Structural requirements for outside-in and inside-out signaling by Drosophila neuroglian, a member of the L1 family of cell adhesion molecules

Expression of the Drosophila cell adhesion molecule neuroglian in S2 cells leads to cell aggregation and the intracellular recruitment of ankyrin to cell contact sites. We localized the region of neuroglian that interacts with ankyrin and investigated the mechanism that limits this interaction to cell contact sites. Yeast two-hybrid analysis and expression of neuroglian deletion constructs in S2 cells identified a conserved 36-amino acid sequence that is required for ankyrin binding. Mutation of a conserved tyrosine residue within this region reduced ankyrin binding and extracellular adhesion. However, residual recruitment of ankyrin by this mutant neuroglian molecule was still limited to cell contacts, indicating that the lack of ankyrin binding at noncontact sites is not caused by tyrosine phosphorylation. A chimeric molecule, in which the extracellular domain of neuroglian was replaced with the corresponding domain from the adhesion molecule fasciclin II, also selectively recruited ankyrin to cell contacts. Thus, outside-in signaling by neuroglian in S2 cells depends on extracellular adhesion, but does not depend on any unique property of its extracellular domain. We propose that the recruitment of ankyrin to cell contact sites depends on a physical rearrangement of neuroglian in response to cell adhesion, and that ankyrin binding plays a reciprocal role in stabilizing the adhesive interaction.     

PECAM-1 is required for transendothelial migration of leukocytes

Platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) is crucial to the process of leukocyte transmigration through intercellular junctions of vascular endothelial cells. A monoclonal antibody to PECAM, or recombinant soluble PECAM, blocks transendothelial migration of monocytes by 70-90%. Pretreating either the monocytes or the endothelial junctions with antibody blocks transmigration. If the endothelium is first activated by cytokines, anti-PECAM antibody or soluble recombinant PECAM again block transmigration of both monocytes and neutrophils. Anti-PECAM does not block chemotaxis of either cell type. Light and electron microscopy reveal that leukocytes blocked in transmigration remain tightly bound to the apical surface of the endothelial cell, precisely over the intercellular junction. Thus, the process of leukocyte emigration can be dissected into three successive stages: rolling, mediated by the selectin class of adhesion molecules; tight adhesion, mediated by the leukocyte integrins and their endothelial cell counter-receptors; and now transmigration, which, based on these studies, requires PECAM-1.     

Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.     

The protein-tyrosine phosphatase SHP-2 associates with tyrosine-phosphorylated adhesion molecule PECAM-1 (CD31)

Aggregation of many cell-surface receptors results in tyrosine phosphorylation of numerous proteins. We previously observed the tyrosine phosphorylation of the platelet/endothelial cell adhesion molecule, PECAM-1 (CD31), after FcepsilonRI stimulation in rat basophilic leukemia RBL-2H3 cells. Here we found that PECAM-1 was also transiently tyrosine-phosphoryated after adherence of these cells to fibronectin. Similarly aggregation of the T cell receptor on Jurkat cells also induced this tyrosine phosphorylation. The protein-tyrosine phosphatase SHP-2 is a widely expressed cytosolic enzyme with two Src homology 2 (SH2) domains. SHP-2, but not the related protein-tyrosine phosphatase SHP-1, associated with PECAM-1. This association of the two proteins correlated with the extent of the tyrosine phosphorylation of PECAM-1. A fusion protein containing the two SH2 domains of SHP-2 precipitated PECAM-1 from cell lysates and also directly bound to phosphorylated PECAM-1. In immune precipitate phosphatase assays, there was tyrosine dephosphorylation of PECAM-1. Therefore, integrin and immune receptor activation results in tyrosine phosphorylation of PECAM-1 and the binding of the protein-tyrosine phosphatase SHP-2, which could regulate receptor-mediated signaling in cells.     

Engagement of human PECAM-1 (CD31) on human endothelial cells increases intracellular calcium ion concentration and stimulates prostacyclin release

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily that plays a role in a number of endothelial cell (EC) functions including migration, angiogenesis, and transmigration of leukocytes across endothelium. We postulated that one way PECAM-1 might exert its effects was by regulating intracellular EC levels of calcium. Using single-cell fluorometry, we found that engagement of PECAM-1 by mAbs induced a slow but sustained increase in intracellular calcium, both in EC and in an adherent PECAM-1-transfected cell line that models endothelium. Generation of this signal was specific for certain anti-PECAM-1 antibodies, required the presence of the cytoplasmic domain, depended on extracellular calcium and on tyrosine phosphorylation, but did not require cross-linking; in fact, calcium increases were stimulated by certain Fab fragments. Activation of EC by PECAM-1 also caused a time-dependent increase in prostacyclin release. Given the importance of intracellular calcium and prostacyclin release as signaling molecules, engagement of PECAM-1 during cell-cell interactions may alter a number of EC functions including secretion of vasoactive mediators.     

Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors

The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for vascular endothelial cells and a potent angiogenic factor. Expression of a chimeric receptor containing the extracellular domain of the flk-1 receptor fused to the transmembrane and intracellular domains of the human c-fms receptor in NIH-3T3 cells, resulted in the appearance of high affinity binding sites for 125I-VEGF165 on transfected cells. The binding of 125I-VEGF165 to the flk-1/fms chimeric receptor of the transfected cells as well as the VEGF165-induced autophosphorylation of the chimeric receptors were inhibited in the presence of low concentrations of heparin (1-10 micrograms/ml). In contrast, similar concentrations of heparin potentiated the binding of 125I-VEGF165 to the endogenous VEGF receptors of the transfected cells, indicating that to some extent, the effect of heparin on 125I-VEGF165 binding is receptor type-dependent. A soluble fusion protein containing the extracellular domain of flk-1 fused to alkaline phosphatase (flk-1/SEAP) was used to study the effects of heparin on the binding of 125I-VEGF165 to flk-1 in a cell-free environment. The fusion protein specifically inhibited VEGF165-induced proliferation of vascular endothelial cells, but bound 125I-VEGF165 inefficiently in the absence of heparin. Addition of low concentrations of heparin or heparan sulfate (0.1-1 microgram/ml) resulted in a strong potentiation of 125I-VEGF165 binding, whereas higher heparin or heparan sulfate concentrations inhibited the binding. The effect of heparin on the binding of 125I-VEGF165 to flk-1/SEAP could not be mimicked by desulfated heparin or by chondroitin sulfate. Both bFGF and aFGF inhibited the binding when low concentrations of heparin were added to the binding reaction. However, higher concentrations of heparin abolished the inhibition, indicating that the inhibition is probably caused by competition for available heparin. Taken as a whole, these results indicate that heparin-like molecules regulate the binding of VEGF165 to its receptors in complex ways which depend on the heparin binding properties of VEGF165, on the specific VEGF receptor type involved, and on the amount and composition of heparin-like molecules that are present on the cell surface of VEGF receptor containing cells.     

Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial cell adhesion molecule-1 (PECAM-1). Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates

Stimulation of platelet aggregation leads to tyrosine phosphorylation of a number of receptors and signaling molecules including platelet endothelial cell adhesion molecule-1 (PECAM-1). In this report, we demonstrate that both protein-tyrosine phosphatases SHP-1 and SHP-2 physically associate with different kinetics of assembly with tyrosine-phosphorylated human PECAM-1 during integrin alphaIIbbeta3-mediated platelet aggregation. Peptido-precipitation analysis revealed that tyrosine-phosphorylated peptides encompassing residues 658-668 and 681-691 of PECAM-1 bound specifically to both protein-tyrosine phosphatases SHP-1 and SHP-2. We further show that the association of SHP-1 with PECAM-1 occurs through the direct interaction of the src homology region 2 domains of SHP-1 with two highly conserved phosphotyrosine binding motifs within PECAM-1 having the sequences NSDVQpY663TEVQV and DTETVpY686SEVRK (where pY represents phosphotyrosine). In vitro dephosphorylation experiments using phosphotyrosyl PECAM-1 peptides encompassing either Tyr-663 or Tyr-686 revealed induction of SHP-1 catalytic activity, suggesting that PECAM-1 serves as a SHP-1 substrate. Surface plasmon resonance studies reveal that recombinant SHP-2 binds PECAM-1 phosphopeptides with 5-fold higher affinity than recombinant SHP-1. These data suggest that in hematopoietic cells such as platelets, PECAM-1 cellular signaling is regulated by the selective recruitment and activation of two distinct protein-tyrosine phosphatases, SHP-1 and SHP-2, via a common immunoreceptor tyrosine-based inhibitory-like motif.     

