Role of Lu/BCAM glycoproteins in red cell diseases

Lu/BCAM glycoproteins (gps) are the unique erythroid receptors of laminin α5 chain, a major component of the extracellular matrix. They interact with the membrane skeleton by binding directly to spectrin via the Lu/BCAM RK573-574 motif. Lu/BCAM gps are involved in abnormal sickle red blood cell (RBC) adhesion to components of the vascular wall. This adhesion is activated by the phosphorylation of the Lu/BCAM long isoform Lu in a protein kinase A-dependent manner. A similar high adhesion to laminin was also observed with RBCs from Hereditary Spherocytosis (HS) patients suffering from haemolytic anaemia subsequent to spectrin deficiencies. We investigated the molecular mechanisms responsible for the Lu/BCAM-mediated abnormal RBC adhesion to laminin in sickle cell disease (SCD) and HS. We showed that SCD patients treated with hydroxycarbamide (HC) had a diminished RBC adhesion to laminin that was associated with reduced levels of the PKA upstream effector cAMP and a severe decrease in Lu isoform phosphorylation. On the other hand, we showed that increased Lu/BCAM-mediated HS RBC adhesion to laminin was independent of Lu/BCAM phosphorylation. A cellular model expressing the RK573-574AA Lu/BCAM mutant, which is unable to bind to spectrin, showed increased Lu/BCAM detergent extractability and enhanced cell adhesion to laminin. Similar results were obtained with HS RBCs, strongly suggesting that their increased adhesion could result from alteration of the Lu/BCAM-spectrin interaction following the severe spectrin deficiency.     

Role and regulation of sickle red cell interactions with other cells: ICAM-4 and other adhesion receptors

Erythrocytes containing primarily hemoglobin S (SS RBCs) are abnormally adherent. We now know that SS RBCs express numerous adhesion molecules, and that many of these can undergo activation. SS RBCs exposed briefly to epinephrine show markedly increased adhesion to both laminin and endothelial cells. In vivo, infusion of epinephrine-activated but not unstimulated SS RBCs causes RBC adhesion, vaso-occlusion, organ trapping, and shortened RBC survival in the circulation. Epinephrine treatment of SS RBCs before infusion also induces adhesion of murine leukocytes to vascular walls. Indeed, in vitro, SS RBCs can activate leukocyte adhesion and cytokine production. We now have demonstrated both in vitro and in vivo evidence for the importance of RBC signaling and have also shown that SS RBC adhesion is determined by genetic polymorphisms in the signaling pathway that activates adhesion. These advances will hopefully lead to new therapeutic modalities for sickle cell disease.     

Erythroid adhesion molecules in sickle cell disease: Effect of hydroxyurea

In sickle cell disease, the complex scenario of vaso-occlusive crisis (VOC) typical of this disease is clearly multifactorial and not fully understood. Cell-cell and cell-cell matrix interactions mediated by adhesive molecules present on blood cells and endothelial cells (ECs) are thought to play an important role. Early studies have shown that sickle red blood cells (RBCs) are abnormally adherent to ECs and some of the molecules involved in these interactions have been identified, such as the alpha4beta1 integrin and CD36, exclusively present on stress reticulocytes, and CD47 on mature RBCs. More recently, attention focused on Lu/BCAM, the unique RBC receptor for laminin, and on ICAM-4, a red cell-specific adhesion receptor, which is a ligand for a large repertoire of integrins (alphaLbeta2, alphaMbeta2, alphaxbeta2, alphaVbeta3). The counter-receptors on ECs and the role of plasma proteins forming bridges between blood cells and ECs have been clarified in part. It has also been shown that reticulocytes from SCD patients express higher levels of alpha4beta1 integrin and CD36, and that under hydroxyurea (HU) therapy, both cell adhesion to ECs or extracellular matrix proteins and the levels of these adhesion molecules are reduced. These findings are consistent with the view that enhanced adhesion of blood cells to ECs is largely determined by the membrane expression level of adhesion molecules and could be a crucial factor for triggering or aggravating vaso-occlusion. In SCD patients, membrane expression of Lu/BCAM (and perhaps ICAM-4) is enhanced on RBCs whose adherence to laminin or ECs is also increased. Interestingly, Lu/BCAM- and ICAM-4-mediated adhesion are enhanced by the stress mediator epinephrine through a PKA-dependent pathway initiated by a rise in intracellular cAMP and leading to receptor activation by phosphorylation according to the same signaling pathway. More recently, studies based on quantitative expression analysis of adhesion molecules on RBCs and during erythroid differentiation in patients undergoing HU therapy, surprisingly revealed that Lu/BCAM level was enhanced, although alpha4beta1, CD36 and ICAM-4 (to a lower extent) levels were indeed reduced. CD47 and CD147 expression were also enhanced in HU-treated patients. Based on these findings we suggest that the signalization cascade leading to receptor activation rather than the expression level only of adhesion molecules may be the critical factor regulating cell adhesion, although both mechanisms are not mutually exclusive.     

