgrC1 in the establishment of a signalling centre during mound morphogenesis .
Our previous study shows that TgrC1 is cleaved at the juxta-membrane position, releasing a 145-kDa fragment which becomes part of the extracellular matrix . The tgrC1− cells are incapable of synthesizing the extracellular matrix, which forms part of the sheath that wraps around the multicellular aggregate to prevent it from dissociation. In the absence of the sheath, the disintegration of aggregates becomes inevitable because of the random cell movements and the gradual turnover of csA/gp80.
Both tgrB1− and tgrC1− strains are arrested at a stage when TgrB1 and TgrC1 undergo rapid accumulation in cells . Immunostaining studies show that the expression of TgrC1 is more abundant in cells in the periphery of the mound [8,19], suggesting that this adhesion system may serve functions other than cell–cell adhesion. Indeed, most of the peripheral cells move to the apical region forming the prestalk zone of the slug. Consistently, the mRNA level of tgrC1 in prestalk cells is approximately 3-fold higher than prepore cells . The higher level of TgrB1/TgrC1 expression may account for the greater resistance of prestalk cells to the inhibitory effects of anti-TgrC1 antibodies. These antibodies also inhibit the sorting out of prespore and prestalk cells  and block terminal differentiation. Additionally, the loss of TgrC1 expression is accompanied by the repression of post-aggregation gene expression, resulting in the inability of mutant cells to form viable spores .
Mapping studies of the active binding sites of TgrB1 and TgrC1 have highlighted the role of the IPT/TIG domains in the interactions between this pair of adhesion molecules. Both TgrB1 and TgrC1 contain three extracellular IPT/TIG domains. These conserved domains are found across all phyla, and it has been proposed that they participate in both protein–DNA binding and protein–protein interactions . Multiple IPT/TIG domain-containing proteins are encoded by the human genome, including plexins, fibrocystin, fibrocystin-L, NF-κB (nuclear factor κB), the cell-surface receptors of Ron and Met, and the polycystic kidney/hepatic disease gene [45,46]. The structure of the IPT/TIG domain has been determined for the C-terminal DNA-binding domain of the transcription factors of the rel/dorsal/NF-κB family and it reveals a seven-stranded β-sandwich [47,48]. Two highly similar IPT/TIG domain structures have been solved for another category of human transcription factors, the early B-cell factors EBF1 and EBF3 . The IPT/TIG domains have been postulated to be involved in DNA binding and/or protein–protein interactions to mediate dimerization of transcription factors.
In a variety of developmental, regenerative and pathological processes, plexins have emerged as important regulatory proteins. The plexin transmembrane proteins are known to function as receptors of semaphorin ligands and their co-receptors include neuropilin, integrin, VEGFR2 (vascular endothelial growth factor receptor 2), ErbB2 and Met kinase . The IPT/TIG domains have been implicated in the Met and Ron receptor tyrosine kinase pathways . Deletion of IPT/TIG domains in the extracellular region of the Ron receptor tyrosine kinase causes conformational changes leading to constitutive phosphorylation and activation of downstream signals . Although the IPT/TIG domains have been implicated in protein–protein interactions, direct evidence is still lacking. In the present study, we demonstrate for the first time that IPT/TIG domains can bind to one another and that the IPT/TIG-3 domain of TgrB1 undergoes trans-interaction with the IPT/TIG-2 domain of TgrC1 leading to cell–cell adhesion. Dictyostelium cells undergo constant transitions between the formation and the breaking of cell–cell contacts during rotational movements in cell mounds and in slugs . There must be mechanisms which facilitate the rapid assembly and disassembly of adhesion complexes during morphogenesis. Although lipid rafts are known to promote the assembly of the homophilic adhesion complexes of csA/gp80, TgrB1 and TgrC1 do not reside in lipid rafts . However, glycine zipper motifs are present in the transmembrane segment of TgrC1, which may induce cis-dimerization . Whether TgrC1 dimers exist on the cell surface and how these dimers, if present, may initiate the assembly process remain to be determined. The identification of the heterophilic binding sites in TgrB1 and TgrC1 should facilitate the future analysis of the assembly of the TgrB1–TgrC1 adhesion complexes.
Gong Chen and Chi-Hung Siu conceived and planned the research, and wrote the paper. Gong Chen performed most of the experimental work. Jun Wang mapped the heterophilic binding site of TgrC1. Xiaoqun Xu made some of the mutant constructs and performed some of the pull-down experiments. Xiangfu Wu and Ruihan Piao contributed to different extents to the research.
This work was supported by an Operating Grant from the Canadian Institutes of Health Research (CIHR) [grant number FRN-6140 (to C.-H.S.)] and a research grant from the Natural Science Foundation of Shandong Province [grant number ZR2010CM067 (to X.X.)] G.C. is the recipient of a University of Toronto Open Scholarship.
We thank Dr Reinhardt Reithmeier and Dr Tony Harris for advice and invaluable suggestions and members of the Siu laboratory for discussion. We thank Mr Li Zhang and Dr Paul Taylor of the Mass Spectrometry Facility at the Advanced Protein Technology Centre of the Hospital for Sick Children for performing the MS analysis. Confocal microscopy was performed at the Microscopy Imaging Laboratory of the Faculty of Medicine, University of Toronto.
Abbreviations: CBB, Coomassie Brilliant Blue; ConA, concanavalin A; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; DiO, 3,3′-dioctadecyloxa-carbocyanine perchlorate; DTT, dithiothreitol; GST, glutathione transferase; IPT/TIG, immunoglobulin, plexin, transcription factor-like/transcription factor immunoglobulin; LC-MS/MS, liquid chromatography–tandem MS; NF-κB, nuclear factor κB; Ni-NTA, Ni2+-nitrilotriacetic acid; Tgr, transmembrane, IPT, IG, E-set, repeat protein
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