Molecular cloning and expression of Pgp-1. The mouse homolog of the human H-CAM (Hermes) lymphocyte homing receptor

Mouse phagocytic glycoprotein-1 (Pgp-1; Ly-24) is a 95-kDa glycoprotein of unknown function that has served as an important T cell/leukocyte differentiation marker. Recent work has suggested that it may be related to a human 85- to 95-kDa glycoprotein (termed variously the Hermes Ag/lymphocyte homing receptor, ECMRIII, P80, and CD44) that is involved in lymphocyte binding to high endothelial venules in the process of lymphocyte homing, and has been implicated in other cell adhesion events. The widespread expression of this molecular class in diverse organ systems suggests a broad role in cellular adhesion, and has led to the unifying designation homing-cellular adhesion molecule (H-CAM). By using human H-CAM cDNA probes, we have isolated a full-length cDNA for the mouse homolog. Comparison of the human and mouse sequences reveals that an N-terminal domain homologous to cartilage proteoglycan core and link proteins, as well as the C-terminal transmembrane and cytoplasmic sequences, are highly conserved (89% and 86% identity, respectively). In contrast, a proximal extracellular domain thought to serve as a target for O-glycosylation and chondroitin sulfate attachment has undergone substantial divergence (only 42% identity). Transient expression of the cDNA in CHO cells followed by immunologic staining confirms that this mouse H-CAM cDNA encodes Pgp-1.1, one of two known Pgp-1 alloantigens.     

Structure of the genes encoding the CD19 antigen of human and mouse B lymphocytes

CD19 is a B lymphocyte cell-surface marker that is expressed early during pre-B-cell differentiation with expression persisting until terminal differentiation into palsma cells. CD19 is a member of Ig gnee superfamily with two extreacellular Ig-like domains separated amino acid cytoplasmic domain. In this study, Southern blot analysis revelaed that the human and mouse CD19 genes were compact single copy genes. Both the human and mouse CD19 genes were isolated and the nucleotide sequences flanking each exon were determined. Both genes were composed of 15 exons and spanned 8 kilobases (kb) of DNA in human and 6 kb in mouse. The positions of exon-intron boundaries were identical between human and mouse and correlated with the putative functional domains of the CD19 protein. The 200 bp region 5 of the putative translation initiation AUG codon as well conserved in sequence between human and mouse and contained potential trasncription regulatory elements. In addition, the 3 untranslated regions (UT) of the CD19 genes following the termination codon were conservedf in sequence. The high level conservation of nucleotide sequences between species in all exons and 5 and 3 UT suggests that expression of the CD19 gene may be regulated in a similar fashion in human and mouse.The nucleotide sequence database reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers: human CD19 gnee, M62544 to M62550; mouse CD19 gene, M62551 to M62553.     

CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.     

Alternative splicing of pre-mRNA encoding the <Emphasis Type="Italic">Musca domestica</Emphasis> latrophilin-like protein: Primary structures of four spliced forms of mRNA and their protein products

An internal DNA fragment (2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5- and 3-ends (5- and 3-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (6000 nt) differ in the structures of both the regions encoding signal peptides and 5-terminal untranslated regions. They encode large proteins (1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane strands followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severely truncated at their C-termini (900 aa). They are composed of the domain with only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 175–185.Original Russian Text Copyright © 2005 by Andreev, Danilevich, Grishin.     

Role of the extracellular and cytoplasmic domains of CD44 in the rolling interaction of lymphoid cells with hyaluronan under physiologic flow