Red cell and endothelial Lu/BCAM beyond sickle cell disease

Recent studies shed new lights on the biological function of blood group antigens, such as the adhesion properties of the Lutheran (Lu) blood group antigens carried by the Lu/BCAM glycoproteins. The Lu/BCAM adhesion glycoproteins were first identified as laminin-10/11 erythroid receptors involved in RBC adhesion to endothelium in sickle cell anemia. Lu/BCAM mediated cell adhesion to laminin is stimulated by epinephrine, a physiological stress mediator, and is dependent of phosphorylation by protein kinase A. More recently, we demonstrated that constitutive phosphorylation of Lu/BCAM is also involved in abnormal RBC adhesion to endothelium in patients with polycythemia vera (PV), a frequent myeloproliferative disorders associated with the V617F mutation of the tyrosine kinase JAK2 leading to continuous stimulation of erythropoiesis. This observation suggests that Lu/BCAM could participate to the high incidence of vascular thrombosis that also characterizes PV disease. In mice, which do not express Lu/BCAM in erytroid tissues, invalidation of the Lu/BCAM gene provided evidence that Lu/BCAM gps, as laminin-α5 receptors, are involved in vivo in the maintenance of normal basement membrane organization in different non erythroid tissues since up to 90% of the mutant kidney glomeruli exhibited a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane, while intestine exhibited smooth muscle coat thickening and disorganization. All these results further illustrate that minor blood group antigens might have important role under physiological and physiopathological conditions in erythroid and non erythroid tissues as well.     

Mechanisms behind adhesive erythrocytes in sickle-cell disease

Erythrocytes containing primarily hemoglobin S (SS RBCs) are abnormally adherent to a number of ligands, including normal constituents of the extracellular matrix as well as those present on the surfaces of other blood cells and endothelial cells. However, SS RBCs are not really very different from normal (AA) RBCs, except that they are younger. In general, they express the same range of adhesion receptors, and the levels of expression of these receptors, although increased in some cases, are not sufficient to explain the difference between the adhesivity of SS and AA RBCs. However, SS RBCs have activated adhesion receptors, and the signaling pathways responsible for this activation are also upregulated in SS RBCs. In addition, SS RBCs convey different extracellular signals, which are also likely to affect their adhesion to other blood cells and endothelial cells. Together, SS RBCs induce cell–cell interactions mediated by the B-CAM/LU, ICAM4 (LW), CD44 (In), CD47, α4β1 integrin, and CD36 adhesion molecules on SS RBCs with target ligands on leukocytes and endothelial cells. Other, as yet unidentified cell surface moieties on SS RBCs are probably also responsible for SS RBC interactions with P-selectin. Interestingly, at least some of these RBC adhesion receptors may also mediate adhesion and adverse physiologic events in the setting of transfusion of stored normal RBCs. Therefore, understanding the mechanisms behind SS RBC adhesion may offer opportunities for improving both therapy of sickle-cell disease as well as transfusion practices.     

Developmentally regulated interactions of human thymocytes with different laminin isoforms

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different alpha, three beta and three gamma subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin alpha1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the alpha2 chain (LN-2/4) or the alpha5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin alpha4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the alpha3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4- CD8- thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin alpha6beta1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin alpha6beta4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.     