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) tyrosine phosphorylation state changes during vasculogenesis in the murine conceptus

Vasculogenesis, the differentiation of mesodermal cells to angioblasts and the subsequent formation of blood islands and blood vessels by angioblasts in the conceptus, is a dynamic process modulated, in part, by cell-extracellular matrix and cell-cell interactions in the presence of a variety of growth factors and morphogens. In this report we demonstrate differential tyrosine phosphorylation of platelet-endothelial cell adhesion molecule-1 (PECAM-1) during the formation of blood islands and vessels from clusters of extraembryonic and embryonic angioblasts in the murine conceptus. In addition, we identify the phosphorylation of a particular tyrosine residue in the PECAM-1 cytoplasmic domain, Tyr686, which has the potential of mediating binding to Src homology 2 domain-containing proteins, affecting PECAM-1 cellular localization and endothelial cell migration.     

The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties

Neurotactin (Nrt), a Drosophila transmembrane glycoprotein which is expressed in neuronal and epithelial tissues during embryonic and larval stages, exhibits heterophilic adhesive properties. The extracellular domain is composed of a catalytically inactive cholinesterase-like domain. A three-dimensional model deduced from the crystal structure of Torpedo acetylcholinesterase (AChE) has been constructed for Nrt and suggests that its extracellular domain is composed of two sub-domains organized around a gorge: an N-terminal region, whose three-dimensional structure is almost identical to that of Torpedo AChE, and a less conserved C-terminal region. By using truncated Nrt molecules and a homotypic cell aggregation assay which involves a soluble ligand activity, it has been possible to show that the adhesive function is localized in the N-terminal region of the extracellular domain comprised between His347 and His482. The C-terminal region of the protein can be removed without impairing Nrt adhesive properties, suggesting that the two sub-domains are structurally independent. Chimeric molecules in which the Nrt cholinesterase-like domain has been replaced by homologous domains from Drosophila AChE, Torpedo AChE or Drosophila glutactin (Glt), share similar adhesive properties. These properties may require the presence of Nrt cytoplasmic and transmembrane domains since authentic Drosophila AChE does not behave as an adhesive molecule when transfected in S2 cells.     

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2

Recent studies have shown that the Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase, SHP-2, associates with the cytoplasmic domain of PECAM-1 as it becomes tyrosine-phosphorylated during platelet aggregation: a process that can be mimicked in part by small synthetic phosphopeptides corresponding to the cytoplasmic domain of PECAM-1 encompassing tyrosine residues Tyr-663 or Tyr-686. To further examine the molecular requirements for PECAM-1/SHP-2 interactions, we generated human embryonic kidney (HEK)-293 cell lines that stably expressed mutant forms of PECAM-1 harboring tyrosine to phenylalanine (Tyr --> Phe) mutations in the cytoplasmic domain. Y663F and Y686F forms of PECAM-1 were tyrosine-phosphorylated to a somewhat lesser extent than wild-type PECAM-1, and a doubly substituted Y663,686F form of PECAM-1 failed to become tyrosine-phosphorylated, suggesting that the PECAM-1 cytoplasmic domain tyrosine residues 596, 636 and 701 do not serve as substrates for cellular kinases. Interestingly, SHP-2 binding was lost when either Tyr-663 or Tyr-686 were changed to phenylalanine, indicating that both residues are required for SHP-2/PECAM-1 association. Although PECAM-1 phosphopeptides NSDVQpY663TEVQV and DTETVpY686SEVRK stimulated the catalytic activity of the phosphatase to a similar extent, surface plasmon resonance studies revealed that the Tyr-663-containing peptide had approximately 10-fold higher affinity for SHP-2 than did the Tyr-686 peptide. Finally, peptido-precipitation analysis showed that the NH2-terminal SH2 domain of SHP-2 reacted preferentially with the Tyr-663 PECAM-1 phosphopeptide, while the Tyr-686 phosphopeptide associated only with the COOH-terminal SH2 domain of the phosphatase. Together, these data provide a molecular model for PECAM-1/SHP-2 interactions that may shed light on the downstream events that follow PECAM-1-mediated interactions of vascular cells.     

A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.     

