Common Cell-Surface Receptors for Discoidin I and Discoidin II in Dictyostelium discoideum

Following nutrient depletion, cells of the cellular slime mould Dictyostelium discoideum become cohesive and aggregate to form multicellular complexes. Several proteins that accumulate on the cell surface during this period have been implicated in mediating aggregative-phase cell cohesion, namely contact sites A (CsA), gp 150, and two endogenous lectins (discoidin I and discoidin II). The aggregating cells also possess receptors for both discoidin I and discoidin II but these have not yet been isolated and characterised for both lectins.
In the present study we investigated the relationship between the receptors for these lectins, in particular to what extent discoidin I and discoidin II receptors are common. Radio-iodinated discoidin I and discoidin II were purified and used in binding assays for lectin receptors on the surface of aggregated (10 h stage of development) D. discoideum NC4 cells. Sugar competition of ¹²⁵I-labelled discoidin I and ¹²⁵I-labelled discoidin II binding indicated distinct but overlapping sugar specificities for these lectins when binding to their in vivo receptors. Competition of the binding of radio-iodinated lectin with either unlabelled discoidin I or unlabelled discoidin II showed that at least 50% of the cell-surface binding sites for these lectins are in common and for these receptors the binding affinity of discoidin I is 9–20 times higher than for discoidin II. Approximately 35% of discoidin II binding sites appear to be unavailable for discoidin I binding.     

Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk-specific proteins. In addition, anterior-like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti-discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium .     

Immunochemical Analysis of Discoidins I and II at the Cell Surface in Wild Type and Aggregation-Defective Mutants of Dictyostelium discoideum

The endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two-stage specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with ¹²⁵I-Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by pread-sorption of the antiserum with discoidin-Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified ¹²⁵I-discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells. These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation-defective mutants. Strain EB-32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB-18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.     

Monoclonal antibodies against discoidin I and discoidin II of the cellular slime mold, Dictyostelium discoideum

The preparation and properties of monoclonal antibodies against carbohydrate-binding proteins (discoidin I and discoidin II) in the cellular slime mold, Dictyostelium discoideum are described. Monoclonal antibody (mAb) ndI,II-1 bound both discoidins I and II specifically. mAb nI-1 and mAb dI-1 bound only discoidin I but their binding specificities were different: nI-1 recognized the native form and dI-1 the denatured form. mAb dII-1 bound only denatured discoidin II. In preliminary work mAbs dII-1 and nI-1 were found to be useful for localizing discoidins I and II immunohistochemically.     

Discoidin I-membrane interactions I. Discoidin I binds to two types of receptor on fixed Dictyostelium discoideum cells

Under physiological buffer conditions (17 mM P_i, pH 6.3), the endogenous lectin of Dictyostelium discoideum, discoidin I, binds to two types of receptors on the surface of glutaraldehyde-fixed, wild-type (NC-4) D. discoideum cells. We have designated these two types of receptors the carbohydrate or C sites and the ionic or I sites. Binding to the C sites is saturable with respect to discoidin I and is inhibited by hapten sugars (such as $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{galactosamine}$), but not by increasing buffer ionic strength with NaCl or polyelectrolytes. The number of C sites increases about 4-fold during the first 8.5 h of suspension differentiation, reaching a capacity for about 2–10⁴ discoidin I tetramers per cell. The binding activity of the C sites is reduced about 50% by sequential NaIO₄ oxidation/NaBH₄ reduction of the fixed cells, but it is not reduced by CHCl₃-CH₃OH extraction of the fixed cells. In marked contrast, binding to the I sites appears nonsaturable with respect to discoidin I, and it is inhibited by increasing buffer ionic strength with NaCl or polyelectrolytes (such as poly-l-glutamic acid or heparin), but not by hapten sugars. The I sites are present on both vegetative and differentiated fixed cells and can bind more than 10⁶ discoidin I tetramers per cell. The binding activity of the I sites on fixed cells is not reduced by sequential NaIO₄ oxidation/NaBH₄ reduction, but is reduced 70 to 90% by CHCl₃-CH₃OH extraction. The data suggest that the I sites represent ionic lipids that bind discoidin I electrostatically.     

