Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding.     

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex,as Revealed by Small Angle Neutron Scattering

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes.     

V3 versican isoform alters the behavior of human melanoma cells by interfering with CD44/ErbB-dependent signaling

Versican is a hyaluronan-binding, extracellular chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The short V3 isoform contains the G1 and G3 terminal domains of versican that may potentially interact directly or indirectly with the hyaluronan receptor CD44 and the EGFR, respectively. We have previously described that overexpression of V3 in MeWo human melanoma cells markedly reduces tumor cell growth in vitro and in vivo. In this study we have investigated the signaling mechanism of V3 by silencing the expression of CD44 in control and V3-expressing melanoma cells. Suppression of CD44 had the same effects on cell proliferation and cell migration than those provoked by V3 expression, suggesting that V3 acts through a CD44-mediated mechanism. Furthermore, CD44-dependent hyaluronan internalization was blocked by V3 expression and CD44 silencing, leading to an accumulation of this glycosaminoglycan in the pericellular matrix and to changes in cell migration on hyaluronan. Furthermore, ERK1/2 and p38 activation after EGF treatment were decreased in V3-expressing cells suggesting that V3 may also interact with the EGFR through its G3 domain. The existence of a EGFR/ErbB2 receptor complex able to interact with CD44 was identified in MeWo melanoma cells. V3 overexpression resulted in a reduced interaction between EGFR/ErbB2 and CD44 in response to EGF treatment. Our results indicate that the V3 isoform of versican interferes with CD44 and the CD44-EGFR/ErbB2 interaction, altering the signaling pathways, such as ERK1/2 and p38 MAPK, that regulate cell proliferation and migration.     

Immunolocalization of the hyaluronan receptor CD44 in the reproductive tract of the mare

Hyaluronan (HA), a glycosaminoglycan, is a major component of the pericellular matrix which envelopes mammalian cells. Binding of hyaluronan to one of its specific receptors, CD44, modulates transduction of intracellular signals which direct a variety of processes, including embryogenesis, wound healing, inflammation, and neoplasia. Since regulation of these processes is critical to equine reproductive success, localization of constitutive CD44 expression was evaluated by immunohistochemical methods in ovarian, oviductal, and uterine tissues from healthy mares. Ovarian stroma contained thecal cells with varying CD44 immunopositivity. Follicular and granulosa cells of some antral and atretic follicles were positive for CD44. In the oviduct, the luminal epithelium was variably positive for CD44, with overall decreasing intensity of immunostaining from the infundibulum to the isthmus. The CD44 molecule was expressed strongly by surface epithelial cells of the uterine endometrium, but was present only rarely among cells of uterine glands. In addition, CD44 was expressed by smooth muscle cells of vascular walls, oviduct, and uterus. Since CD44 is known to modulate cell movement and differentiation, and was present at multiple sites in the reproductive tract of normal mares, we inferred there may be an important role for the HA-CD44 signaling pathway in reproductive function and inflammation.     

Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells

Proinflammatory cytokines such as TNF-alpha up-regulate the expression of the cell adhesion molecule, CD44, and induce hyaluronan (HA) binding in peripheral blood monocytes (PBM). Here we show that in PBM, TNF-alpha induced cytoskeletal rearrangement, increased threonine phosphorylation of ERM proteins, and induced the redistribution and colocalization of phospho-ERM proteins (P-ERM) with CD44. In the myeloid progenitor cell line, KG1a, hyaluronan binding occurred in the pseudopod where CD44, P-ERM, and F-actin were highly localized. Hyaluronan binding correlated with high expression of both CD44 and P-ERM clustered in a single pseudopod. Disruption of polymerized actin reduced hyaluronan binding in both PBM and KG1a cells and abolished CD44 clustering and the pseudopod in KG1a cells. The pseudopod was not required for the clustering of CD44, the colocalization with P-ERM, or hyaluronan binding. However, treatment with a kinase inhibitor abolished ERM phosphorylation and reduced hyaluronan binding. Furthermore, expression of CD44 lacking the putative ERM binding site resulted in reduced hyaluronan binding. Taken together, these data suggest that CD44-mediated hyaluronan binding in human myeloid cells is regulated by P-ERM and the actin cytoskeleton.     

A Novel CD44-binding Peptide from the Pro-Matrix Metalloproteinase-9 Hemopexin Domain Impairs Adhesion and Migration of Chronic Lymphocytic Leukemia (CLL) Cells

(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4β1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC₅₀ value of 90 μm. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4β1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences.     

Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44

The tumor microenvironment makes a decisive contribution to the development and dissemination of cancer, for example, through extracellular matrix components such as hyaluronan (HA), and through chemokines that regulate tumor cell behavior and angiogenesis. Here we report a molecular link between HA, its receptor CD44 and the chemokine CXCL12 in the regulation of cell motility and angiogenesis. High-molecular-weight HA (hHA) was found to augment CXCL12-induced CXCR4 signaling in both HepG2iso cells and primary human umbilical vein endothelial cells, as evidenced by enhanced ERK phosphorylation and increased cell motility. The augmentation of CXCR4 signaling translated into increased vessel sprouting and angiogenesis in a variety of assays. Small HA oligosaccharides (sHA) efficiently inhibited these effects. Both siRNA-mediated reduction of CD44 expression and antibodies that block the interaction of CD44 with HA provided evidence that CXCL12-induced CXCR4 signaling depends on the binding of hHA to CD44. Consistently, CD44 and CXCR4 were found to physically interact in the presence of CXCL12, an interaction that could be inhibited by sHA. These findings provide novel insights into how microenvironmental components interact with cell surface receptors in multi-component complexes to regulate key aspects of tumor growth and progression.     

Hepatocyte growth factor enhances adhesion of breast cancer cells to endothelial cells in vitro through up-regulation of CD44

For cancer metastasis, tumor cells present in the circulation must first adhere to the endothelium. Integrins play a central role in leukocyte adhesion to the endothelium and subsequent migration into tissues. The majority of tumor cells derived from solid cancers, including breast cancer, do not express integrins. We investigated the mechanisms of adhesion and transendothelial migration of cancer cells using breast carcinoma cell lines. Our results showed the following features of breast cancer cells: (1) HGF stimulated breast cancer cells by up-regulating CD44 expression in a concentration-dependent manner. (2) the maximum level of HGF-induced CD44 up-regulation on breast cancer cell lines occurred within 3 h. (3) HGF-induced up-regulation of CD44 was mediated by the tyrosine kinase signaling pathway. (4) HGF induced CD44-mediated adhesion of tumor cell lines to bone marrow-derived endothelial cells. (5) HGF did not change rolling of breast cancer cell lines on bone marrow-derived endothelial cells, but enhanced firm adhesion of cancer cells on endothelial cells under shear stress conditions. (6) HGF increased transendothelial migration of cancer cells. Our results indicate that HGF stimulates CD44-mediated adhesion of breast cancer cells to bone marrow-derived endothelial cells, which subsequently results in transendothelial migration of tumor cells. These results suggest that CD44 may confer the metastatic properties of breast cancer cells and, therefore, could be used as a target in future molecular cancer therapy.     

Primary mesenchymal stem and progenitor cells from bone marrow lack expression of CD44 protein

Despite significant progress in our understanding of mesenchymal stem cell (MSC) biology during recent years, much of the information is based on experiments using in vitro culture-selected stromal progenitor cells. Therefore, the natural cellular identity of MSCs remains poorly defined. Numerous studies have reported that CD44 expression is one of the characteristics of MSCs in both humans and mice; however, we here have prospectively isolated bone marrow stromal cell subsets from both human and mouse bone marrow by flow cytometry and characterized them by gene expression analysis and function assays. Our data provide functional and molecular evidence suggesting that primary mesenchymal stem and progenitor cells of bone marrow reside in the CD44(-) cell fraction in both mice and humans. The finding that these CD44(-) cells acquire CD44 expression after in vitro culture provides an explanation for the previous misconceptions concerning CD44 expression on MSCs. In addition, the other previous reported MSC markers, including CD73, CD146, CD271, and CD106/VCAM1, are also differentially expressed on those two cell types. Our microarray data revealed a distinct gene expression profile of the freshly isolated CD44(-) cells and the cultured MSCs generated from these cells. Thus, we conclude that bone marrow MSCs physiologically lack expression of CD44, highlighting the natural phenotype of MSCs and opening new possibilities to prospectively isolate MSCs from the bone marrow.     

Binding of CD157 Protein to Fibronectin Regulates Cell Adhesion and Spreading

CD157/BST-1 behaves both as an ectoenzyme and signaling receptor and is an important regulator of leukocyte trafficking and ovarian cancer progression. However, the molecular interactions underpinning the role of CD157 in these processes remain obscure. The biological functions of CD157 and its partnership with members of the integrin family prompted us to assume the existence of a direct interaction between CD157 and an unknown component of the extracellular matrix. Using solid-phase binding assays and surface plasmon resonance analysis, we demonstrated that CD157 binds fibronectin with high affinity within its heparin-binding domains 1 and 2. Furthermore, we found that CD157 binds to other extracellular matrix proteins containing heparin-binding domains. Finally, we proved that the CD157-fibronectin interaction occurs with living cells, where it elicits CD157-mediated cell responses. Indeed, knockdown of CD157 in Met-5A mesothelial cells changed their morphology and cytoskeleton organization and attenuated the activation of intracellular signaling pathways triggered by fibronectin. This led to impaired cell spreading and adhesion to selected extracellular matrix proteins. Collectively, these findings indicate a central role of CD157 in cell-extracellular matrix interactions and make CD157 an attractive therapeutic target in inflammation and cancer.     