A role for polysialic acid in neural cell adhesion molecule heterophilic binding to proteoglycans

The neural cell adhesion molecule (NCAM) is known to participate in both homophilic and heterophilic binding, the latter including mechanisms that involve interaction with proteoglycans. The polysialic acid (PSA) moiety of NCAM can serve as a negative regulator of homophilic binding, but indirect evidence has suggested that PSA can also be involved in heterophilic binding. We have examined this potential positive role for PSA in terms of the adhesion of PSA-expressing mouse F11 cells and chick embryonic brain cells to substrates composed of the purified heparan sulfate proteoglycans agrin and 6C4. This adhesion was specifically inhibited by polyclonal anti-NCAM Fab antibodies, monoclonal anti-PSA antibodies, PSA itself, and enzymatic removal of either PSA or heparan sulfate side chains. By contrast, the adhesion was not affected by chondroitinase, and cell binding to laminin was not inhibited by any of these treatments. A specific NCAM-heparan sulfate interaction in this adhesion was further indicated by its inhibition with monoclonal anti-NCAM Fab antibodies that recognize the known heparin-binding domain of NCAM and with the HBD-2 peptide derived from this region, but not with antibodies directed against other regions of the protein including the homophilic binding region. Together, the results suggest that PSA can act in vitro either as a receptor in NCAM heterophilic adhesion or as a promoter of binding between heparan sulfate proteoglycans and the NCAM heparin-binding domain.     

Ligation of CD31/PECAM-1 modulates the function of lymphocytes, monocytes and neutrophils

CD31 or platelet/endothelial cell adhesion molecule (PECAM-1) is a 130-kDa glycoprotein expressed on endothelial cells, granulocytes, a subset of lymphocytes and platelets. In this study, we examined the ability of four monoclonal antibodies (mAb) against different domains of CD31 to modulate the function of T lymphocytes, monocytes and neutrophils. Engagement of CD31 on T lymphocytes results in co-stimulation of T lymphocyte proliferation to suboptimal doses of anti-CD31 mAb. This proliferation is accompanied by secretion of numerous cytokines and chemokines, up-regulation of CD25 and an increase in cell size. Purification of T lymphocytes into CD45RO and CD45RA subsets showed that only naive CD45RA T lymphocytes are co-stimulated by anti-CD31 mAb. Further studies on neutrophils show that engagement of CD31 results in down-regulation of CD62L and up-regulation of CD11b/CD18 as well as oxidative burst, as assessed by superoxide release. In addition, ligation of CD31 on monocytes results in TNF-alpha secretion, and studies with various cell signaling inhibitors indicate that tyrosine kinases and cAMP-dependent kinases are involved in monocyte activation via CD31. Of the four mAb used in this study, only two activated human leukocytes. These mAb were PECAM-1.3 and hec7, which bind to domains 1 and 2 of CD31. We conclude that engagement of domains 1 and 2 of CD31 results in outside-in signaling in leukocytes.     

The lymphocyte function-associated antigen 1 I domain is a transient binding module for intercellular adhesion molecule (ICAM)-1 and ICAM-3 in hydrodynamic flow

The I domain of lymphocyte function-associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.     

Prognostic factors for swallowing rehabilitation following head and neck cancer surgery

MUC18/MCAM is a cell-surface glycoprotein that is strongly expressed on advanced human melanomas. Transfection of 3 MCAM-negative melanoma cell lines with MCAM cDNA led to cell-surface expression and to a MCAM-dependent homotypic adhesion. This adhesion was independent of divalent cations and was inhibited at 4 degrees C. Mixed aggregation assays with MCAM-expressing and non-expressing cells revealed that MCAM can function as a heterophilic cell adhesion molecule interacting with a non-MCAM ligand. Although MCAM contains a potential glycosaminoglycan-binding site, cell-surface glycosaminoglycans do not appear to be involved in the heterophilic adhesion observed here since these molecules were not able to influence the adhesion. Using a functional adhesion assay, 4/4 melanoma cell lines examined were found to express an MCAM ligand. In contrast, no evidence for an MCAM ligand was found on the 2 carcinoma or 2 hematopoietic cell lines examined. Stable transfection of an MCAM ligand-negative colorectal cell line resulted in MCAM surface expression but not in homotypic adhesion, indicating that homophilic MCAM-MCAM adhesive interactions may not occur. Our results suggest that MCAM expression by melanoma cells is associated with increased homotypic adhesion, an event that may support tumor cell survival and growth in vivo.     

Identification of the 2B4 molecule as a counter-receptor for CD48

The CD48 molecule belongs to a subfamily of the Ig superfamily that also includes the CD2, CD58, 2B4, Signaling lymphocyte activation molecule (SLAM), and Ly-9 molecules. Receptor-ligand interactions are known to occur between several members of this family, and these interactions can strengthen cell to cell adhesion. In mice, the CD48 molecule can bind to CD2. To search for additional ligands of murine CD48, we have generated a chimeric fusion protein consisting of the extracellular domain of murine CD48 and the C region of human IgG1. The results of immunofluorescence and immunoprecipitation experiments in which this reagent was used identify the 2B4 molecule as a novel counter-receptor of CD48.     

