Proteomic identification of CD44 interacting proteins (original) (raw)
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Molecular mechanisms regulating the hyaluronan binding activity of the adhesion protein CD44
Journal of Neuro-Oncology, 1995
In the present study, we describe the isolation and characterization of a cDNA clone designated B6F1.3, that appears to 'activate' the hyaluronan-binding capacity of CD44 upon transfection into the murine fibroblastoid cell line MOP8. Sequence analysis indicates that the putative regulatory molecule encoded by this clone is identical to the murine interleukin-2 receptor ? chain (mIL-2Ry), a recently described type i transmembrane protein that constitutes an integral component of the cell surface receptors that bind a number of cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and perhaps also IL-13. Mutations in this molecule have been shown to be responsible for X-linked severe combined immunodeficiency (XSCID) in humans. With the exception of bone marrow, the mIL-2Ry chain was found to be expressed at high levels on all hemopoietic cell lines and tissue types examined. Non-hemopoietic tissues are generally negative. FACS analysis and Western blot analysis indicated respectively that B6F1.3 does not mediate its effects by upregulating the expression of CD44 or by altering the alternative splicing of the molecule. Removal of the cytoplasmic tail of the mIL-2R? chain, including a Src homology region 2 (SH2) subdomain, abolished its ability to enhance CD44-mediated binding to hyaluronan suggesting the involvement of signal transduction events triggered via the cytoplasmic domain in the 'activation' process. Determining whether activating molecules such as B6F1.3 are co-expressed within tumor cells may help improve the potential value of CD44 as a diagnostic marker of metastatic disease.
Mechanisms regulating the binding activity of CD44 to hyaluronic acid
Frontiers in bioscience : a journal and virtual library, 1998
CD44 is a cell surface glycoprotein present on many cell types. Many CD44 isoforms have been identified. All CD44 isoforms utilize identical transmembrane and cytoplasmic domains. The hematopoietic form of CD44 (CD44H) is the major CD44 protein present on normal human lymphocytes and monocytes. One of the ligands for CD44 is hyaluronic acid (HA), a polymer consisting of repeat units of disaccharide; N-acetyl-D-glucosamine and N-acetyl-D-glucuronic acid. Since HA is present ubiquitously in extracellular matrix and in circulation, promiscuous binding of HA to CD44 may have undesirable affect. Similar to other adhesion molecules, binding of HA to cell surface CD44 requires regulation. In this review, we summarized our studies using a human lymphoma cell line, Jurkat. We found that binding of CD44+ Jurkat transfectants to HA requires cellular activation. Cellular activation induces the reorganization of the cytoskeleton proteins. Reorganization of cytoskeletal proteins results in cluste...
CD44H regulates tumor cell migration on hyaluronate-coated substrate
Journal of Cell Biology, 1992
CD44 is a broadly distributed cell surface glycoprotein expressed in different isoforms in various tissues and cell lines. One of two recently characterized human isoforms, CD44H, is a cell surface receptor for hyaluronate, suggesting a role in the regulation of cell-cell and cell-substrate interactions as well as of cell migration. While CD44H has been shown to mediate cell adhesion, direct demonstration that CD44H expression promotes cell motility has been lacking. In this work we show that a human melanoma cell line, stably transfected with CD44H, displays enhanced motility on hyaluronate-coated surfaces while transfectants expressing an isoform that does not bind hyaluronate, CD44E, fail to do so. Migration of CD44H-expressing transfectants is observed to be blocked by a soluble CD44-immunoglobulin fusion protein as well as by anti-CD44 antibody, and to depend on the presence of the cytoplasmic domain of CD44. However, cells expressing CD44H cytoplasmic deletion mutants retain s...
Identification of CD44 Residues Important for Hyaluronan Binding and Delineation of the Binding Site
Journal of Biological Chemistry, 1998
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).