CD44与HA特异性结合在肿瘤靶向给药中的应用

设计肿瘤靶向性抗癌药物一直是研究热点。因为CD44在许多肿瘤细胞表面过表达,所以基于CD44与其配体透明质酸（hyaluronic acid,HA）的相互作用设计肿瘤靶向药物成为一个新的研究方向。本文介绍了CD44的基本结构7LCD44与HA之间的相互作用,并对以CD44为靶点的靶向抗癌药物研究进展进行了简介。     

The C-terminal Peptide of Chondroadherin Modulates Cellular Activity by Selectively Binding to Heparan Sulfate Chains

Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH³⁵⁹, now shown to bind to heparin with a K_D of 13 μm. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparan sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their β₁ integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.     

MOC-31和CD44v6在良恶性腹水鉴别诊断中的应用

目的探讨检测肿瘤标志物MOC-31和CD44v6对鉴别良恶性腹水的诊断价值。方法利用液基薄层细胞学自动涂片技术方法筛查出查到肿瘤细胞的恶性腹水标本390例以及良性腹水标本100例,分别采用酶联免疫吸附法(ELISA)和免疫细胞化学染色检测MOC-31和CD44v6的含量和表达情况。结果 ELISA结果显示MOC-31和CD44v6在良性腹水中的含量分别为21±4ng/ml和291±32ng/ml,在恶性腹水中的含量分别为98.1±19.3ng/ml和891±116ng/ml,差异均具有显著性(P<0.05);免疫细胞化学染色显示MOC-31在恶性腹水细胞中阳性表达250例,良性腹水细胞中阳性表达5例;CD44v6在恶性腹水细胞中阳性表达266例,良性腹水细胞中阳性表达3例,差异均具有显著性(P<0.05)。结论 MOC-31和CD44v6可以做为良恶性腹水鉴别诊断的重要指标,值得在临床病理工作中推广应用。     

Glycated albumin and cross-linking of CD44 induce scavenger receptor expression and uptake of oxidized LDL in human monocytes

Functional role of CD44, a principal receptor of hyaluronan, and glycated albumin for differentiation of resting human monocytes isolated by counterflow centrifugal elutriation was investigated. Flow cytometric analysis revealed that amadori-modified glycated albumin induced expression of CD44 as well as macrophage scavenger receptors (MSRs) such as CD36 and CD68 on resting monocytes. Crosslinking of CD44 on monocytes also induced MSR expression. Furthermore, CD44 crosslinking and/or glycated albumin enhanced the uptake of oxidized-low density lipoprotein in monocytes and foam cell formation. Taken together, engagement of CD44 (e.g., hyaluronan) and glycated albumin induced the differentiation of resting monocytes into foam macrophages through the induction of MSRs, implying that CD44 could be involved in atherosclerotic lesions of those such as diabetic patients.     

HIV-1 p17 matrix protein interacts with heparan sulfate side chain of CD44v3, syndecan-2, and syndecan-4 proteoglycans expressed on human activated CD4+ T cells affecting tumor necrosis factor alpha and interleukin 2 production

HIV-1 p17 contains C- and N-terminal sequences with positively charged residues and a consensus cluster for heparin binding. We have previously demonstrated by affinity chromatography that HIV-1 p17 binds strongly to heparin-agarose at physiological pH and to human activated CD4(+) T cells. In this study we demonstrated that the viral protein binds to heparan sulfate side chains of syndecan-2, syndecan-4, and CD44v3 purified from HeLa cells and that these heparan sulfate proteoglycans (HSPGs) co-localize with HIV-1 p17 on activated human CD4(+) T cells by confocal fluorescence analysis. Moreover, we observed a stimulatory or inhibitory activity when CD4(+) T cells were activated with mitogens together with nanomolar or micromolar concentrations of the matrix protein.     

Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor

The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited by uPA binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexibility by covalently tethering domains DI and DIII via a non-natural interdomain disulfide bond (uPAR(H47C-N259C)). The corresponding soluble receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered binding kinetics towards uPA. Most importantly, the purified uPAR(H47C-N259C) also displays a gain in affinity for the somatomedin B domain of vitronectin compared with uPAR(wt), thus recapitulating the improved affinity that accompanies uPA-uPAR(wt) complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus bypasses the regulatory role of uPA binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.     

