No abstract

CD45 is a family of high molecular weight leukocyte cell surface glycoproteins. Recently, two related subregions ofthe cytoplasmic domain ofCD45 have been shown to have 3040% amino acid identity with a human placental protein phosphotyrosine phosphatase, and CD45 isolated from human spleen was found to exhibit intrinsic protein phosphotyrosine phosphatase (EC 3.1.3.48) activity. In the present studies, we demonstrate that each of the known isoforms of murine CD45 has an equivalent basal level of protein phosphotyrosine phosphatase activity and establish that this enzymatic activity is associated with the cytoplasmic domain of the glycoprotein. Studies with three independent sets of wellcharacterized parental CD45+, mutant CD45-, and revertant CD45+ lymphoma cell lines indicate that loss of CD45 increases the phosphorylation of the src-related leukocyte-specific tyrosine protein kinase p56Ick on tyrosine-505, a putative negative regulatory site. This suggests that CD45 may play a role in leukocyte growth regulation by altering the kinase activity of ps6ck.CD45 (T200 or L-CA) is a family of major leukocyte-specific cell surface glycoproteins expressed exclusively-on hematopoietic cells (reviewed in ref. 1). Isoforms of CD45 are generated by the alternative splicing of three exons, each encoding '50 amino acids that are inserted near the amino terminus of the molecule. Of the eight possible mRNAs that can be generated by the differential use ofthe three exons, six have been identified by sequencing CD45 cDNAs from mouse, human, and rat (2-6). The extra sequences of CD45 encoded by the alternatively spliced exons contain multiple sites for O-linked oligosaccharides, and at least one extra segment is known to be extensively glycosylated (7). Different isoforms of CD45 are selectively expressed on specific subpopulations of hematopoietic cells, and changes in the pattern of expression of CD45 isoforms in T cells also occur upon antigenic stimulation (8-10). CD45 is also distinguished by a large, highly conserved, cytoplasmic domain of 705 amino acids that can be subdivided into two related subdomains of '300 amino acids. Recently, each of these subdomains was shown to have 30-40% amino acid identity with a soluble human placental protein phosphotyrosine phosphatase (PTPase; protein-tyrosine phosphatase, EC 3.1.3.48), and CD45 isolated from human spleen was found to have intrinsic PTPase activity (11,12). We have extended these observations by analyzing the PTPase activity of individual isoforms of murine CD45 and truncated forms of the molecule. Further, the availability of three independent sets of parental CD45+, mutant CD45-, and revertant CD45+ lymphoma cells has allowed us to search for in vivo substrates of the CD45 PTPase activity by comparing the phosphotyrosine-containing proteins in CD45' and CD45-cells. The results ofthis analysis indicate that loss of CD45 in the mutant lymphoma cells correlates with increased phosphorylation of the src-related leukocyte-specific tyrosine protein kinase p56Ick (13...

The c-kit-encoded transmembrane tyrosine kinase receptor for stem cell factor (Kit/SCF-R) is required for normal haematopoiesis, melanogenesis and gametogenesis. However, the roles of individual Kit/SCF-R-induced signalling pathways in the control of developmental processes in the intact animal are completely unknown. To examine the function of SCF-induced phosphatidylinositol (PI) 3'-kinase activation in vivo, we employed the Cre-loxP system to mutate the codon for Tyr719, the PI 3'-kinase binding site in Kit/SCF-R, to Phe in the genome of mice by homologous recombination. Homozygous (Y719F/Y719F) mutant mice are viable. The mutation completely disrupted PI 3'-kinase binding to Kit/SCF-R and reduced SCF-induced PI 3'-kinase-dependent activation of Akt by 90%. The mutation induced a gender- and tissue-specific defect. Although there are no haematopoietic or pigmentation defects in homozygous mutant mice, males are sterile due to a block in spermatogenesis, with initially decreased proliferation and subsequent extensive apoptosis occurring at the spermatogonial stem-cell level. In contrast, female homozygotes are fully fertile. This is the first report so far demonstrating the role of an individual signalling pathway downstream of Kit/SCF-R in the intact animal. It provides the first in vivo model for male sterility caused by a discrete signalling pathway defect affecting early germ cells.

No abstract

Interactions between CD44 and hyaluronan are implicated in the primary adhesion of lymphocytes to endothelium at inflammatory locations. Here we show that preincubation of hyaluronan with full-length recombinant TSG-6 or its Link module domain (Link_TSG6) enhances or induces the binding of hyaluronan to cell surface CD44 on constitutive and inducible cell backgrounds, respectively. These effects are blocked by CD44-specific antibodies and are absent in CD44-negative cells. Enhancement of CD44-mediated interactions of lymphoid cells with hyaluronan by TSG-6 proteins was seen under conditions of flow at shear forces that occur in post-capillary venules. Increases in the number of rolling cells were observed on substrates comprising TSG-6-hyaluronan complexes as compared with a substrate containing hyaluronan alone. In ligand competition experiments, cell surface-bound TSG-6-hyaluronan complexes were more potent than hyaluronan alone in inhibiting cell adhesion to immobilized hyaluronan. Link_TSG6 mutants with impaired hyaluronan binding function had a reduced ability to modulate ligand binding by cell surface CD44. However, some mutants that exhibited close to wild-type hyaluronan binding were found to have either reduced or increased activity, suggesting that some amino acid residues outside of the hyaluronan binding site might be involved in protein self-association, potentially leading to the formation of cross-linked hyaluronan fibers. In turn, cross-linked hyaluronan could increase the binding avidity of CD44 by inducing receptor clustering. The ability of TSG-6 to modulate the interaction of hyaluronan with CD44 has important implications for CD44-mediated cell activity at sites of inflammation, where TSG-6 is expressed.

