The putative counterparts of human plasmacytoid pre-dendritic cells (pDCs) have been described in vivo in mouse models and very recently in an in vitro culture system. In this study, we report that large numbers of bone marrow-derived murine CD11c+B220+ pDCs can be generated with Flt3 ligand (FL) as the sole exogenous differentiation/growth factor and that pDC generation is regulated in vivo by FL because FL-deficient mice showed a major reduction in splenic pDC numbers. We extensively analyzed bone marrow-derived CD11c+B220+ pDCs and described their immature APC phenotype based on MHC class II, activation markers, and chemokine receptor level of expression. CD11c+B220+ pDCs showed a nonoverlapping Toll-like receptor pattern of expression distinct from that of classical CD11c+B220− dendritic cells and were poor T cell stimulators. Stimulation of CD11c+B220+ pDCs with oligodeoxynucleotides containing certain CpG motifs plus CD40 ligand plus GM-CSF led to increased MHC class II, CD80, CD86, and CD8α expression levels, to a switch in chemokine receptor expression that affected their migration, to IFN-α and IL-12 secretion, and to the acquisition of priming capacities for both CD4+ and CD8+ OVA-specific TCR-transgenic naive T cells. Thus, the in vitro generation of murine pDCs may serve as a useful tool to further investigate pDC biology as well as the potential role of these cells in viral immunity and other settings.

CD44 is a widely distributed cell surface protein that plays a role in cell adhesion and migration. As a proteoglycan, CD44 is also implicated in growth factor and chemokine binding and presentation. The extracellular region of CD44 is variably spliced, giving rise to multiple CD44 isoforms. All isoforms contain an amino-terminal domain, which is homologous to cartilage link proteins. The cartilage link protein-like domain of CD44 is important for hyaluronan binding. The structure of the link protein domain of TSG-6 has been determined by NMR. Based on this structure, a molecular model of the linkhomologous region of CD44 was constructed. This model was used to select residues for site-specific mutagenesis in an effort to identify residues important for ligand binding and to outline the hyaluronan binding site. Twentyfour point mutants were generated and characterized, and eight residues were identified as critical for binding or to support the interaction. In the model, these residues form a coherent surface the location of which approximately corresponds to the carbohydrate binding sites in two functionally unrelated calcium-dependent lectins, mannose-binding protein and E-selectin (CD62E).CD44 is a type I transmembrane protein encoded by a gene containing 19 exons (1, 2). Ten of these exons are variably spliced (V1-V10), giving rise to multiple CD44 isoforms. All CD44 isoforms contain at their amino terminus a domain of ϳ100 residues, which is homologous to cartilage link protein domains (3, 4). The link homology domain of CD44 has been implicated in the hyaluronan (HA) 1 binding activity of CD44 (5, 6). In different CD44 isoforms, polypeptides encoded by the variably spliced exons are inserted following exon E5. The functional relevance of different CD44 isoforms is still under investigation, but the following observations have been made. (a) Inclusion of exon V3 results in the modification of CD44 with heparan sulfate (HS) added to an SGSG site contained in this exon (7,8). These CD44 isoforms can interact with HSbinding growth factors and chemokines. (b) Inclusion of exon V6 renders tumor cells expressing this CD44 isoform aggressively metastatic (9, 10). (c) Inclusion of variably spliced exons results in a increase in the number of O-linked carbohydrates in CD44 (11). This change in glycosylation has been proposed to modulate the ability of CD44 to bind HA and is consistent with the finding that N-linked glycosylation can also modulate HA binding (12, 13). The variably spliced region of CD44 is followed by a stalk encoded by exons E15 and E16, a hydrophobic transmembrane domain, and a cytoplasmic domain that can engage in intracellular signaling pathways (2).CD44 is expressed by a large number of different cell types. Leukocytes predominantly express the standard form of CD44 (CD44H). This isoform contains no variably spliced exons and binds HA on activated leukocytes (3,5,6). This interaction has been shown to play an important role in leukocyte adhesion and migration at sites of inflammation (14). Acti...

Abstract. The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resuiting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.

Background: JAK JH2s (pseudokinase domains) mediate important regulatory functions; it is unclear whether TYK2 JH2 binds ATP and possesses enzymatic activity. Results: TYK2 JH2 binds ATP, but is catalytically inactive; ATP stabilizes JH2 and modulates TYK2 activity. Conclusion: ATP binding to JH2 is functionally important; the rigid activation loop probably hinders substrate phosphorylation. Significance: The TYK2 JH2 domain can be targeted with ATP-competitive compounds for therapeutics.

