Localization of T25 glycoprotein in wild-type and Thy 1- mutant cells by immunofluorescence and immunoelectron microscopy.

The wild-type BW5147 (Thy 1+) cell line and its Thy 1- mutant derivative BW5147 (Thy 1-a) were examined by immunofluorescence and immunoelectron microscopy for the presence of T25, the glycoprotein which bears the Thy 1 alloantigen. The wild-type cell had T25 predominantly localized on the cell surface. In the mutant cell line, T25 accumulated intracellularly and was present in a clustered distribution throughout the cytoplasm. T25 was not present on the surface of the mutant cell line in significant amount.

Biochemical analysis of ligand-induced receptor patching and capping using a novel immunolactoperoxidase iodination technique.

A novel approach for the analysis of membrane proteins involved in ligand-induced surface receptor patching and capping is described. The technique is based on the use of immunolactoperoxidase (immuno-LPO) conjugates which catalyze the iodination of those surface proteins with available tyrosine groups that are located in the immediate vicinity of the patch or cap of a particular antigen. We have used the patching and capping of the H-2 (histocompatibility) antigen on mouse thymocytes to illustrate this method. However, this technique should be generally applicable to any cell surface proteins which can be induced to form patches or caps by a specific ligand. Cytochemical analysis indicates that the immuno-LPO conjugates induce the same patching and capping of the H-2 antigen as does the unconjugated antibody. Biochemical analysis of the 125I-labeled proteins by SDS polyacrylamide gel electrophoresis indicates that a large membrane protein (mol wt of approximately 200,000 daltons) is closely associated with H-2 patches and caps. Since a number of other prominent membrane proteins are not labeled by this procedure, selective redistribution of certain surface proteins must be occurring during H-2 antibody-induced patching and capping.

Mouse T lymphoma cells contain a transmembrane glycoprotein (GP85) that binds ankyrin.

In this study we have used complementary biochemical and immunological techniques to establish that the lymphoma GP85 membrane glycoprotein is a transmembrane protein with a cytoplasmic domain that binds directly to ankyrin, a molecule known to link the membrane to the cytoskeleton. The evidence supporting our conclusion that the GP85 is a transmembrane glycoprotein is as follows: (a) GP85 can be surface-labeled with Na 125I and contains wheat germ agglutinin-binding sites, indicating that it has an extracellular domain; (b) GP85 can be phosphorylated by intracellular kinases, indicating that it has an intracellular domain; and (c) GP85 can be successfully incorporated into phospholipid vesicles, indicating the existence of a hydrophobic domain in the molecule. Further studies show that GP85 displays immunological cross-reactivity with the lymphocyte Pgp-1 (differentiation-specific) membrane glycoprotein, and with the erythrocyte anion transport membrane protein, band 3. Immunocytochemical studies indicate that an ankyrin-like protein accumulates underneath the lymphoma GP85 cap structure, suggesting an association of the ankyrin-like protein and GP85. This relationship has been further confirmed by the following results of binding and reconstitution experiments: (a) purified GP85 binds directly to an ankyrin-Sepharose column; (b) purified GP85 inserts into phospholipid vesicles in both the normal (right side out) and reversed (inside out) orientation (and with only the reversed configuration permits binding of ankyrin to GP85); and (c) cleavage of GP85 with trypsin yields a 40-kD peptide fragment that is part of the cytoplasmic domain and contains the ankyrin binding site(s). Based on these findings, we suggest that the lymphoma GP85 transmembrane glycoprotein contains a cytoplasmic domain that is directly involved in linking ankyrin to the cytoskeleton. This transmembrane linkage may play a pivotal role in receptor capping and cell activation in lymphocytes.

Ankyrin-binding domain of CD44(GP85) is required for the expression of hyaluronic acid-mediated adhesion function.

GP85 is one of the most common hemopoietic isoforms of the cell adhesion molecule, CD44. CD44(GP85) is known to contain at least one ankyrin-binding site within its 70 aa cytoplasmic domain and to bind hyaluronic acid (HA) with its extracellular domain. In this study we have mapped the ankyrin-binding domain of CD44(GP85) by deleting various portions of the cytoplasmic region followed by expression of these truncated cDNAs in COS cells. The results of these experiments indicate that the ankyrin-binding domain resides between amino acids 305 and 355. Biochemical analyses, using competition binding assays and a synthetic peptide (NGGNGT-VEDRKPSEL) containing 15 aa between aa 305 and aa 320, support the conclusion that this region is required for ankryin binding. Furthermore, we have constructed a fusion protein in which this 15 aa sequence of CD44(GP85) is transplanted onto another transmembrane protein which does not bind ankyrin. Our results show that this fusion protein acquires the ability to bind ankyrin confirming that the sequence (306NGGNGTVEDRKPSE320L) is a critical part of the ankryin-binding domain of CD44(GP85). In addition, we have demonstrated that deletion of this 15 aa ankyrin-binding sequence from CD44(GP85) results in a drastic reduction (> or = 90%) of HA-binding and HA-mediated cell adhesion. These findings strongly suggest that ankyrin binding to the cytoplasmic domain of CD44(GP85) plays a pivotal role in regulating hyaluronic acid-mediated cell-cell and cell-extracellular matrix interactions.