Review: Lutheran/B-CAM: a laminin receptor on red blood cells and in various tissues

The Lutheran blood group glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is a transmembrane receptor with five immunoglobulin-like domains in its extracellular region; it is therefore classified as a member of the immunoglobulin (Ig) gene family. Lu/B-CAM is observed not only on red blood cells, but also on a subset of muscle and epithelial cells in various tissues. Recently, several groups have reported that Lu/B-CAM is a novel receptor for laminin a5. The laminin a5 chain is a component of the laminin-511 (alpha 5 beta 1 gamma 1), -521 (alpha 5 beta 2 gamma 1), and -523 (alpha 5 beta 2 gamma 3) heterotrimers and is expressed throughout the mammalian body. We also have shown that Lu/B-CAM is co-localized with laminin alpha 5 in various tissues. Although the biological role of Lu/B-CAM remains unclear, the specific binding of Lu/B-CAM to laminin alpha 5 suggests that it plays an important role in developmental and physiological processes. It also is necessary to investigate further the interaction between Lu/B-CAM and laminin a5 in pathological processes, including sickle cell disease and cancer.     

The α4β1 integrin in sickle cell disease

The α4β1 integrin is an adhesion receptor expressed on reticulocytes in sickle cell disease (SCD) and mediates the adhesion of these cells to sub-endothelial matrix proteins and the endothelium. In this review, we describe the mechanism of activation of the α4β1 integrin on sickle reticulocytes and discuss novel roles for this integrin in SCD as a result of this activation. We also illustrate novel therapies in SCD that may target the integrin and alleviate vaso-occlusion.     

Adhesion of sickle red cells to endothelium: Myths and future directions

Sickle RBC are abnormally adherent to vascular endothelial cells. We briefly review the mechanisms that underlie this type of cell/cell adhesion, expose a number of extant myths about RBC adhesion, and discuss some aspects that need consideration via future experimentation. The relationship of this phenomenon of RBC-endothelial adhesion to the cytoadherence of parasitized sickle RBC is not yet clear.     

L'adhérence des érythrocytes à l'endothélium vasculaire

Blood cells are in continuous contact with the vascular endothelium. Endothelial cell culture, intravital videomicroscopy allowed the investigation of blood cell-endothelium interactions in dynamic conditions.In the various diseases, diabetes mellitus, sickle cell anemia and malaria, erythrocytes have an increased adhesion to endothelial cells. The presence of advanced glycation end products (AGE) on erythrocytes of diabetics is responsible for their binding to the receptor RAGE present on the endothelium. The AGE-RAGE binding provokes an oxidant stress and induces the expression of the adhesion molecule. Furthermore, erythrocyte AGE induce an increase in vascular permeability. In sickle cell anemia, the increased adhesiveness and the sickling of red blood cells are responsible for thrombosis. Plasmodium falciparum infestation of erythrocytes induces knob formation at the cell surface and the P. falciparum protein binding to CD36, ICAM-1 and thrombospondin present on the endothelium, and facilitates the parasite dissemination. © 2999 Éditions scientifiques et médicales Elsevier SAS     

Characterization of adhesive interactions between mast cells and laminin isoforms: evidence of a principal role for alpha 6 integrin.

Mast cells are known to adhere to laminin, although there is limited information on the characteristics of this event. To further examine this adhesive interaction, we thus determined the adherence of murine mast cell lines and primary bone marrow cultured mast cells (BMCMC) to murine laminin (mLN), human placental laminin-1 (hLN), merosin (laminin-2) and various laminin fragments, concentrating on activating stimuli, the involvement of integrins, and the effect on mast cell activation. Murine mast cells were found to adhere to both mLN and hLN and to merosin, not only following exposure to phorbol 12-myristate 13-acetate (PMA), but also after Fc epsilon RI aggregation or addition of stem cell factor (SCF). Adhesion to laminin was partially inhibited by soluble E8 and PA22-2, both fragments of laminin that promote mast-cell adhesion when bound on surfaces. Mast-cell lines and BMCMC consistently expressed high levels of alpha 6 integrin. Antibody to alpha 6 blocked spontaneous and inhibited activated mast-cell adhesion to hLN, and inhibited mast-cell adhesion to mLN and its fragment E8. Mast-cell adhesion to both laminin isoforms increased Fc epsilon RI-mediated mast-cell secretion. These observations demonstrate that mast-cell attachment to laminin is promoted by physiological stimuli, is mediated principally by alpha 6 integrin, and results in enhanced cell activation.     