CD44 can function as an adhesion receptor that mediates leukocyte rolling on hyaluronan (HA). To study the contributions of different domains of the standard isoform of CD44 to cell rolling, a CD44-negative mouse T lymphoma AKR1 was transfected with wild type (WT) or mutated cDNA constructs. A parallel flow chamber was used to study the rolling behavior of CD44 transfectants on immobilized HA. For CD44WT transfectants, the fraction of cells that rolled and the rolling velocity was inversely proportional to the amount of cell surface CD44. When the cytoplasmic domain distal to Gly(305) or sequences that serve as binding sites for cytoskeletal linker proteins, were deleted or replaced with foreign sequences, no significant changes in the rolling behavior of mutant cells, compared with the transfectant expressing CD44WT, were observed. Transfectants lacking 64 amino acids of the cytoplasmic tail distal to Cys(295) adhered to HA but showed enhanced rolling at low shear forces. When 83 amino acids from the "non-conserved" membrane-proximal region of the CD44 extracellular domain were deleted, cells adhered firmly to the HA substrate and did not roll at any fluid shear force tested. Unlike wild type cells that exhibited a nearly homogeneous distribution of CD44 on a smooth cell surface, cells expressing the non-conserved region deletion mutant accumulated CD44 in membrane protrusions. Disruption of the actin cytoskeleton with cytochalasin B precluded the formation of membrane protrusions, however, treated cells still adhered firmly to HA and did not roll. We conclude that interaction between the cytoplasmic domain of CD44 and the cytoskeleton is not required for cell rolling on immobilized ligand. The strong effect of deletion of the non-conserved region of the extracellular domain argues for a critical role of this region in CD44-dependent rolling and adhesion interactions with HA under flow.     

The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differently expressed

A Nicotiana tabacum thioredoxin h gene (EMBL Accession No. Z11803) encoding a new thioredoxin (called h₂) was isolated using thioredoxin h₁ cDNA (X58527), and represents the first thioredoxin h gene isolated from a higher plant. It encodes a polypeptide of 118 amino acids with the conserved thioredoxin active site Trp-Cys-Gly-Pro-Cys. This gene comprises two introns which have lengths of 1071 and 147 by respectively, and three exons which encode peptides of 29, 41 and 48 amino acids, respectively. This thioredoxin h shows 66% identity with the amino acid sequence of thioredoxin h₁ (X58527) and only around 35% with the choroplastic thioredoxins. The two thioredoxins, h₁ and h₂, do not have any signal peptides and are most probably cytoplasmic. Using the 3 regions of the mRNAs, two probes specific for thioredoxins h₁ and h₂ have been prepared. Southern blot analysis shows that thioredoxin sequences are present in only two genomic EcoRI fragments: a 3.3 kb fragment encodes h₁ and a 4.5 kb fragment encodes h₂. Analysis of the ancestors of the allotetraploid N. tabacum shows that thioredoxin h₂ is present in N. sylvestris and N. tomentosiformis but that thioredoxin h₁ is absent from both putative ancestors. Thus, the thioredoxin h₁ gene has probably been recently introduced in to N. tabacum as a gene of agronomic importance, or linked to such genes. Northern blot analysis shows that both genes are expressed in N. tabacum, mostly in organs or tissues that contain growing cells. Thioredoxin h₁ is always expressed at a lower level than h₂ in tobacco plants. In contrast, the thioredoxin hl gene is abundantly expressed in freshly isolated protoplasts, while h₂ mRNAs are not detectable.     

The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins [published erratum appears in J Cell Biol 1991 Feb;112(3):following 513]

The present study was undertaken to determine the relationship between the hyaluronate receptor and CD44 (H-CAM), cell-surface glycoproteins of similar molecular weights that have been implicated in cell adhesion. In initial experiments, a panel of monoclonal antibodies directed against CD44 were tested for their ability to cross react with the hyaluronate receptor. These antibodies immunoprecipitated [3H]hyaluronate binding activity from detergent extracts of both mouse and human cells, indicating that the hyaluronate receptor is identical to CD44. In addition, one of these antibodies (KM-201 to mouse CD44) directly blocked the binding of labeled hyaluronate to the receptor and inhibited hyaluronate dependent aggregation of SV-3T3 cells. CD44 has also been implicated in lymphocyte binding to high endothelial venules during lymphocyte homing. Interestingly, the monoclonal antibody Hermes- 3, which blocks lymphocyte binding to the high endothelial venules of mucosal lymphoid tissue, had no effect on the binding of labeled hyaluronate. Furthermore, the binding of lymphocytes to high endothelial cells of lymph nodes and mucosal lymphoid tissue was not significantly affected by treatment with agents that block the binding of hyaluronate (hyaluronidase, excess hyaluronate and specific antibodies). Thus, CD44 appears to have at least two distinct functional domains, one for binding hyaluronate and another involved in interactions with mucosal high endothelial venules.     