Journal of Biological Chemistry, 2006
In this study we have investigated the interaction of hyaluronan (HA) and CD44 with the neuronal Wiskott-Aldrich syndrome protein (N-WASP) in regulating actin polymerization and ErbB2/-catenin signaling in human ovarian tumor cells (SK-OV-3.ipl cells). Biochemical and immunological analyses indicate that N-WASP is expressed in SK-OV-3.ipl cells and that the binding of HA stimulates N-WASP association with CD44 and Arp2/Arp3 leading to filamentous actin formation and ovarian tumor cell migration. In addition, HA binding promotes CD44-N-WASP association with ErbB2 and activates ErbB2 kinase activity that in turn increases phosphorylation of the cytoskeletal protein, -catenin. Subsequently, phosphorylated -catenin is transported into the nucleus leading to -cateninmediated TCF/LEF-transcriptional co-activation. Because HAinduced -catenin phosphorylation, nuclear translocation, and TCF/LEF transcriptional activation is effectively blocked by the ErbB2 inhibitor, AG825, we conclude that HA/CD44-N-WASPassociated ErbB2 activation is required for -catenin-mediated signaling events. Transfection of SK-OV-3.ipl cells with N-WASP-VCA (verpolin homology, cofilin homology, and acidic domain) fragment cDNA not only blocks HA/CD44-induced N-WASP-Arp2/3 complex formation but also inhibits actin polymerization/F-actin assembly and tumor cell migration. Overexpression of the N-WASP-VCA domain also significantly reduces HA-induced ErbB2 recruitment to CD44, diminishes -catenin phosphorylation/nuclear translocation, and abrogates TCF/LEF-specific transcriptional co-activation by -catenin. Taken together, our findings strongly suggest that N-WASP plays a pivotal role in regulating HA-mediated CD44-ErbB2 interaction, -catenin signaling, and actin cytoskeleton functions that are required for tumor-specific behaviors and ovarian cancer progression. Ovarian carcinoma is the most lethal tumor of the female genital tract and continues to be a major cause of mortality in female cancer patients. A common mechanism for the spread of ovarian cancer is the shedding of cells from the primary tumor into the peritoneal cavity (1). This event is then followed by the formation of secondary tumor masses attached to the bowel and omental surface, both of which are covered by a single layer of mesothelial cells (2). Extracellular matrix components, such as hyaluronan (HA), 2 are present in large amounts in the mesothelial lining of the peritoneum (3, 4). HA is often bound to CD44, which is a ubiquitous, abundant, and functionally important surface receptor that displays HA-binding site(s) (5). Both CD44 and HA are overexpressed at sites of tumor attachment and are involved in tumor cell-specific functions (3, 6-9). Thus, it has been postulated that CD44 interaction with HA may be one of the important requirements for the spread of ovarian cancer. CD44 denotes a family of cell-surface glycoproteins that are expressed in a variety of human solid neoplasms, particularly those of gynecologic origin (e.g. ovarian cancers) (6, 7). One of the distinct features of CD44 is the enormous heterogeneity in the molecular masses of this family of proteins. It is known that CD44 is encoded by a single gene that contains 19 exons (10), and out of the 19 exons, 12 exons can be alternatively spliced (10). Most often, the alternative splicing occurs between exons 5 and 15 leading to an insertion in tandem of one or more variant exons (v1-v10, involving exons 6-14 in human cells) within the membrane proximal region of the extracellular domain (10). The binding of HA to CD44 isoforms triggers oncogenic signals (9) and direct "cross-talk" between two different tyrosine kinase-linked (ErbB2 (p185 HER2)/EGFR tyrosine kinases (11, 12) and c-Src kinase (13)) signaling pathways (cell growth versus cell migration, respectively). Most importantly, the cytoplasmic domain of CD44 isoforms selects its unique downstream effectors (e.g. cytoskeletal proteins,
Hyaluronan and CD44 control of cell fate
2016
Fibrosis can be charactorised as abberent wound healing resulting from an increased presence of α-smooth muscle actin (αSMA)-rich, myofibroblasts and a continued influx of immune cell mediators. The pro-fibrotic and pro-inflammatory cytokines TGF-β1 and IL-1β, respectivley, have been implicated in fibrotic progression by activating hyaluronan (HA)/CD44-mediated pathways. CD44, the principal HA receptor, exists as multiple spliced variants which mediate multiple celluar functions through their association with HA. The aim of this Thesis was to investigate the expression and interactions of CD44 variants asociated with fibroblast activation induced by TGF-β1 or IL-1β. Multiple forms of CD44 spliced variants were identified in fibroblasts. Stimulation with TGF-β1 decreased the expression of all variants, whereas IL-1β-increased global CD44 expression. CD44s was the variant identified as essential for both TGF-β1 induction of myofibroblasts and IL-1β-induced monocyte binding to fibrobla...
