Agrin-induced phosphorylation of the acetylcholine receptor regulates cytoskeletal anchoring and clustering

At the developing neuromuscular junction, a motoneuron-derived factor called agrin signals through the muscle-specific kinase receptor to induce postsynaptic aggregation of the acetylcholine receptor (AChR). The agrin signaling pathway involves tyrosine phosphorylation of the AChR beta subunit, and we have tested its role in receptor localization by expressing tagged, tyrosine-minus forms of the beta subunit in mouse Sol8 myotubes. We find that agrin-induced phosphorylation of the beta subunit occurs only on cell surface AChR, and that AChR-containing tyrosine-minus beta subunit is targeted normally to the plasma membrane. Surface AChR that is tyrosine phosphorylated is less detergent extractable than nonphosphorylated AChR, indicating that it is preferentially linked to the cytoskeleton. Consistent with this, we find that agrin treatment reduces the detergent extractability of AChR that contains tagged wild-type beta subunit but not tyrosine-minus beta subunit. In addition, agrin-induced clustering of AChR containing tyrosine-minus beta subunit is reduced in comparison to wild-type receptor. Thus, we find that agrin-induced phosphorylation of AChR beta subunit regulates cytoskeletal anchoring and contributes to the clustering of the AChR, and this is likely to play an important role in the postsynaptic localization of the receptor at the developing synapse.     

Cell surface beta-1,4-galactosyltransferase is associated with the detergent-insoluble cytoskeleton on migrating mesenchymal cells.

Cell surface beta-1,4-galactosyltransferase (GalTase) partially mediates a variety of cell interactions with laminin-containing matrices, including mesenchymal cell spreading and migration and neurite initiation, by binding to N-linked oligosaccharides within the E8 domain of laminin. Previous studies using indirect immunofluorescence have suggested that some surface GalTase colocalizes with actin-containing microfilaments in migrating cells. In this study, we present more direct biochemical evidence showing that surface GalTase is associated with the detergent-insoluble cytoskeleton and that this association is dependent upon the integrity of the cytoskeleton, valency of the anti-GalTase antibody, and migratory status of the cell. Two-thirds of the surface GalTase was associated with the detergent-insoluble cytoskeleton when assayed either by monovalent anti-GalTase Fab fragments or by extracting any detergent-soluble GalTase prior to labeling with intact anti-GalTase IgG. However, 80-100% of the surface GalTase could be induced to associate with the cytoskeleton when cross-linked with anti-GalTase IgG prior to detergent extraction. Destabilizing cytoskeleton-protein interactions with high levels of KCl, elevated pH, or cytochalasin B reduced the amount of surface GalTase retained in the detergent-insoluble cytoskeleton fraction. Finally, we have shown previously that laminin induces the expression of GalTase onto lamellipodia of migrating cells, and in this study, we show that the laminin-induced increase in surface GalTase is cytoskeletally associated. Collectively, these data suggest that cell surface GalTase participates in cell spreading and migration on laminin by virtue of its association with the cytoskeleton.     

Laminin polymerization induces a receptor-cytoskeleton network

The transition of laminin from a monomeric to a polymerized state is thought to be a crucial step in the development of basement membranes and in the case of skeletal muscle, mutations in laminin can result in severe muscular dystrophies with basement membrane defects. We have evaluated laminin polymer and receptor interactions to determine the requirements for laminin assembly on a cell surface and investigated what cellular responses might be mediated by this transition. We found that on muscle cell surfaces, laminins preferentially polymerize while bound to receptors that included dystroglycan and alpha7beta1 integrin. These receptor interactions are mediated through laminin COOH-terminal domains that are spatially and functionally distinct from NH2-terminal polymer binding sites. This receptor-facilitated self-assembly drives rearrangement of laminin into a cell-associated polygonal network, a process that also requires actin reorganization and tyrosine phosphorylation. As a result, dystroglycan and integrin redistribute into a reciprocal network as do cortical cytoskeleton components vinculin and dystrophin. Cytoskeletal and receptor reorganization is dependent on laminin polymerization and fails in response to receptor occupancy alone (nonpolymerizing laminin). Preferential polymerization of laminin on cell surfaces, and the resulting induction of cortical architecture, is a cooperative process requiring laminin- receptor ligation, receptor-facilitated self-assembly, actin reorganization, and signaling events.     