Characterization ofVitis vinifera L. glutamine synthetase and molecular cloning of cDNAs for the cytosolic enzyme

Grapevine (Vitis vinifera L.) glutamine synthetase (GS) was analysed into two distinct classes of isoforms; one of them was present in both leaf and root tissues while the other one showed leaf specificity. Western blot analysis revealed that grapevine GS consists of three types of polypeptides of distinct size and differential tissue specificity. Two structurally distinct cDNA clones, pGS1;1 and pGS1;2, encoding grapevine GS were isolated from a cell suspension library and characterized. Both clones contained open reading frames encoding for polypeptides of 356 amino acids with a predicted molecular mass of about 39 kDa. Although the coding sequences of pGS1;1 and pGS1;2 were 84% similar, their 5-and 3-untranslated sequences showed only 40% similarity. The coding sequences of the two clones and the derived amino acid sequences showed higher homology to cytosolic than to chloroplastic GSs of other higher plants indicating that the cDNAs isolated encode for cytosolic isoforms of grapevine GS. Southern blot analysis suggested the existence of more than two GS genes in the grapevine genome. In northern blots both clones were hybridized to mRNAs of about 1.4 kb that are differentially expressed in the various tissues. Supply of nitrate or ammonium in the cell suspension culture medium, as a sole nitrogen source, resulted in differential response of the pGS1;1-and pGS1;2-related genes.     

Amplification and expression of the TGF-α, EGF receptor and c-myc genes in four human oesophageal squamous cell carcinoma lines

We have previously shown that four human oesophageal squamous cell carcinoma (SCC) cell lines secrete significant quantities of transforming growth factor alpha (TGF-)in vitro. Three of these lines are known to produce supernumary low-affinity epidermal growth factor receptors (EGF-Rs). Using an¹²⁵I-EGF compeitive binding assay and Scatchard analysis, we show that the fourth also overproduces low-affinity receptors. According to slot blot DNA analyses, the secretion of high levels of TGF- is not associated with amplification of the TGF- gene, and hyperproduction of the EGF-R is correlated with receptor gene amplification. Western blot analyses show that the c-Myc protein is overexpressed in two of the cell lines; and Southern and Northern blot analyses indicate that this overexpression occurs independently of c-myc gene amplification. Our results are consistent with an autocrine role for TGF- and EGF-R in oesophageal carcinogenesis and support the possibility that c-myc overexpression may be required for thein vivo tumourigenicity of cells that produce high levels of TGF- and the EGF-R.     

Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allelespecific residue: arginine at position 81 located on the 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the ul helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and-Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the 1 and 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.     

Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the <Emphasis Type="Italic">gpd</Emphasis> promoter for transformation in <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

The glyceraldehyde-3-phosphate dehydrogenase gene of Flammulina velutipes was isolated. The complete gpd sequence (from ATG to TAA) was 1,489 bp in length and contained nine introns. The locations of these nine introns were similar to those of other basidiomycetes, which might reflect the evolutionary divergence of these mushrooms. The F. velutipes gpd gene was found to encode a protein of 339 amino acids and its putative amino acid sequence revealed a high similarity to an analogous protein deriving from other basidiomycetes. Results of Southern blot analysis suggested that there existed only one copy of the gpd gene in the genome of F. velutipes and that there was one typical TATA box and two CAAT boxes located in the 5 flanking region. The F. velutipes gpd promoter was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selection marker. Using the resulting construction, hph was efficiently transformed into F. velutipes by basidiospore electroporation. No false-positive antibiotic-resistant cultures were detected by PCR amplification and the hygromycin resistance trait was maintained stably during mitotic cell division for 3 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. This rapid and convenient electroporation procedure offers new prospects for the genetic manipulation and a tool for tagging genes of this important edible mushroom species. Sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AF515622.     