Expression of integrin family of cell adhesion receptors in rheumatoid synovium. Alpha 6 integrin subunit in normal and hyperplastic synovial lining cell layer.

Integrins are heterodimeric cell adhesion receptors. The beta 1 integrin subunit can be in a complex with multiple a subunits and form receptors for collagen, laminin, fibronectin, and vitronectin. We have characterized the distribution of eight integrin subunits in rheumatoid synovium, with special interest in the lining cell layer. The beta 1 integrin subunit was found in abundance in synovial stroma and in lining cells. The only alpha subunit seen constantly in lining cells was alpha 6. In complex with alpha beta subunit, alpha 6 forms a laminin receptor usually seen in epithelial or endothelial cells or in macrophages. The fact that laminin was found in the extracellular matrix around synovial cells suggests the importance of alpha 6 integrin in the adhesion of synovial lining cells. Furthermore, alpha 6 expression was noticeably weaker in strongly proliferative lining cell layers, indicating that the inflammatory process may regulate integrin expression. A potential connection between altered expression of cell adhesion receptors and the pathological behavior of rheumatoid lining cells is suggested.     

Oxidized laminin‐1 induces increased monocyte attachment and expression of ICAM‐1 in endothelial cells

Atherosclerosis has been associated with increased oxidative stress and monocyte recruitment by endothelial cells. Sub‐endothelial basement membrane proteins, such as laminins that play a central role in cell adhesion, are exposed to reactive oxygen species. In the present study monocyte attachment on human umbilical cord vein endothelial cells (HUVEC) that were preattached to oxidized or native laminin, was investigated. Intracellular cell adhesion molecule‐1 (ICAM‐1) expression by HUVEC was estimated by an enzyme‐linked immunosorbent assay. HUVEC attachment to oxidized or native laminin‐1 was examined using the Hemacolor kit. Anti‐alphaL, anti‐alphaM, anti‐alpha2 and anti‐beta2 integrin subunit antibodies were used in order to further investigate the above phenomena. HUVEC that were preattached to oxidized laminin expressed higher levels of ICAM‐1 and monocytes attached at a higher degree to these cells as compared to HUVEC that were preattached to native laminin. Incubation of monocytes with monoclonal antibodies against the alphaM and beta2 integrin subunits equalized the above mentioned differences. Moreover, HUVEC attached to oxidized laminin at a higher degree as compared to native laminin. This difference was equalized after incubation with the antibody against the alpha2 integrin subunit. These results indicate a modified interaction between HUVEC and the basement membranes in cases where laminin is oxidatively modified. This modified interaction results in increased ICAM‐1 expression by endothelial cells and consequently increased monocyte recruitment capacity.     

Localization of Lutheran, a novel laminin receptor, in normal, knockout, and transgenic mice suggests an interaction with laminin alpha5 in vivo.

Laminins are major components of all basement membranes. One laminin that has garnered particular interest, due to its widespread expression pattern and importance during development, is the laminin alpha5 chain. In vitro studies have suggested that the Lutheran blood group glycoprotein/basal cell adhesion molecule (Lu), an Ig superfamily transmembrane protein, is a receptor for laminins containing the alpha5 chain. However, there are no in vivo studies showing that these proteins are capable of interacting in tissues. We have isolated the mouse ortholog of Lu and characterized its expression and localization in mouse tissues. Lu was primarily found on the basal surface of epithelial cells and on muscle cells adjacent to basement membranes containing laminin alpha5. In addition, there was both a dramatic reduction in the basal concentration of Lu in mice lacking laminin alpha5, and a significant increase in Lu protein in transgenic mice overexpressing laminin alpha5. Together, these data provide the first in vivo evidence for an interaction between Lu and laminin alpha5 and support the hypothesis that Lu is a laminin alpha5 receptor. We propose that laminin alpha5 is involved in concentrating Lu on the basal surface of epithelial cells. This may be one mechanism by which basement membrane signals are transmitted to the cell.     