Interactions between laminin receptor and the cytoskeleton during translation and cell motility

Human laminin receptor acts as both a component of the 40S ribosomal subunit to mediate cellular translation and as a cell surface receptor that interacts with components of the extracellular matrix. Due to its role as the cell surface receptor for several viruses and its overexpression in several types of cancer, laminin receptor is a pathologically significant protein. Previous studies have determined that ribosomes are associated with components of the cytoskeleton, however the specific ribosomal component(s) responsible has not been determined. Our studies show that laminin receptor binds directly to tubulin. Through the use of siRNA and cytoskeletal inhibitors we demonstrate that laminin receptor acts as a tethering protein, holding the ribosome to tubulin, which is integral to cellular translation. Our studies also show that laminin receptor is capable of binding directly to actin. Through the use of siRNA and cytoskeletal inhibitors we have shown that this laminin receptor-actin interaction is critical for cell migration. These data indicate that interactions between laminin receptor and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer, cellular translation and migration.     

High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells.

Sindbis virus is an alphavirus with a very wide host range, being able to infect many birds and mammals as well as mosquitoes. We have isolated a monoclonal antibody that largely blocks virus binding to mammalian cells. This antibody was found to be directed against the C-terminal domain of the high-affinity laminin receptor, a 67-kDa protein present on the cell surface that binds with high affinity to basement membrane laminin and that is known to be important in development and in tumor invasion. This receptor is believed to be formed from a 295-amino-acid polypeptide that is modified in some unknown way after translation. The primary sequence of this 295-amino-acid protein is highly conserved among mammals. We found the hamster amino acid sequence to be identical to a mouse sequence and to differ at only two amino acids from a human sequence and at two amino acids from a bovine sequence. To verify the importance of the laminin receptor for infection by Sindbis virus, hamster cells were stably transfected with the gene encoding the 295-amino-acid protein under the control of a high-efficiency promoter. Such transfected hamster cells overexpressed the laminin receptor at the cell surface, bound severalfold more Sindbis virions than did the parental cells, and became infected by Sindbis virus with a higher efficiency. In contrast, cells transfected with the antisense gene expressed less laminin receptor on the surface and were less susceptible to the virus. Binding of the virus varied linearly with the amount of laminin receptor on the cell surface, whereas infectivity measured with a plaque assay varied with the 1.4 power of the receptor concentration, suggesting that interaction with more than one receptor aids virus penetration. By these criteria, the laminin receptor functions as the major receptor for Sindbis virus entry into mammalian cells. We also found that the anti-laminin receptor antibody partially blocked Sindbis virus binding to mosquito cells, suggesting that the laminin receptor is conserved in mosquitoes and functions as a Sindbis virus receptor in this host. The wide distribution of this highly conserved receptor may be in part responsible for the broad host range exhibited by the virus, which infects a wide range of mammals and birds as well as its mosquito vector and can infect many different tissues within these hosts.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel cell binding site in the coiled-coil domain of laminin involved in capillary morphogenesis

Recently, we reported the isolation and characterization of an anti-laminin antibody that modulates the extracellular matrix-dependent morphogenesis of endothelial cells. Here we use this antibody to precisely map the binding site responsible for mediating this biologically important interaction. By using a phage display-assisted mapping strategy to preserve protein structure, we demonstrate for the first time that the coiled-coil region of laminin contains a cell binding site. The adhesion motif is formed by residues contributed by both alpha and gamma chains, and is located in the middle part of the rod-like portion in a highly flexible area, which corresponds to a protease-susceptible site. Based on this information, a peptide mimotope was used to characterize the cognate receptor. Although we can not rule out the implication of other receptors, our results demonstrate that the laminin helical rod active site interacts with alpha2beta1 integrin on the surface of endothelial cells. These findings provide new insight into the complex mechanisms regulating capillary morphogenesis.     

Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells

The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross- linked by antibodies, they initially assimilate into detergent- resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets.     

Transmembrane control of laminin, lectin receptors and surface villi in teratocarcinoma-derived endodermal cells

Distribution of laminin on the surface of teratocarcinoma-derived parietal endoderm cells was studied by immuno-histochemical staining of the fixed specimen using affinity-purified anti-laminin antibody. Laminin was distributed on the basal surface of the cells, while treatment either with colchicine or with cytochalasin D (CD) resulted in a severely polarized distribution; laminin was seen only at one end of the cell. Treatment with both the reagents did not cause the severe polarization. Receptors for lectins and cell surface villi were polarized by treatment with CD but not by treatment with colchicine. These results suggest that laminin--or its cell surface receptor--is linked to both microfilament and microtubules and that the mode of transmembrane control for laminin is different from certain other cell surface components of the cells.     