Class- and splice variant-specific association of CD98 with integrin beta cytoplasmic domains

CD98 is a type II transmembrane protein involved in neutral and basic amino acid transport and in cell fusion events. CD98 was implicated in the function of integrin adhesion receptors by its capacity to reverse suppression of integrin activation by isolated integrin beta(1A) domains. Here we report that CD98 associates with integrin beta cytoplasmic domains with a unique integrin class and splice variant specificity. In particular, CD98 interacted with the ubiquitous beta(1A) but not the muscle-specific splice variant, beta(1D), or leukocyte-specific beta(7) cytoplasmic domains. The ability of CD98 to associate with integrin cytoplasmic domains correlated with its capacity to reverse suppression of integrin activation. The association of CD98 with integrin beta(1A) cytoplasmic domains may regulate the function and localization of these membrane proteins.     

A mutant lymphoma cell line with a defectiveThy-1 glycoprotein gene

We characterized a mutant T -cell lymphoma line selected for the inability to express the Thy-1 glycoprotein. This cell line is a member of the D complementation class of Thy-1^– somatic cell mutants, and it lacks detectable cell-surface Thy-1.1 glycoprotein and detectable cytoplasmic Thy-1 mRNA. Southern blot analysis using a number of probes isolated from the clonedThy-1.2 gene demonstrated that, in the mutant, one copy of theThy-1 gene is absent from the genome and the other has undergone rearrangement. This rearrangement results from a deletion of the 5 portion of the gene removing the first two alternate exons and promoters and a portion of the second intron. The deletion breakpoint within the mutantThy-1 gene was localized to within 400 nucleotides by Southern blot analysis. The breakpoint is near two classes of mouse repetitive elements-a mouse B1-family repetitive element and a simple repetitive sequence-suggesting a mechanism of rearrangement leading to the mutation. Southern blot analysis demonstrated that two closely linked molecular markers on chromosome 9 are unaltered, demonstrating that the deletion in this mutant cell line is subchromosomal.     

Isolation and characterization of mouse CD7 cDNA

The human CD7 antigen is a glycoprotein, M _r 40 000, expressed on the surface of peripheral blood T-lymphocytes and thymocytes, and is the earliest surface antigen to appear on T-cell lineage cells. In this study, putative mouse CD7 cDNA was identified based on its similarities with human CD7. Five independent clones originating from the same mRNA species were isolated (designated as mCD7) by screening a mouse thymocyte cDNA library with human CD7 cDNA, J61, under moderate stringency. The longest insert of a 995 base pair had an open reading frame of 210 amino acids. Northern blot analysis using the mouse CD7 cDNA probe demonstrated a single 1.2 kilobase mRNA ni the thymus, spleen, bone marrow, and small intestine. The protein deduced from mCD7 cDNA consisted of the leader, extracellular, transmembrane, and cytoplasmic domains of 24, 126, 21, and 39 amino acids, respectively, based on the hydrophobicity plot and the structure of human CD7. The extracellualr domain contained three potential N-glycosilation sites, while the cytoplasmic domain contained one potential protein kinase C phosphorylation site. The amino acid sequence had 45.5% similarity with human CD7, while the similarities for the individual domains ranged from 49.2% to 63.2%. The six highly conserved regions, which may possibly be involved with still unknown CD7-mediated functions, were located in the extracellular and cytoplasmic domains.The nucleotide sequence data reported in this paper have seen submitted to the GenBank, DDBJ, and EMBL nucleotide sequence database and have been assigned the accession number D10329.     