Molecular analysis of collagen binding by the human discoidin domain receptors,DDR1 and DDR2. Identification of collagen binding sites in DDR2

The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site.     

Mota1552, a Mutation of Dictyostelium Discoideum Having Pleiotropic Effects on Motility and Discoidin I Regulation

The Dictyostelium discoideum mutant MC2 exhibits temperature-sensitive growth, temperature-sensitive motility, and temperature induction of discoidin I synthesis. These three phenotypes of MC2 were not separated in the genetic experiments reported here. They were therefore assigned to the mutation motA1552, which was mapped to linkage group II by segregation analysis and by analysis of mitotic recombinant diploids. In one motA1552 strain, loss of motility preceded accumulation of discoidin I by 3 hr, indicating that discoidin I is not involved in generation of the motility defect. Expression of motA1552 phenotypes varied both among strains carrying the mutation, and among different clones of a particular strain. MC2 and its derivatives displayed elevated levels of recombination between whiA and acrA on linkage group II, and yielded highly unstable mutations at the acrA locus. Accumulation of large amounts of discoidin I during axenic growth of strain AX3 was found to depend on the presence of a second linkage group II mutation, daxA1551. This mutation was already present in the strain mutagenized to isolate motA1552, complicating explication of motA1552 action.     

Receptor for the cell binding site of discoidin I

Discoidin I, a developmentally regulated lectin in Dictyostelium discoideum, has been implicated in cell-substratum adhesion and ordered cell migration during aggregation. This depends on the cell binding site of discoidin I, which is distinct from its carbohydrate binding site. We have isolated a receptor for the cell binding site by affinity chromatography. The receptor binds immobilized discoidin I in the presence of 0.3 M galactose and can be eluted with gly-arg-gly-asp-his-asp, a synthetic peptide the sequence of which is found in discoidin I, and which blocks cell migration into aggregates. The receptor is a developmentally regulated cell-surface glycoprotein of apparent Mr approximately 67,000. Univalent antibodies specific for this glycoprotein block aggregation.     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

Molecular Cloning and Characterization of the cDNA for Discoidin II of Dictyostelium discoideum

By using monoclonal antibodies directed against discoidin II,we have isolated cDNA clones from axenically grown Ax-2 cells.On cDNA clone (D2) condtained a 1.2-k.b insert encoding theentire discoidin II protein, which is conposed of 257 aminoacid residuces and has a calculated molecular mass of 28,574.The amino acid sequences, determined by Edman degradation ofsix tryptic peptides of discoidin II, were identical to thosededuced from the cDNA sequences. The protein bears no resemblanceto any proteins in the data banks, except that its sequenceis 49% identical with the amino acid sequence of discoidin I.Discoidin II shares with discoidin I both a carbohydratebindingsite and an Arg-Gly-Asp (RGD) sequence, which has been foundin fibronectin in mammalian cells. With the onset of aggregation(8 h of development), a 1.3-kb discoidin II mRNA begins to accumulate.A similar pattern of regulation occurs at the protein level. ¹Present address: MRC Laboratory for Molecular Cell Biology,University College London, Gower Street, London, WC1E 6BT UnitedKingdom     

Discoidin I and discoidin II are localized differently in developing Dictyostelium discoideum

The distribution of discoidin I and discoidin II, developmentally regulated lectins in Dictyostelium discoideum, was determined immunohistochemically at various stages of development. Discoidin I was first prominent as focal clumps in aggregating cells, then accumulated on the surface of aggregates and around them. Discoidin II became prominent later and ultimately localized in what appear to be prespore vesicles. The results indicate that discoidin I and discoidin II have different and possibly multiple functions.     