Coelomocyte locomotion in the sipunculan Themiste petricola induced by exogenous and endogenous chemoattractants: role of a CD44-like antigen--HA interaction

Cell migration is a key event in the invertebrate immuno-defense system. Microbial products like lipopolysacharide (LPS) and formyl-methyl-leucyl-phenylalanine (fMLP) promote cell recruitment to sites of infection. In mammals, complement activation by factors such as zymosan induces C5a production, which influences leukocyte migration. The endogenous factor hyaluronic acid (HA), an extracellular matrix component, also promotes cell migration through its receptor CD44. We evaluated whether coelomocytes from the sipunculan worm T. petricola migrated towards LPS, fMLP, or zymosan treated plasma (ZTP) and if HA was involved in coelomocyte migration and adhesion. We also evaluated if antibodies specific for mouse HA receptor CD44 inhibited any of the effects induced by HA. Using microchemotaxis chambers we found that coelomocytes migrated towards exogenously and endogenously derived chemoattractants. We also observed that HA was a potent chemotactic signal and that coelomocytes adhered strongly to plates coated with LMW-HA but not with HMW-HA. In addition we found that these HA mediated effects were blocked by the monoclonal antibody IM7 directed to mouse CD44, suggesting that a CD44-like cross-reactive antigen might play a role in HA mediated coelomocyte locomotion.     

The hyperthermia-enhanced association between tropoelastin and its 67-kDa chaperone results in better deposition of elastic fibers

The results of our in vitro experiments indicate that exposing cultured human aortic smooth muscle cells and dermal fibroblasts to 39 to 41 °C induces a significant up-regulation in the net deposition of elastic fibers, but not of collagen I or fibronectin, and also decreases the deposition of chondroitin sulfate-containing moieties. We further demonstrate that mild hyperthermia also rectifies the insufficient elastogenesis notable in cultures of fibroblasts derived from the stretch-marked skin of adult patients and in cultures of dermal fibroblasts from children with Costello syndrome, which is characterized by the accumulation of chondroitin 6-sulfate glycosaminoglycans that induce shedding and inactivation of the 67-kDa elastin-binding protein. We have previously established that this protein serves as a reusable chaperone for tropoelastin and that its recycling is essential for the normal deposition of elastic fibers. We now report that hyperthermia not only inhibits deposition of chondroitin 6-sulfate moieties and the consequent preservation of elastin-binding protein molecules but also induces their faster recycling. This, in turn, triggers a more efficient preservation of tropoelastin, enhancement of its secretion and extracellular assembly into elastic fibers. The presented results encourage using mild hyperthermia to restore elastic fiber production in damaged adult skin and to enhance elastogenesis in children with genetic elastinopathies.     

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.     

Topography of amphiregulin expression in cultured human keratinocytes: Colocalization with the epidermal growth factor receptor and CD44

Summary Much of the autonomous growth of cultured keratinocytes is attributable to the signaling of amphiregulin, a heparin-binding autocrine growth factor, through the epidermal growth factor receptor. Emerging evidence suggests, moreover, that the membrane proteoglycan, CD44, is a cofactor for the interaction of heparin-binding ligands with their receptors. This model was evaluated by characterizing the patterns of the immunolabeled molecules in cultured human neonatal keratinocytes, to test the hypothesis that involvement in a common function results in coordinate segregation within or on the cell. The molecules were localized by double immunofluorescence labeling to detect amphiregulin and either the epidermal growth factor receptor or CD44, and the immunostained products were imaged by scanning laser confocal microscopy. Both amphiregulin and the epidermal growth factor receptor segregated to a perinuclear distribution and to intercellular contacts. In addition, amphiregulin localized to the outer leading edge of colonies and focally to intranuclear sites. Metabolic blockade of proteoglycan sulfation with sodium chlorate inhibited growth of the cells and concurrently enhanced the nuclear, but decreased the outer leading edge, labeling for amphiregulin. There was no nuclear or perimeter labeling for the epidermal growth factor receptor. Cultures co-immunolabeled for CD44 and amphiregulin exhibited variable perinuclear staining for both, but otherwise CD44 was distributed to intercellular contacts. The intercellular localizations of CD44 with amphiregulin and of amphiregulin with the epidermal growth factor receptor were strongly concordant. These data are consistent with a concerted function at intercellular contacts, where cytokine signaling is mediated via receptor binding and possibly regulated by the CD44 proteoglycan as cofactor. The intranuclear and perimeter labeling of amphiregulin, however, suggests that this cytokine has additional functions, both in the nucleus and as a matrix receptor.