Incorporation of polypeptides into thylakoid membranes of Chlamydomonas reinhardtii. Cyclic variations.

A purified fraction of unstacked thylakoid membranes (TMF1u) has been obtained from homogenates of Chlamydomonas reinhardtii (wild type 137+) by using repeated centrifugates in sucrose density gradients and low salt treatment. The contaminants of the fraction are reduced to a few mitochondria (approximately 3% of the total mitochondrial population), a few osmiophilic granules, and fragments of chloroplast envelopes. By SDS-polyacrylamide gel electrophoresis the polypeptide components of TMF1u were resolved into at least 30 bands. To determine the relative rates of assembly of newly synthesized polypeptides into thylakoid membranes, synchronized algal cells were doubly labeled in vivo with L-[14C] and L-[3H]arginine--used for long- and short-term labeling, respectively. TMF1u's were isolated from the labeled cells at selected time points during the cycle and the distribution of radioactivity was assayed in the gel electrophoretograms of their solubilized polypeptides. Incorporation of newly synthesized polypeptides into the bands of the gels was found to occur continuously but differentially throughout the cycle. Maximal rates of incorporation for the majority of the polypeptides were detected shortly after cell division (6D-7D; equivalent to early G1 phase). The rates of radioactive labeling decreased gradually to a low level at the end of the dark period and then rose slightly at the beginning of the next light period. The findings suggest that, in addition to the light/dark control postulated in the past, assembly of newly synthesized proteins into thylakoid membranes is activated by signals at work in the early G1 phase.

Lymphoma Thy-1 glycoprotein is linked to the cytoskeleton via a 4.1-like protein.

In this study we have found that the phosphoprotein doublet of 68,000 and 65,000 daltons (68/65 kD) in mouse T-lymphoma cells shares several structural and functional similarities with erythrocyte band 4.1. Our evidence for identifying the 68/65-kD doublet as a lymphoma 4.1-like protein is as follows: it displays an immunological cross-reactivity with anti-erythrocyte band 4.1 antibody; it exhibits a Svedberg unit of sedimentation coefficient of 4 S; it is phosphorylated in the presence of phorbol ester (phorbol-12-O-tetradecanoylphorbol-13-acetate) and its phosphorylation requires Ca2+; it is phosphorylated primarily at serine residues; and it can bind directly to fodrin (a spectrin-like actin-binding protein). In addition, this lymphoma 4.1-like protein can be both colocalized and coisolated with the major T-lymphocyte-specific glycoprotein, Thy-1 (gp 25). Therefore, all of these results strongly suggest that the lymphoma 4.1-like protein (68/65-kD doublet) may play a pivotal role in linking the Thy-1 (gp 25) glycoprotein to fodrin which, in turn, binds to the actin filaments that are responsible for recruiting Thy-1 antigens into cap structures.

A lymphoma plasma membrane-associated protein with ankyrin-like properties.

In this study we have used several complementary techniques to isolate and characterize a 72-kD polypeptide that is tightly associated with a major mouse T-lymphoma membrane glycoprotein, gp 85 (a wheat germ agglutinin-binding protein), in a 16 S complex. These two proteins do not separate in the presence of high salt but can be dissociated by treatment with 2 M urea. Further analysis indicates that the 72-kD protein has ankyrin-like properties based on the following criteria: (a) it cross-reacts with specific antibodies raised against erythrocyte and brain ankyrin; (b) it displays a peptide mapping pattern and a pI (between 6.5 and 6.8) similar to that of the 72-kD proteolytic fragment of erythrocyte ankyrin; (c) it competes with erythrocyte ghost membranes (spectrin-depleted preparations) for spectrin binding; and (d) it binds to purified spectrin and fodrin molecules. Most importantly, in intact lymphoma cells this ankyrin-like protein is localized directly underneath the plasma membrane and is found to be preferentially accumulated beneath receptor cap structures as well as associated with a membrane-cytoskeleton complex preparation. It is proposed that the ankyrin-like 72-kD protein may play an important role in linking certain surface glycoprotein(s) to fodrin which, in turn, binds to actin filaments required for lymphocyte cap formation.

Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration.

Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11-amino acid sequence between amino acids 717 and 727 of Tiam1 ((717)GEGTDAVKRS(727)L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1). Using an Escherichia coli-derived calmodulin-binding peptide (CBP)-tagged recombinant Tiam1 (amino acids 393-728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393-738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1-ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)-tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1-ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin-Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.

Phosphorylation of myosin light chain during capping of mouse T-lymphoma cells.

Colchicine induces the clustering of at least three different T-lymphoma surface antigens (T200, Thy-1, and gp 69/71) into a cap structure in the absence of any external ligand. In addition, colchicine induces the intracellular accumulation of actin and myosin directly beneath the surface cap structure. We have discovered that myosin molecules (both heavy and light chains) are closely associated with the plasma membrane of T-lymphoma cells. Most importantly, we have found that the 20,000-dalton light chain of lymphocyte myosin is both phosphorylated and preferentially accumulated in the plasma membrane of colchicine-induced capped cells. It is proposed that myosin light chain is directly involved in the activation of membrane-associated actomyosin required for the collection of surface proteins into a cap structure (analogous to muscle cell sliding filament contraction).

Association of myosin light chain kinase with lymphocyte membrane-cytoskeleton complex.

A specific antibody against myosin light chain kinase (MLCK) was used to identify the presence of a Ca2+-calmodulin-activated MLCK in mouse 1-lymphoma cells. With a double immunofluorescence technique, MLCK was determined to be accumulated directly under Con A-capped structures in a manner similar to that of previously described accumulation of actomyosin. The lymphocyte MLCK was phosphorylated in the uncapped cell and, by immunoprecipitation with a specific MLCK antibody, was shown to possess a Mr of 130,000. The MLCK was also found to constitute a major fraction of the phosphoproteins present in the plasma membrane associated-cytoskeleton. Myosin light chain kinase catalyzed the phosphorylation of both endogenous lymphocyte myosin light chains and those from smooth and skeletal muscle. The enzyme activity was dependent on the presence of Ca2+-calmodulin and was inhibited by the calmodulin-binding drug, trifluoperazine. These data suggest that the membrane-cytoskeleton-associated MLCK activity may be important in regulation of the actinmyosin contraction which is believed to be required for the collection of surface receptors into capped structures.

A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin.

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.

A CD44-like endothelial cell transmembrane glycoprotein (GP116) interacts with extracellular matrix and ankyrin.

We used complementary biochemical and immunological techniques to establish that an endothelial cell transmembrane glycoprotein, GP116, is a CD44-like molecule and binds directly both to extracellular matrix components (e.g., hyaluronic acid) and to ankyrin. The specific characteristics of GP116 are as follows: (i) GP116 can be surface labeled with Na 125I and contains a wheat germ agglutinin-binding site(s), indicating that it has an extracellular domain; (ii) GP116 displays immunological cross-reactivity with a panel of CD44 antibodies, shares some peptide similarity with CD44, and has a similar 52-kDa precursor molecule, indicating that it is a CD44-like molecule; (iii) GP116 displays specific hyaluronic acid-binding properties, indicating that it is a hyaluronic acid receptor; (iv) GP116 can be phosphorylated by endogenous protein kinase C activated by 12-O-tetradecanoylphorbol-13-acetate and by exogenously added protein kinase C; and (v) GP116 and a 20-kDa tryptic polypeptide fragment of GP116 from the intracellular domain are capable of binding the membrane-cytoskeleton linker molecule, ankyrin. Furthermore, phosphorylation of GP116 by protein kinase C significantly enhances GP116 binding to ankyrin. Together, these findings strongly suggest that phosphorylation of the transmembrane glycoprotein GP116 (a CD44-like molecule) by protein kinase C is required for effective GP116-ankyrin interaction during endothelial cell adhesion events.

Further characterization of a fodrin-containing transmembrane complex from mouse T-lymphoma cells.

A transmembrane complex containing fodrin (an actin-binding protein) and a major surface glycoprotein (GP 180) was previously isolated from mouse T-lymphoma cells by the complementary techniques of non-ionic detergent extraction and sucrose gradient centrifugation (Bourguignon et al. (1985) J. Cell Biol. 101, 477-487). The analysis of this complex has been extended to verify the structural association and further define the interaction between fodrin and GP 180. The association between fodrin and GP 180 has been confirmed by the following evidence: co-sedimentation of fodrin and GP 180 in a single peak on a sucrose gradient with a sedimentation coefficient of 20 S; a constant ratio of fodrin and GP 180 across the 20 S peak; the specific co-precipitation of GP 180 with fodrin from the 20 S peak using anti-fodrin antibody; and the colocalization of fodrin and GP 180 from the 20 S peak on actin filaments using an immuno-electron microscopic technique. Furthermore, this fodrin-GP 180 complex can be readily dissociated and reassembled in the presence and absence of 0.6 M NaCl, respectively. The fact that this fodrin-GP 180 complex displays actin-binding ability indicates that this transmembrane complex may play an important role in the linking event between receptors and the cytoskeleton during lymphocyte patching and capping.