Reconstitution of a pentameric complex of dimeric transforming growth factor beta ligand and a type I,II, III receptor in baculoviral-infected insect cells

Summary Two transmembrane serine-threonine kinases (type I and II receptors), a membrane-anchored proteoglycan (type III), and a homodimeric ligand participate in the transforming growth factor beta type on (TGFβ1) signal transduction complex. The expression of recombinant receptors in insect cells co-infected with up to three recombinant baculoviruses was employed to study interactions among the ectodomains of the three types of receptors and the TGFβ1 ligand in absence of uncontrollable extrinsic factors in mammalian cells. Multi-subunit complexes were assembled in intact cells and purified on glutathione-conjugated beads for analysis by tagging one of the subunits with glutathione S-transferase (GST). Intrinsic ligand-independent interactions were observed among receptor subunits as follows: type III–III type I–I, type III-I, and type II-I. The homeotypic complex of type II–II receptors and the heterotypic type III-II interaction was ligand dependent. The type I, but not the type III, subunit displaced about 50% of the type II component in either ligand-dependent homomeric type II-type II complexes or heteromeric type III-type II complexes to form type II-I or type III-II-I oligomers, respectively. The type II subunit displaced type I subunits in oligomers of the type I subunit. Specificity of type I receptors may result from differential affinity for the type II receptor rather than specificity for ligand. A monomeric subunit of the TGFβ1 ligand bound concurrently to type III and type II or type III and type I receptors, but failed to concurrently bind to the type II and type I subunits. The binding of TGFβ1 to the type I kinase subunit appears to require an intact disulfide-linked ligand dimer in the absence of a type III subunit. The combined results suggest a pentameric TGFβ signal transduction complex in which one unit each of the type III, type II, and type I components is assembled around the two subunits of the dimeric TGFβ1 ligand. An immobilized GST-tagged subunit of the receptor complex was utilized to assemble multi-subunit complexesin vitro and to study the phosphorylation events among subunits in the absence of extrinsic cell-derived kinases. The results revealed that (a) a low level of ligand-independent autophosphorylation occurs in the type I kinase; (b) a high level of autophosphorylation occurs in the type II kinase; (c) both the type III and type I subunits aretrans-phosphorylated by the type II subunit; and (d) the presence of both type I and II kinases complexed with the type III subunit and dimeric TGFβ1 ligand in a pentameric complex causes maximum phosphorylation of all three receptor subunits.     

Discoidin-binding polysaccharide from Dictyostelium discoideum

Extracts of Dictyostelium discoideum grown axenically in a chemically defined medium were evaluated for binding to discoidin I and discoidin II, endogenous lectins of this slime mold. Binding activity was measured by competitive inhibition of 125I-lactosyl-bovine serum albumin binding to the immobilized lectins. With the solubilization procedure used extracts of vegetative cells and of early aggregates had no significant inhibitory activity, but an abundant discoidin-binding substance was detected in late aggregates and fruiting bodies. This material was purified by ethanol and acid precipitation followed by precipitation with discoidin. It is a polysaccharide composed of 77% galactose, 15% N-acetylgalactosamine, 5% glucose, and 3% N-acetylglucosamine and may be a biologically functional ligand for the slime mold lectins, in particular discoidin II. Use of axenic cells was critical in these experiments, since extracts of Escherichia coli and Klebsiella aerogenes, commonly used as food for D. discoideum, were found to contain substances that react with discoidin. This would complicate isolation of endogenous discoidin ligands from cells raised on bacteria.     

Monoclonal antibody against cytoplasmic lectins of Dictyostelium discoideum: cross-reactivity with a membrane glycoprotein, contact site A, and with E. coli beta-galactosidase and lac repressor

Monoclonal antibodies were raised against two soluble, galactose-binding lectins from cells of Dictyostelium discoideum, discoidin I and II. These antibodies reacted not only with both discoidins, but also with a plasma membrane glycoprotein of aggregation competent cells, called contact site A, and with two carbohydrate-binding proteins of E. coli, beta-galactosidase and lac repressor. The possibility that the antibody recognizes a structure common to different carbohydrate-binding proteins is discussed. The two carbohydrate-binding proteins of E. coli share with discoidin I the sequence -Ser-X-X-Ile-His(Pro)-Pro(His)-Leu-Thr- which might be responsible for the cross-reactivity.     