SummaryThe CD44-negative T lymphoma AKR1 (CD44.2 genotype) was transfected with a CD44.1 cDNA. The intact eDNA conferred on the transfected cells the ability to bind hyaluronic acid (HA) both from solution and immobilized on culture plates. It also conferred a CD44-dependent and hyaluronidase-sensitive increase in adhesion to a lymph node endothelial cell line. A mutant cDNA which codes for a CD44 molecule lacking most of the cytoplasmic domain of CD44 was also transfected into AKR1, and cell sorting was used to sdect transfectants expressing levels of cell surface CD44 expression comparable with the line transfected with the wild-type CD44 cDNA. The cells transfected with the mutant construct bound fluoresceinated HA from solution very poorly, but did adhere to immobilized HA, though less well than ceils transfected with the wild-type construct. This result indicates that the cytoplasmic domain of CD44 is necessary for binding of HA from solution but is not required for binding to immobilized HA, although it may contribute to adhesion following ligand recognition. A monoclonal antibody (mAb), IILAWB 14, which reacts with CD44 on all CD44 + cells dramatically induced HA binding by some CD44 + cell lines that did not constitutively bind HA. The transfectant expressing a CD44 molecule with a truncated cytoplasmic domain could be induced by this antibody to bind fluoresceinated-HA from solution.' Splenic T cells did not bind fluoresceinated HA constitutively. In the presence of the IKAWB 14 mAb, virtually all CD44 + splenic T cells bound HA. Induction was immediate and occurred equally well at room temperature and at 4~ indicating that the new HA-binding activity was due to preexistent CD44 molecules. These results are compatible with an antibody-induced activation of CD44 by either a conformational change in the CD44 molecule or a change in the distribution of CD44 molecules on the cell surface.

A murine leukocyte surface glycoprotein (Mr = 95 000) has been defined by means of xenogeneic monoclonal antibodies. In normal hematopoietic tissues, the glycoprotein is found in highest amounts in the bone marrow. Flow cytometric analysis shows that essentially all bone-marrow cells express the glycoprotein and that it is a major component of a subpopulation of cells containing predominantly granulocytic precursors. In contrast, only about 5 percent of thymocytes express sufficient glycoprotein to be detected by flow cytometric analysis, although under stringent conditions up to 20 percent of thymocytes are susceptible to complement-mediated cytotoxicity using a monoclonal antibody against the glycoprotein. Functional assays showed that both prothymocytes and colony forming unit-spleen express the glycoprotein which is broadly distributed on murine hematopoietic tumor cell lines. However, although some Thy-1+ (T) cell lymphomas express large amounts of the glycoprotein, others do not express detectable quantities of the molecule. The glycoprotein is not restricted to hematopoietic cells and can be detected on lung, kidney, brain, and liver as well as cultured fibroblasts. Monoclonal antibodies against the glycoprotein cross-react with an antigen present on human cells. As described in the accompanying paper, the glycoprotein exists in two antithetical allelic forms and we show that it is identical to a polymorphic surface molecule independently characterized by Colombatti and co-workers.

SummaryCD44 is a major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA). However, the ability of CD44 to bind ligand is strictly regulated. Three activation states of CD44 have been demonstrated: (a) inactive; (b) inducible (by certain CD44-specific mAb); and (c) constitutively active. Starting with two parental cell lines expressing CD44 in the inactive state, a pre-B cell (RAW 253) and a fibroblast (L cells), we used fluorescence-activated cell sorting with fluorescein-conjugated hyaluronan in the presence of inducing mAb to derive variant cell lines with CD44 in the inducible state. Constitutively active derivatives were isolated from the inducible variants by a further round of fluorescence-activated cell sorting in the absence of inducing antibody. However, constitutively active variants could not be isolated directly from parental cells expressing CD44 in the inactive state. These results suggest that two genetic events must occur to obtain an active CD44-HA receptor from an inactive receptor. Variant and parental cell-derived CD44 molecules exhibited differences in migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were partly attributable to differences in N-linked glycosylation. C D44 is a cell surface receptor for hyaluronan (HA), 1 a glycosaminoglycan (GAG) that is abundantly distributed in extracellular spaces (1-3). CD44 is found on diverse cell types and is thought to be involved in a number of functions, including lymphocyte activation, recirculation and homing, tumor metastasis, hematopoiesis, and HA metabolism (1, 3, 4). The HA receptor function of CD44 appears to be strictly regulated, with many cells that express CD44 failing to exhibit receptor function (1, 5). The mechanisms of this regulation are unknown, but the importance of regulated receptor function may be reflected in the correlation of altered CD44 expression with malignancy, inflammation, and autoimmunity, as well as cell activation in a normal immunological response (1, 4).:Abbreviations used in this paper: fl-v-xyloside, p-nitrophenyl H-Dxylopyranoside; BZ~GalNAc, benzyl 2-acetamido-2-deoxy-ol-v-galactopyranoside; F1-HA, fluorescein-conjugated HA; GAG, glycosaminoglycan; HA, hyaluronan.We have previously described three activation states of CD44 with respect to HA-binding function (1,(6)(7)(8). Here we show that a single cell line can express CD44 in each of the three activation states as the result of a limited number of mutation events. By comparison of variant cell lines differing in CD44-mediated HA-binding function, we define differences in glycosylation that are potentially responsible for differences in CD44-mediated ligand binding. Materials and MethodsCell Lines and Antibodies. RAW 253 (a pre-B lymphoma, 9), L.TK-(K) strain of L cell fibroblasts (10), and the variant cell lines derived from them were cultured in DMEM with 10% horse serum. Tissue culture supernatants of the CD44-specific mAb IM7(11), IRAWB14 (7), and KM81 (12) were used for immunoprecipitation, i...