The participation of adenylate cyclase in lymphocyte capping.

In this study, we have observed that cells increase their intracellular cAMP to relatively high levels during receptor capping induced by either ligand-dependent (anti-Thy-1 antibody) or ligand-independent (colchicine) treatment. In addition, we have found that under capping conditions, membrane-bound adenylate cyclase is induced to co-cap with independent membrane molecules such as Thy-1 antigens. These findings suggest that the binding of anti-Thy-1 to its receptors or treatment with colchicine induces the molecular reorganization of membrane-bound adenylate cyclase which may be responsible for activating the contractile machinery required for the collection of surface receptors into a cap structure.

Isolation and initial characterization of a lymphocyte cap structure.

A method for isolating the cap structure induced by polycationized ferritin on the surface of mouse T-lymphoma cells is described. The procedure, based on the 'density perturbation' approach designed by Wallach and co-workers (Wallach, D.F.H., Kranz, B., Ferber, E. and Fischer, H. (1972) FEBS Lett. 21, 29-33), involves a simple, one-step density gradient centrifugation using metrizamide as the gradient material. The isolated polycationized ferritin cap fraction is approx. 20-fold enriched in plasma membrane relative to the whole cell homogenate and is apparently free of all uncapped membrane. Our initial analysis of the protein composition of the isolated cap structure indicates that there are approx. 30 membrane-bound polypeptides specifically associated with the polycationized ferritin cap fraction. Interestingly, there are at least four phosphorylated membrane-bound polypeptides (mol.wt. approximately 130 000, 100 000, 30 000 and 20 000) which are preferrentially accumulated in the cap fraction. These findings provide further evidence for the selective redistribution of certain surface membrane proteins during lymphocyte capping.

Role of Ca2+ in the regulation of hormone receptor exposure during lymphocyte activation.

Ca2+ is known to be required for mitogen-mediated lymphocyte activation. In order to further define the regulatory role of Ca2+, we have examined the activation events which occur following treatment with ionomycin (a Ca2+ ionophore), as compared to those occurring following concanavalin A (Con A) treatment of mouse splenic T-lymphocytes. Our results indicate that ionomycin and Con A induce the exposure of both interleukin-2 (IL-2) and insulin receptors on the surface of the lymphocytes within the first 5 min of treatment. The exposed insulin and IL-2 receptors have the following properties: (1) they consist of both high- and low-affinity receptors; and (2) they appear on the cell surface in small clusters (i.e., patches) or, occasionally, a large aggregate (i.e., cap). c-myc gene expression and DNA synthesis occur in both the ionomycin and Con A-treated lymphocytes when either IL-2 or insulin is present in the culture medium. Furthermore, the exposure of both hormone receptors can be inhibited by either EGTA (a Ca2+ chelator), bepridil (a Ca2+ channel blocker), W-7 (a calmodulin antagonist) or cytochalasin D (a microfilament inhibitor). Treatment with these inhibitors also blocks the expression of c-myc gene and DNA synthesis which occur at later times during IL-2 and insulin-induced activation of ionomycin- and Con A-treated lymphocytes. These findings suggest that a Ca2+ and calmodulin-mediated contractile system is involved in the exposure of certain hormone receptors which appear to be required for complete lymphocyte activation.

A new splice variant of the inositol-1,4,5-triphosphate (IP3) receptor.

In this study we have identified a new splice variant of the IP3 receptor (IP3R) transcript in a number of mouse cell lines (e.g. mouse T-lymphoma cells, mouse splenic lymphocytes and mouse NIH 3T3 fibroblast cell lines) using the reverse transcriptase-polymerase chain reaction. This variant IP3 receptor (designated as IP3RV-S2, approximately 453 bp) is larger than the non-neuronal form (402 bp) but smaller than the neuronal form (522 bp) of the IP3 receptors. Nucleotide sequencing data indicate that this new isoform (IP3RV-S2) contains a 51 nucleotide insertion within the non-neuronal form of IP3R at the S2 splice site. During mitogenic stimulation by Con A, the ratio between IP3R (non-neuronal form) and IP3RV-S2 (variant isoform) in mouse splenic T-lymphocytes increases approximately 1.5-fold. The change in relative amounts of these two IP3 receptor isoforms during mitogenic-stimulation suggests that T-lymphocytes may have different requirements for the IP3 isoforms in order to control intracellular calcium mobilization. The selective expression of these two IP3R isoforms (IP3RV-S2 and non-neuronal IP3R) may be critically important for the onset of signal transduction and cell activation.