Allelic deletions on chromosome 18q in Ͼ70% of primary colorectal tumors prompted the search for a tumor suppressor gene at that locus. An early result of this search was the cloning of a putative cell-surface receptor, DCC (deleted in colorectal carcinoma) (1). While the cloning of DCC brought much excitement that a tumor suppressor gene responsible for many colorectal cancers had been identified, 7 years later, the evidence that DCC is the putative tumor suppressor located on chromosome 18q remains inconclusive. Nonetheless, a role for DCC in tumor progression is suggested by the large number of different tumor types that have been reported to have lost DCC expression, including carcinomas of the pancreas, breast, prostate, bladder, and stomach; leukemias; neuroblastomas; and gliomas. The evidence for DCC as a tumor suppressor was examined in a recent review by Fearon (2).While the question of DCC as a tumor suppressor is still being debated, understanding of the normal physiological role of DCC has moved forward. Recent studies provide biochemical, functional, and genetic data suggesting that DCC is a receptor for the diffusible neural chemoattractant netrin-1 (3-5). Netrin-1 bound specifically to cells expressing DCC, and DCC mediated netrin-1-dependent outgrowth of commissural axons from dorsal spinal cord explants. This outgrowth was blocked by an anti-DCC mAb 1 that did not block the interaction between netrin-1 and DCC, suggesting that the interaction between netrin-1 and DCC may require additional factors. Furthermore, genetic analysis of UNC-40, a DCC homolog from Caenorhabditis elegans, suggests that there are several developmental functions attributed to UNC-40 that do not require netrin-1 (UNC-6). Taken together, these data suggest that while DCC/netrin-1 interactions are important, all the components for this guidance system have yet to be identified, and the functional role of DCC is not fully understood.A functional role of DCC in epithelial cells has also been suggested. Chuong et al. (6) showed that a Fab fragment of an anti-DCC mAb disrupted normal dermal condensation during feather bud formation in an embryonic chicken dorsal skin explant culture, a process that involves epithelial/mesenchymal cell interaction (6). In addition, the same mAb blocked aggregation of stage 34 (embryonic day 8) skin epithelial cells. These findings suggested that DCC participated in Ca 2ϩ -independent cell/cell interactions.DCC encodes a transmembrane protein with an extracellular domain composed of four Ig C2-like repeats, six fibronectin type III (FNIII)-like repeats, a single membrane-spanning region, and a 325-amino acid cytoplasmic domain (7). This complicated extracellular domain structure of DCC provides many candidate domains for mediating intracellular interactions. In this study, we set out to identify counter cell-surface DCC ligand(s) by using a DCC-Ig fusion protein, which bound to neural and epithelial derived cell lines. We have further demonstrated that the molecular basis for this interaction is ...

Isoforms of CD44 are differentially modified by the glycosaminoglycans (GAGs) chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate. GAG assembly occurs at serines followed by glycines (SG), but not all SG are utilized. Seven SG motifs are distributed in five CD44 exons, and in this paper we identify the HS and CS assembly sites that are utilized in CD44. Not all the CD44 SG sites are modified. The SGSG motif in CD44 exon V3 is the only HS assembly site; this site is also modified with CS. HS and CS attachment at that site was eliminated by mutation of the serines in the V3 motif to alanine (AGAG). Exon E5 is the only other CD44 exon that supports GAG assembly and is modified with CS. Using a number of recombinant CD44 protein fragments we show herein that the eight amino acids located downstream of the SGSG site in V3 are responsible for the specific addition of HS to this site. If the eight amino acids located downstream from the first SG site in CD44 exon E5 are exchanged with those located downstream of the SGSG site in exon V3, the SG site in E5 becomes modified with HS and CS. Likewise if the eight amino acids found downstream from the first SG in E5 are placed downstream from the SGSG in V3, this site is modified with CS but not HS. We also show that these sequences cannot direct the modification of CD44 with HS from a distance. Constructs containing CD44 exon V3 in which the SGSG motif was mutated to AGAG were not modified with HS even though they contained other SG motifs. Thus, a number of sequence and structural requirements that dictate GAG synthesis on CD44 have been identified.

NKG2D is a homodimeric C-type lectin-related receptor expressed on natural killer (NK) cells and T cells. In mice, alternative deoxyribonucleic acid (DNA) splicing generates two isoforms of NKG2D that differ in the length of their cytoplasmic domains. Their ability to induce cellular activation is mediated via association with two membrane-bound, signaling adaptor molecules, DAP10 and DAP12. It has been reported that the long form of NKG2D associates exclusively with DAP10, whereas the short variant can interact with either adaptor. The short isoform was reported to be almost undetectable in naive NK cells. Using two distinct cell types, we demonstrate that like the short isoform, the long variant of NKG2D also associates not only with DAP10 but also with DAP12. Using reporter cells (70Z/3), we demonstrate that DAP12 can compete equally with DAP10 for association with both variants of NKG2D when DAP10 and DAP12 are coexpressed. Cross-linking either isoform of NKG2D induces a calcium flux when associated exclusively with DAP10 or DAP12. Moreover, using quantitative polymerase chain reaction (PCR), we also show that the short isoform of NKG2D is expressed in naive NK cells. Our data suggest that signaling via mouse NKG2D isoforms is more complex than originally presented.

Osteopontin is a secreted glycoprotein with adhesive and migratory functions. Cellular interactions with osteopontin are mediated through integrin receptors which recognize the RGD domain. Recently, CD44, a non-integrin, multifunctional adhesion molecule was identified as an osteopontin receptor. CD44 is a ubiquitous surface molecule that exists as a number of different isoforms, generated by alternative splicing. To analyze which forms of CD44 mediate binding to osteopontin, we used the standard form of CD44 as CD44-human immunoglobulin fusion proteins and several splice variants in enzyme-linked immunosorbant assays. Multiple preparations of osteopontin were used including native osteopontin derived from smooth muscle cells, human urinary osteopontin, full-length recombinant osteopontin, and two recombinant osteopontin fragments expected to be formed following thrombin cleavage. Our data show that although the CD44-hlg fusion proteins could interact with hyaluronic acid as expected, there was no interaction between CD44H, CD44E, CD44v3,v8-v10, or CD44v3 with osteopontin. These studies suggest that CD44-osteopontin interactions may not be common in vivo and may be limited to a specific CD44 isoform(s), and/or a particular modified form of osteopontin.