Assay and characterization of carbohydrate binding by the lectin discoidin I immobilized on nitrocellulose

Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of 125I-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 degrees C stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Discoidin I Gene Family of Dictyostelium Discoideum Is Linked to Genes Regulating Its Expression

The discoidin I protein has been studied extensively as a marker of early development in the cellular slime mold Dictyostelium discoideum. However, like most other developmentally regulated proteins in this system, no reliable information was available on the linkage of the discoidin genes to other known genes. Analysis of the linkage of the discoidin I genes by use of restriction fragment length polymorphisms revealed that all three discoidin I genes as well as a pseudogene are located on linkage group II. This evidence is consistent with the discoidin I genes forming a gene cluster that may be under the control of a single regulatory element. The discoidin I genes are linked to three genetic loci (disA, motA, daxA) that affect the expression of the discoidin I protein. Linkage of the gene family members to regulatory loci may be important in the coordinate maintenance of the gene family and regulatory loci. A duplication affecting the entire discoidin gene family is also linked to group II; this appears to be a small tandem duplication. This duplication was mapped using a DNA polymorphism generated by insertion of the Tdd-3 mobile genetic element into a Tdd-2 element flanking the gamma gene. A probe for Tdd-2 identified a restriction fragment length polymorphism in strain AX3K that was consistent with generation by a previously proposed Tdd-3 insertion event. A putative duplication or rearrangement of a second Tdd-2 element on linkage group IV of strain AX3K was also identified. This is the first linkage information available for mobile genetic elements in D. discoideum.     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Colocalization of discoidin-binding ligands with discoidin in developing Dictyostelium discoideum

The Dictyostelium discoideum lectins, discoidin I and discoidin II, and the endogenous ligands to which they bind were immunohistochemically localized in sections of this organism at successive stages of development. For these studies, an axenic strain, AX3, was grown in a macromolecule-depleted medium rather than on bacteria, which themselves contain discoidin-binding ligands. Discoidin I-binding sites (endogenous ligands) in sections of D. discoideum were concentrated in the slime coat around aggregates, whereas discoidin II-binding sites were observed in a vesicle-like distribution in prespore cells and also in spore coats. In contrast, discoidin II did not bind to the slime coat and discoidin I bound relatively poorly to prespore cells and spore coats. The distributions of the endogenous lectins themselves were the same in axenically grown cells as previously reported for cells raised on bacteria. Discoidin I was concentrated in the slime coat and around stalk cells, and discoidin II was prominent in and around prespore cells. The congruent localization of each lectin with its endogenous ligand suggests that discoidin I normally functions in association with glycoconjugates in the slime around aggregates, and discoidin II with the galactose-rich spore coat polysaccharide.     

Induction of the insulin receptor and other differentiation markers by sodium butyrate in the Burkitt lymphoma cell, Raji

The very low expression of insulin receptors in the Burkitt lymphoma cell Raji was increased 2-fold, 6-fold and 10-fold after 1, 2 and 3 days, respectively, by incubation with the differentiation inducer sodium butyrate. Insulin receptor number was increased without a change in receptor affinity, in association with an increase in the receptor alpha and beta subunits detected after cell-surface labelling and immunoprecipitation. Expression of cell-surface class I and II human leukocyte antigens, the intercellular adhesion molecule-1 and the CD38 leukocyte antigen was also increased, consistent with B cell differentiation. Butyrate effects were not unspecific, as the binding of tumour necrosis factor and growth hormone and the expression of the B cell markers CD20, B5 and CD21 was not increased. The low expression of insulin receptors on Raji cells is therefore a reflection of the less differentiated state of these cells compared to lymphoblastoid cells.