Selective down-regulation of IP(3)receptor subtypes by caspases and calpain during TNF alpha -induced apoptosis of human T-lymphoma cells.

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP(3)R) [IP(3)-gated Ca(2+)channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP(3)R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca(2+)channel properties in an IP(3)-dependent manner. We have also demonstrated that TNFalpha promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca(2+)response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFalpha, several IP(3)R subtype-related changes occur (e.g. proteolysis of IP(3)R subtypes, inhibition of IP(3)binding and impairment of IP(3)-mediated Ca(2+)flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFalpha-mediated perturbation of IP(3)R1 and IP(3)R2 (but not IP(3)R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFalpha on IP(3)R3 function. These findings suggest that IP(3)R1 and IP(3)R2 serve as cellular substrates for caspases, and IP(3)R3 is a substrate for calpain. We propose that the selective down-regulation of IP(3)R subtype-mediated Ca(2+)function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFalpha in human T-cells.

Ca2+ signaling in endothelial cells stimulated by bradykinin: Ca2+ measurement in the mitochondria and the cytosol by confocal microscopy.

In this study we have monitored the change of intracellular Ca2+ concentrations in the cytosol ([Ca2+]c) and the mitochondria ([Ca2+]m) of single bovine endothelial cells following treatment with bradykinin (BK). Using laser scanning confocal microscopy, we have found that the Ca2+ indicator, Fluo-3, is compartmentalized in the mitochondria of endothelial cells loaded with Fluo-3/AM. After BK stimulation, the pattern of Ca2+ increase in the cytosol is different from that in the mitochondria. The amplitude of the Ca2+ rise in the mitochondria is higher than that in the cytosol. Further analysis using rapid scanning measurements indicates that the [Ca2+]c increase is very fast after BK addition and reaches a maxima level within 400 ms. In contrast, the [Ca2+]m increase appears to be biphasic with an initial rapid increase (concomitant with the [Ca2+]c increase) followed by a slower [Ca2+]m increase before reaching a maximal level (within 5 s of BK treatment). The differential Ca2+ signaling pattern between the cytosol and the mitochondria suggests that the intracellular Ca2+ concentrations needed to regulate various Ca(2+)-dependent enzymes located in these two compartments are different during BK-induced endothelial cell activation.

CD44 isoform-cytoskeleton interaction in oncogenic signaling and tumor progression.

CD44, a major hyaluronan receptor, exists as several isoforms and is widely distributed in different cells and tissues. The isoforms of CD44, such as CD44s (the standard form), CD44E (the epithelial form) and CD44v (variant isoforms) (arise from differential splicing of one to ten (or eleven) variable exons that encode portions of the membrane proximal extracellular domain. The molecular diversity of CD44 isoforms is further compounded by differential biosynthetic processes and post-translational modifications [e.g. N-/O-glycosylation or glycosaminoglycan (GAG) addition]. This structural arrangement, which occurs within either the invariant region or the extracellular domain of the variant region, is important for CD44-mediated communication between extracellular matrix materials [ECM-hyaluronic acid (HA), collagen and fibronectin] and intracellular protein components (e.g cytoskeletal proteins and various regulatory enzymes). The 15 amino acid sequence [e.g. NSGNGAVEDRKPSGL (in human) or NGGNGTVEDRKPSEL (in mouse)] residing in the cytoplasmic domain of CD44 isoforms is the ankyrin-binding domain of this family of transmembrane glycoproteins. Biochemical analyses plus in vitro mutagenesis indicate that the ankyrin-binding domain is required for CD44-mediated "outside-in" and "inside-out" cell activation events. Furthermore, CD44s-cytoskeleton interaction is tightly coupled with signal transducing molecules (e.g. p185HER2 or Src kinases) during oncogenic signaling. Moreover, the transmembrane linkage between CD44v isoforms (CD44v10 and CD44v3) and the cytoskeleton up-regulates invasive and metastatic-specific tumor phenotypes [e.g. matrix degradation (MMPs) activities, tumor cell invasion and migration]. These findings strongly suggest that the interaction between CD44 isoforms and the cytoskeleton plays a pivotal role in the onset of oncogenesis and tumor progression.