Platelet endothelial cell adhesion molecule-1 is phosphorylatable by c-Src, binds Src-Src homology 2 domain, and exhibits immunoreceptor tyrosine-based activation motif-like properties

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is 130-kDa member of the immunoglobulin gene superfamily that localizes to cell-cell borders of confluent endothelial cells and has been shown to play a role in the control of endothelial sheet migration and leukocyte transmigration through the endothelium. The cytoplasmic tail plays an important role in the modulation of PECAM-1 function. Mutation of tyrosine 663 or 686 in the cytoplasmic tail reduces phosphorylation and mutation of 686 is associated with a reduction in PECAM-1-mediated inhibition of cell migration (1). We have previously noted that these two tyrosine residues are surrounded by consensus sequences for Src homology 2 (SH2) domain binding (1, 2), and the experiments presented explore the potential for PECAM-1-Src and PECAM-1-SH2 domain interactions. PECAM-1 is more highly phosphorylated in endothelial cells overexpressing c-Src, and in in vitro kinase assays, c-Src can phosphorylate a glutathione S-transferase (GST)-PECAM cytoplasmic tail fusion protein. The phosphorylated fusion protein associates with the bead-bound c-Src. This association appears to be mediated by Src-SH2 domain, because PECAM-1 can be precipitated by a GST-Src-SH2 affinity matrix. The binding to the GST-Src-SH2 affinity matrix correlates directly with the level of PECAM-1 phosphorylation, because more PECAM-1 is precipitated from c-Src overexpressors and from wild-type rather than Tyr663 --> Phe and Tyr686 --> Phe mutant PECAM-1 expressors. Yet unidentified phosphoproteins can also be coimmunoprecipitated with wild-type but not mutant PECAM-1. Finally, we note the similarity of the PECAM-1 cytoplasmic domain sequence to the immunoreceptor tyrosine-based activation motif. Our data begin to delineate how tyrosines 663 and 686 may play a role in mediating PECAM-1 signal transduction.     

Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli

Adhesion to collagens by most cell types is mediated by the integrins alpha1beta1 and alpha2beta1. Both integrin alpha subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified alpha1beta1 and alpha2beta1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the alpha1 and alpha2 I domains in specific collagen adhesion. We find that introducing the alpha2 I domain into alpha1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (alpha1-2-1beta1) is similar to the adhesion profile conferred by alpha2beta1, not alpha1beta1. The presence of alpha2 or alpha1-2-1 results in preferential binding to collagen I, whereas alpha1 expressing cells bind better to collagen IV. In addition, alpha1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas alpha2 or alpha1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by alpha2beta1 or alpha1-2-1beta1, but not by alpha1, requires the presence of Mn2+ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins.     

Right ventricular responses to vagus stimulation of fibers to discrete cardiac regions in dog hearts

OBJECTIVE: To characterize and compare the expression of PECAM-1 in unstimulated and tumor necrosis factor alpha-(TNF-alpha)-stimulated tissues of mice. METHODS: Binding and non-binding monoclonal antibodies (mAb) were radiolabeled and injected into wild-type mice and mice deficient of P-selectin, CD18, or ICAM-1. The relative accumulation of binding mAb (PECAM-1 mAb) was determined in wild-type mice following a 25 micrograms/kg i.p. injection of TNF-alpha and in mutant mice under basal conditions. RESULTS: Under unstimulated conditions, PECAM-1 was significantly expressed in all tissues examined, with no changes occurring after TNF-alpha stimulation. An equivalence of PECAM-1 expression was observed in unstimulated tissues among wild-type mice and mice that are genetically deficient in either CD18, ICAM-1, or P-selectin. The level of PECAM-1 expression in different vascular beds was highly correlated to published values of endothelial surface area. Normalization of previously published values of ICAM-1, VCAM-1, E- and P-selectin expression relative to PECAM-1 expression in the same tissues revealed a diminished organ-to-organ variability in expression of the different adhesion molecules. Estimates of adhesion molecule expression in lung and brain were most profoundly affected by normalization to PECAM-1 expression. CONCLUSIONS: These findings support the use of PECAM-1 expression in regional vascular beds as an indicator of endothelial cell surface area.     

Mutational evidence for control of cell adhesion through integrin diffusion/clustering, independent of ligand binding

Previous studies have shown that integrin alpha chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin alpha4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the alpha4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon alpha4 tail deletion. Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.