The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin

Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation.     

The alpha4 laminin subunit regulates endothelial cell survival

The alpha4 laminin subunit is a major structural component of assembling basement membranes of endothelial cells. We have been investigating its functions with regard to endothelial cell survival. An anti-laminin alpha4 antibody (2A3), directed against the G domain of the alpha4 laminin subunit of laminins-8 and -9, inhibits proliferation and enhances apoptosis of endothelial cells when cells are maintained in vitro. Activation of caspases-9 and -3 plays a role in 2A3 antibody-induced apoptosis, since inhibitors specific for these caspases and overexpression of the anti-apoptotic protein Bcl-X(L), but not c-FLIP, inhibit 2A3 antibody-triggered endothelial cell death. Extracellular matrix is known to play a role in regulating programmed cell death in an integrin-dependent fashion. The alpha4 laminin subunit conforms to this idea since activation of beta1 integrin subunits on endothelial cells blocks the ability of 2A3 antibody to induce endothelial cell death. In summary, our data indicate that complexes composed of alpha4 laminin/beta1 subunit-containing integrins at the cell surface support endothelial cell survival. Furthermore, we propose that antagonists of alpha4 laminin function, including antibody 2A3, have value as angiogenesis inhibitors in a clinical setting where blocking aberrant growth of blood vessel by triggering apoptosis of endothelial cells may be therapeutic.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

Recognition and lysis by natural killers of tumor cells with participation of laminin

According to the data obtained in the present work, the receptor complex of mouse natural killers (NK) includes laminin, antibody to which blocks EK-activity (NKA regardless of the presence of complement. Preincubation of mouse splenocytes with anti-laminin serum led to a decrease in their NKA towards tumor cells-targets (CT), the NKA activity decreasing 2 times with respect to cultivated cells of rat hepatoma HTC, while 10 times - to cultivated cells of human erythroblastosis K562. Pretreatment of aplenocytes with noraml nonimmune serum did not lead to a change of NKA. Quite different was the pattern after the tumor cell preincubation with anti-laminin serum: pretreatment of CT K562 led to a twofold decrease in sensitivity of these cells to NK-lysis, whereas the pretreatment of CT K562, on the contrary, made them twice sensitive to NK-lysis. Electrophoretic separation of protein of CT plasma membranes with subsequent immunoblotting with anti-laminin immune serum revealed the presence oflaminin on HTC cell plasma membrane, which was identified as laminin 8/9 by the mass-spectrometry method, while no laminin was detected on K562 cells. Preincubation of splenocytes with laminin did nor affect NKA with respect to CT K562 and HTC. Pretreatment of CT K562 and HTC with laminin decreased the NKA to zero. The obtained data allow suggesting a doubtless participation of laminin and its receptors in CT cytolysis by NK.     

Mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrin-induced phosphorylation of tyrosine residues in the beta subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate.     

Evidence against a laminin receptor role for calsequestrin

Calsequestrin, a muscle calcium binding protein, has been shown to bind the extracellular matrix protein laminin and evidence has been presented that CAL (initially called aspartactin) is on the cell surface, consistent with a role as a laminin receptor (1). In this report, we present evidence that does not support a laminin receptor function for CAL. We found that CAL immunoreactivity could not be detected on live cultured chick myotubes unless they were permeabilized with detergent. Furthermore, polyclonal anti-CAL antibodies did not perturb myotube adhesion to laminin or the rate of myoblast fusion on laminin. Expression of the CAL cDNA in a melanoma cell line that was poorly adherent to laminin did not increase adhesion to laminin. In these cells, CAL could not be detected on the cell surface, and the majority of CAL was found to be secreted into the media.     

Mechanism of agrin-induced acetylcholine receptor aggregation

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrininduced phosphorylation of tyrosine residues in the β subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate. © 1992 John Wiley & Sons, Inc.     