Expression of three osmotin-like protein genes in response to osmotic stress and fungal infection in potato

We have characterized three cDNAs encoding osmotin-like proteins from potato (Solanum commersonii) cell cultures. These cDNAs (pA13, pA35, and pA81) have extensive nucleotide identity in the coding regions but low homology in the 3 non-coding sequences, and may encode three isoforms of potato pathogenesis-related (PR) type 5 proteins. Using gene-specific probes, RNA gel blot analyses showed constitutive accumulation of osmotin-like protein mRNAs in cell cultures, leaves, stems, roots and flowers, with high abundance in the roots and mature flowers. Treatments with abscisic acid (ABA), low temperature, and NaCl increased the accumulation of all three mRNAs in S. commersonii cell cultures and plants grown in vitro. Salicylic acid (SA), and wounding resulted in a moderate increase in the levels of pA13 and pA81 but not pA35 mRNAs. Infection with the fungus Phytophthora infestans activated strong and non-systemic expression of all three osmotin-like protein genes. The accumulation of osmotin-like proteins, however, was detected only in P. infestans-infected tissues but not in plants treated with ABA, SA, NaCl, low temperature, or wounding.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

Monoclonal antibodies to human lymphocyte homing receptors define a novel class of adhesion molecules on diverse cell types

A 90-kD lymphocyte surface glycoprotein, defined by monoclonal antibodies of the Hermes series, is involved in lymphocyte recognition of high endothelial venules (HEV). Lymphocyte gp90Hermes binds in a saturable, reversible fashion to the mucosal vascular addressin (MAd), a tissue-specific endothelial cell adhesion molecule for lymphocytes. We and others have recently shown that the Hermes antigen is identical to or includes CD44 (In[Lu]-related p80), human Pgp-1, and extracellular matrix receptor III-molecules reportedly expressed on diverse cell types. Here, we examine the relationship between lymphoid and nonlymphoid Hermes antigens using serologic, biochemical, and, most importantly, functional assays. Consistent with studies using mAbs to CD44 or Pgp-1, mAbs against five different epitopes on lymphocyte gp90Hermes reacted with a wide variety of nonhematolymphoid cells in diverse normal human tissues, including many types of epithelium, mesenchymal elements such as fibroblasts and smooth muscle, and a subset of glia in the central nervous system. To ask whether these non-lymphoid molecules might also be functionally homologous to lymphocyte homing receptors, we assessed their ability to interact with purified MAd using fluorescence energy transfer techniques. The Hermes antigen isolated from both glial cells and fibroblasts--which express a predominant 90-kD form similar in relative molecular mass, isoelectric point, and protease sensitivity to lymphocyte gp90Hermes--was able to bind purified MAd. In contrast, a 140-160-kD form of the Hermes antigen isolated from squamous epithelial cells lacked this capability. Like lymphocyte binding to mucosal HEV, the interaction between glial gp90Hermes and MAd is inhibited by mAb Hermes-3, but not Hermes-1, suggesting that similar molecular domains are involved in the two binding events. The observation that the Hermes/CD44 molecules derived from several nonlymphoid cell types display binding domains homologous to those of lymphocyte homing receptors suggests that these glycoproteins represent a novel type of cell adhesion/recognition molecule (H-CAM) potentially mediating cell-cell or cell-matrix interactions in multiple tissues.     

MYCN-Related Suppression of Functional CD44 Expression Enhances Tumorigenic Properties of Human Neuroblastoma Cells

Highly malignant neuroblastoma tumors with MYCN amplification have been shown to downregulate the expression of the CD44 adhesion receptor. We have previously shown that MYCN amplified neuroblastoma cell lines either lack CD44 expression or express a nonfunctional, nonhyaluronic acid-binding CD44 receptor. By analysis of cells with manipulated expression of either CD44 or MYCN, we demonstrate that transfection of cells with a CD44 full-length cDNA construct produced a functional receptor in single copy MYCN cells and a nonfunctional CD44 receptor in MYCN amplified cells, similar to the CD44 receptor expressed by cells with enforced MYCN. Analysis of the in vivo growth properties of the transfectants revealed that the restoration of a functional CD44 receptor in nonamplified cells resulted in the suppression of in vivo cell growth, therefore linking the MYCN-related lack of hyaluronic acid-binding function of CD44 to the highly tumorigenic properties of a subset of neuroblastoma cells.