Cytokines regulate the affinity of soluble CD44 for hyaluronan
FEBS Letters, 2004
CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan, has been implicated in many adhesion-dependent cellular processes including tumor growth and metastasis. Soluble CD44 has been identi¢ed in the serum of normal individuals. Furthermore, tumor progression is often associated with marked increases in plasma levels of soluble CD44. Release of soluble CD44 by proteolytic cleavage (shedding) of membrane-anchored CD44 is likely to alter cellular responses to the environment due to modi¢cation of the cell surface and the potential for soluble CD44 to in£uence CD44mediated hyaluronan binding to cell surfaces. Cellular activation is typically required to induce hyaluronan binding to cell surface CD44 but the a⁄nity of endogenous soluble CD44 for hyaluronan remains unknown. In this study, we demonstrate that oncostatin M and transforming growth factor L L1 (TGF-L L1) which stimulate hyaluronan binding to HTB58 lung epithelial-derived tumor cells, also induce the release of soluble CD44. Interestingly, soluble CD44 released by oncostatin M-treated cells retained the ligand-binding properties of the membrane-anchored receptor. In contrast, soluble CD44 released from TGF-L L1treated HTB58 cells di¡ered in its hyaluronan-binding capacity from cell surface CD44 expressed on TGF-L L1-stimulated cells. These data indicate that the mechanisms that regulate the generation of soluble CD44 may also govern the binding of the released receptor to hyaluronan and therefore determine the impact on CD44-dependent physiologic and pathologic processes. .pl (J. Cichy).
Biochemical Journal, 2001
CD44 is the principal cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and binding to this ligand underlies CD44-mediated cell attachment and migration. As would be expected for a widely expressed adhesion receptor, CD44 is subject to complex regulatory events, and mis-regulation of the receptor has been associated with a number of disease pathologies, including chronic inflammatory conditions and the progression of metastatic tumours. In previous studies we have demonstrated that a key control point for this receptor is the phosphorylation of CD44 on a conserved cytoplasmic serine residue, Ser$#&. This modification is not required for efficient ligand binding, but is an essential component of CD44-dependent Abbreviations used : CaMKII, Ca 2 + /calmodulin-dependent protein kinase II ; FCS, fetal calf serum ; GST, glutathione S-transferase ; HRP, horseradish peroxidase ; mAb, monoclonal antibody ; WT, wild type.
Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity
The Journal of Cell Biology, 1996
Cell contact with the extracellular matrix component hyaluronic acid (HA) plays an important role in many developmental, physiological, and pathological processes, although the regulation of this contact is poorly understood. CD44 proteins carry an amino acid motif that mediates affinity to HA. Artificial clustering of the smallest 85-kD isoform of CD44 (CD44s) has previously been shown to promote binding of the protein to soluble HA (Lesley, J., R. Hyman, and P.W. Kincade. 1993. Adv. Immunol. 54:271-335; Persche, A., J. Lesley, N. English, I. Trowbridge, and R. Hyman. 1995. Eur. J. Immunol. 25:495-501). Here we show that in rat pancreatic carcinoma cells, splice variants of CD44 (CD44v), but not CD44s, form molecular aggregates in the plasma membrane. We demonstrate that reduction-sensitive dimerization of CD44v occurs, and also that larger aggregations of the protein can be stabilized by chemical cross-linking. Different CD44v proteins present on the same cell exclusively form hom...