Laminin isoforms of lymph nodes and predominant role of alpha5-laminin(s) in adhesion and migration of blood lymphocytes

During extravasation and within lymph nodes (LNs), blood lymphocytes interact with laminins (Lms), major components of vascular basement membranes (BMs) and of reticular fibers (RFs), a fibrillar extracellular matrix. However, the identity and role of these laminin isoform(s) are poorly known. By using confocal microscopy examination of human LNs, we show that BMs of high endothelial venules (HEVs) express laminin alpha3, alpha4, alpha5, beta1, beta2, and gamma1 chains and that the same chains, in addition to alpha2, are found in RFs. In functional studies with laminin isoforms covering all Lm alpha chains, alpha5-laminin (Lm-511) was the most adhesion- and migration-promoting isoform for human blood lymphocytes, followed by alpha3- (Lm-332) and alpha4- (Lm-411) laminins, and the lymphocytes used the alpha6beta1 integrin as the primary receptor for the alpha5-laminin. Moreover, Lm-511 strongly costimulated T cell proliferation, and blood lymphocytes were able to secrete alpha4- and alpha5-laminins following stimulation. The LN cell number in laminin alpha4-deficient mice compared with wild-type did not differ significantly. This study demonstrates a predominant role for alpha5-laminin(s) in blood lymphocyte biology and identifies LN laminins and their integrin receptors in blood lymphocytes.     

The alpha4beta1 integrin in sickle cell disease.

The alpha4beta1 integrin is an adhesion receptor expressed on reticulocytes in sickle cell disease (SCD) and mediates the adhesion of these cells to sub-endothelial matrix proteins and the endothelium. In this review, we describe the mechanism of activation of the alpha4beta1 integrin on sickle reticulocytes and discuss novel roles for this integrin in SCD as a result of this activation. We also illustrate novel therapies in SCD that may target the integrin and alleviate vaso-occlusion.     

Differential expression of laminin chains and their integrin receptors in human gastric mucosa.

The proliferating cells of the gastric mucosa are found among the pit and mucous neck cells. These cells migrate upward to renew the surface epithelium and downward to restitute the glandular cells. As the epithelial basement membranes (BMs) function as substrate for cell adhesion and migration as well as signals for their differentiation, we studied, by indirect immunofluorescence microscopy, the distribution of different laminin chains and their integrin receptors in adult human stomach. The immunoreactivity for laminin alpha 2 chain localized to the BMs of glands and the lower parts of the gastric pits whereas the laminin alpha 3 chain (laminin-5/kalinin) immunoreactivity was strictly confined to BMs underneath the surface epithelium and the upper parts of the pits. Proliferating mucosal epithelial cells, identified by Ki-67 antibodies, were confined to the areas containing both alpha 2 and alpha 3 laminin chains. The alpha 1, beta 1, and gamma 1 laminin chains were found in all BMs of the mucosa whereas the beta 2 chain was prominent in mucosal blood vessels and also detectable in some glands. Among the laminin integrin receptors, the alpha 3 and beta 4 subunits were seen to be expressed in cells along the BMs with the alpha 3 laminin chain. The alpha 6 integrin, on the other hand, was seen in all gastric epithelia. The present results demonstrate that in the adult human stomach laminin alpha 2 and alpha 3 chains show zonal distribution in BM underlying gastric mucosal epithelium whereas other laminin chains show a more general distribution.     