Laminin 5 Deposition Promotes Keratinocyte Motility

We examined the role of individual integrins in promoting human keratinocyte migration. In short-term assays on collagen type I- or fibronectin-coated substrates, migration was blocked by antibody to the α2 integrin and the α5 integrin, respectively. Unexpectedly, antibodies to integrin α3 also significantly inhibited cell locomotion on both ligands. Time-course immunofluorescence staining revealed that keratinocyte migration was accompanied by deposition of endogenous laminin 5. Since α3β1 is a known receptor for this ligand, this observation suggested that migrating keratinocytes use freshly deposited laminin 5 in locomotion. Indeed, further investigation showed that anti-laminin 5 blocking antibodies effectively inhibited keratinocyte motility on both collagen and fibronectin substrates. Furthermore, cell migration on laminin 5-coated substrates was blocked by both anti-α3 and anti-laminin 5 antibodies. Laminin 5 did not appear important in the initial attachment of keratinocytes, since adhesion of cells to collagen type I- or fibronectin-coated surfaces was not blocked by antibody to α3 integrin or to laminin 5, but could be inhibited by antibody to α2 or α5, respectively. Using anin vitrowound assay, blocking antibodies to α3 integrin and to laminin 5 also blocked reepithelization of the denuded monolayer. These results show that α3β1 integrin plays an important role in the migration of keratinocytes via their interaction with laminin 5. Furthermore, they suggest that cell migration is dependent not only on exogenous ligands but, importantly, on endogenously secreted laminin 5. Finally, the data are consistent with our earlier finding that laminin 5 is the first extracellular matrix component to be expressed and deposited by migrating keratinocytes during wound healingin vivo[1].     

Mobility and detergent extractability of acetylcholine receptors on cultured rat myotubes: a correlation

On aneurally cultured rat primary myotubes, 10% of the acetylcholine receptors (AChR) are found aggregated and immobilized in endogenous clusters. The remaining receptors are diffusely distributed over the cell membrane and the majority of these are free to diffuse in the plane of the membrane. This study correlates the mobility of AChR (as measured with the fluorescence photobleaching recovery technique, FPR) with the detergent extractability of this receptor. Gentle detergent extraction of the cells removes the lipid membrane and the soluble cytoplasmic proteins but leaves an intact cytoskeletal framework on the substrate. Two studies indicate a correlation between mobility and extractability: (a) mobility of diffusely distributed AChR decreases as myotubes age in culture; previous work showed that extractability of AChR decreases as myotubes age in culture (Prives, J., C. Christian, S. Penman, and K. Olden, 1980, In Tissue Culture in Neurobiology, E. Giacobini, A. Vernadakis, and A. Shahar, editors, Raven Press, New York, 35-52); (b) mobility of clustered AChR increases when cells are treated with metabolic inhibitors such as sodium azide (NaN3); extractability of clustered AChR also increases with this treatment. From these results we suggest the involvement of a cytoskeletal framework in the immobilization of AChR on the cell surface.     

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.     

Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton

Tumor endothelial marker 8 (TEM8) is induced in tumor-associated vasculature and acts as a receptor for Protective Antigen (PA), the cell-binding component of the anthrax toxin determinant for toxin entrance into cells. However, the normal function for TEM8 remains unknown. We show that TEM8 functions as an adhesion molecule mediating cell spreading on immobilized PA and collagen I. The mechanism for TEM8 interaction with collagen I was cell type-specific, because binding to collagen I was abrogated by beta1 integrin function blocking antibody in HEK293 cells, but not in primary synovial rabbit fibroblasts. Binding to PA remained unaffected by the addition of beta1 integrin function blocking antibody. Whereas the extracellular and transmembrane domains of TEM8 were sufficient to provide cell attachment, the intracellular domain was critical for spreading. Fusion of the cytosolic domain of TEM8 to the IL-2 receptor, conferred cell-spreading capability on IL-2 receptor antibody substrates. The cytoplasmic domain mediated linkage with the actin cytoskeleton as it co-precipitated actin and determined partitioning of TEM8 to the actin-containing detergent insoluble cellular fraction. TEM8 anchorage to actin was relevant as spreading was inhibited by the cytoskeleton-disrupting drug cytochalasin D, but persisted in the presence of the microtubule-depolymerizing drug nocodazole, and in cells lacking intermediate filaments. Thus, our results indicate that TEM8 is a new adhesion molecule linking collagen I or PA to the actin cytoskeleton.