Mouse models of sickle cell disease

In the absence of a natural animal model for sickle cell disease, transgenic mouse models have been generated to better understand the complex pathophysiology of the disease and to evaluate potential specific therapies. In the early nineties, the simple addition of human globin genes induced the expression of hemoglobin S (HbS) or HbS-related human hemoglobins in mice still expressing mouse hemoglobin. To increase the proportion of human hemoglobin and the severity of the mouse sickle cell syndrome, the proportion of mouse hemoglobin could be decreased by a combination of mouse alpha- and beta-thalassemic defects, leading to complex genotypes and mild disease. Following the discovery of gene targeting in the mouse embryonic stem cells (ES cells), it was made possible to knock out all mouse adult globin genes (2alpha and 2beta) and to add the human homologous genes elsewhere in the mouse genome. In addition, the human gamma gene of fetal hemoglobin was protecting the fetus from HbS polymer formation. Accordingly, the resulting adult mouse models obtained in 1997, expressing human HbS-only, had a very severe anemia (Hb=5-6 g/dL). In order to survive, these "HbS-only mice" had to reduce the HbS concentration within the red blood cells. The phenotype could be less severe by adding modified human gamma genes, still expressed in adult mice. In 2006, a last "S-only" model was obtained by homologous knock in, replacing the mouse globin genes by human genes. This array of models contributes to better understand the role of different interacting factors in the complexity of sickle cell events, such as red cell defects, changes in blood flow and vaso-occlusion, hyperhemolysis, vascular tone dysregulation, oxidations, inflammation, activation and adhesion of cells, ischemia, reperfusion... In addition, each model has an appropriate usefulness to evaluate experimental therapies in vivo and to perform preclinical studies.     

Integrin alpha2beta1 recognizes laminin-2 and induces C-erb B2 tyrosine phosphorylation in metastatic human melanoma cells

Previous studies have shown that tumor cells with metastatic propensity secrete more of the laminin alpha2 chain than non-metastatic tumor cells do, and that laminin-2, which contains the alpha2 chain, promotes cell adhesion better than laminin-1 (Jenq et al. (1994). Differentiation, 58, 29-36). The current studies were designed to determine whether a correlation exists between the expression of the laminin-2 isoform and the metastatic phenotype in melanoma cells. We found that expression of the laminin-2 isoform was upregulated in the metastatic melanoma cell lines tested. Cell attachment studies showed that metastatic melanoma cells attached more efficiently to laminin-2 substrates. Studies on integrin expression revealed that the presence of alpha2beta1 integrin correlated with expression of the laminin-2 isoform in metastatic melanoma cells; anti-integrin alpha2 antibody prevented cell attachment to laminin-2 substrates. The data suggest that the alpha2beta1 integrin is the receptor mediating cell attachment to the laminin-2 isoform. This interaction, mediated by the alpha2beta1 integrin, stimulates secretion of the 72 kD type IV collagenase and induces a specific 185 kD protein tyrosine phosphorylation. The 185 kD tyrosine-phosphorylated protein was identified as the p185/C-erb B2 oncoprotein by immunoprecipitation. These studies suggest that upregulation of expression of the laminin-2 chain correlates with the metastatic phenotype of melanoma cells and provides evidence that the specific p185/C-erb B2 tyrosine phosphorylation may be involved in integrin-mediated signaling during tumor cell invasion and metastasis.     

Characterization of glomerular epithelial cell matrix receptors.

Integrin matrix receptors on glomerular epithelial cells (GEC) may play an important role in adhesion of GEC to the glomerular basement membrane (GBM) and in the maintenance of normal glomerular permeability. Therefore, the author determined the types of matrix receptors present on cultured rat GEC and examined their interactions with several components of the extracellular matrix. Beta 1 integrin matrix receptors were detected on all three glomerular cell types in rat kidney in vivo and at areas of cell-cell contact on cultured GEC. Glomerular epithelial cell adhesion to types I and IV collagen was slightly greater than to laminin and fibronectin. Adhesion to fibronectin was significantly inhibited by a synthetic peptide containing the RGD adhesion sequence. Immunoprecipitation of lysates of surface-iodinated GEC showed the presence of alpha 3 beta 1 integrin. Chromatography of lysates on immobilized collagen showed alpha 3 beta 1 integrin and a 70- to 75-kd protein band as the collagen receptors on GEC. Chromatography on the 120-kd cell-binding fragment of fibronectin disclosed only alpha 3 beta 1 as a specific fibronectin receptor. Antibody to the beta 1 integrin chain inhibited adhesion to laminin and collagen. These studies demonstrate that in vitro, as in vivo, GEC appear to express only alpha 3 beta 1 integrin. Furthermore, this matrix receptor is capable of mediating GEC adhesion to collagen, fibronectin, and laminin, components of the GBM, and presumably plays a similar role in promoting GEC adhesion to GBM in vivo.