Integrin αvβ1 promotes infection by human metapneumovirus

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for αvβ1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with αv or β1 cDNAs confers hMPV infectivity, whereas reduction of αv and β1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that αvβ1 integrin is a functional receptor for hMPV.     

Expression of αv and β3 integrin chains on murine lymphocytes

The vitronectin receptor is a member of the integrin family of adhesion protein receptors and binds a broad spectrum of ligands, including fibronectin and fibrinogen in addition to vitronectin. We have generated four mAbs that recognize the murine αvβ3 vitronectin receptor. Biochemical and expression analyses showed that two of the mAbs are specific for the αv chain, and two are specific for the β3 chain. The mAbs are effective blocking reagents and inhibited cell adhesion to vitronectin, fibrinogen, and fibronectin. Staining analysis revealed expression of αv and β3 on certain populations of murine thymocytes, splenocytes, and bone marrow cells. The expression of αv and β3 appeared to be modulated at specific stages of thymocyte development, suggesting a possible function for the αvβ3 vitronectin receptor in T cell development.     

αVβ3 Integrin Expression Is Essential for Replication of Mosquito and Tick-Borne Flaviviruses in Murine Fibroblast Cells

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3^−/−), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3^−/− on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication.     

Expression of α and β subunits of the integrin superfamily in articular cartilage from macroscopically normal and osteoarthritic human femoral heads

OBJECTIVE—The objective of this study was to detail the topographical and zonal distribution of α and β subunits of the integrin superfamily in normal and osteoarthritic cartilage.
METHODS—Immunohistochemistry utilising antibodies towards α and β subunits was performed on cryostat sections of human articular cartilage from macroscopically normal (n = 6) and osteoarthritic (n = 6) femoral heads. Samples of articular cartilage were obtained from 12 topographically distinct sites from each femoral head. Each section was divided into zones (superficial, middle, deep) and staining scores were recorded.
RESULTS—Normal cartilage stained for integrin subunits α1, α5, αV, β1, β4, and β5, but not for α2, α3, α4, α6, β2, β3, and β6. Intact and non-intact residual cartilage from osteoarthritic femoral heads stained for α1, α2, α5, αV, β1, β4, and β5. Staining was occasionally seen for α4 and β2, but not for α3, α6, β3, and β6. There was no topographical variation in the staining for any of the subunits in either normal or osteoarthritic cartilage. The only subunit that displayed a zonal variation was αV; staining for this subunit was most pronounced in the superficial zone compared with the middle and deep zones.
CONCLUSION—Chondrocytes in normal and osteoarthritic cartilage express the integrin subunits α1, α5, αV, β1, β4, and β5. Chondrocytes in osteoarthritic cartilage, in addition, express the α2, α4, and β2 subunits. The αv subunit is expressed by more chondrocytes in the superficial zone in comparison with cells in the deeper zones. None of the subunits display topographical variation in expression.

Keywords: cartilage; integrins; immunohistochemistry; osteoarthritis     

An Improved αvβ6-Receptor-Expressing Suspension Cell Line for Foot-and-Mouth Disease Vaccine Production

Foot-and-mouth disease (FMD) is endemic in large parts of sub-Saharan Africa, Asia and South America, where outbreaks in cloven-hooved livestock threaten food security and have severe economic impacts. Vaccination in endemic regions remains the most effective control strategy. Current FMD vaccines are produced from chemically inactivated foot-and-mouth disease virus (FMDV) grown in suspension cultures of baby hamster kidney 21 cells (BHK-21). Strain diversity means vaccines produced from one subtype may not fully protect against circulating disparate subtypes, necessitating the development of new vaccine strains that “antigenically match”. However, some viruses have proven difficult to adapt to cell culture, slowing the manufacturing process, reducing vaccine yield and limiting the availability of effective vaccines, as well as potentiating the selection of undesired antigenic changes. To circumvent the need to cell culture adapt FMDV, we have used a systematic approach to develop recombinant suspension BHK-21 that stably express the key FMDV receptor integrin αvβ6. We show that αvβ6 expression is retained at consistently high levels as a mixed cell population and as a clonal cell line. Following exposure to field strains of FMDV, these recombinant BHK-21 facilitated higher virus yields compared to both parental and control BHK-21, whilst demonstrating comparable growth kinetics. The presented data supports the application of these recombinant αvβ6-expressing BHK-21 in future FMD vaccine production.     

A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins αvβ3 and αIIbβ3

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin α_vβ₃ (also known as the vitronectin receptor) or α_IIbβ₃ (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin α_IIbβ₃. Defined β₃ mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing α_vβ₃ and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.     

Loss of laminin and of the laminin receptor integrin subunit α6 in situ correlates with cytokine induced down regulation of α6 on fibroblast-like synoviocytes from rheumatoid arthritis

OBJECTIVE—To investigate in situ the expression of the integrin receptor subunits α6 and β1 and the distribution of the ligand laminin in the synovia from osteoarthritis (OA) and rheumatoid arthritis (RA) patients and to study the effect of cytokines and antirheumatic drugs on the expression of the α6 and β1 integrin subunits on long term cultures of fibroblast-like synoviocytes (FBS) derived from OA and RA.
METHODS—The expression of the α6 and β1 integrin subunits and the distribution of laminin were examined immunohistochemically in normal synovia and in synovia from patients with OA and RA. The effect of proinflammatory cytokines (IL1β and TNFα), and of antirheumatic drugs (salicylic acid, dexamethasone, and methotrexate) on the α6 and β1 expression of cultured normal FBS and FBS from patients with OA and RA was determined by flow cytometry.
RESULTS—In normal synovia and in OA synovia samples with a low grade of inflammation, synovial lining cells (SLC) showed a parallel expression and distribution of α6 and laminin. In synovia samples of OA with a higher grade of inflammation and in the majority of RA synovia samples laminin was pericellularly distributed in a low number of SLC, whereas α6 was expressed on the surface of a high number of SLC. In RA synovia samples with severe inflammatory changes the gradual loss of laminin generally corresponded to a decrease of the α6 integrin subunit. β1 was always strongly expressed in all synovia samples detected. Proinflammatory cytokines up regulated the expression of α6 and β1 on OA-FBS, whereas these effectors decreased α6 and β1 on RA-FBS. In contrast, antirheumatic drugs, in particular methotrexate and dexamethasone, reduced the expression of α6 and β1 on OA-FBS, whereas the same treatment on RA-FBS stimulated the expression of these integrin subunits.
CONCLUSION—The gradual loss of laminin in chronic synovitis may contribute to the altered expression of α6 in SLC. IL1β and TNFα down regulated the expression of the α6 and β1 integrin subunits on long term cultures of FBS derived from RA. Therefore, these cytokines may be among the effectors regulating the expression of the α6 integrin subunit in SLC in vivo. As antirheumatic drugs increase the expression of α6 on RA-FBS, the presence of the laminin receptor may confer a protective effect on the synovia in vivo.

Keywords: laminin; alpha 6; integrins; rheumatoid arthritis; osteoarthritis     

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of G_s, the stimulatory regulator of adenylyl cyclase, promotes dissociation of α_s from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α_s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α_s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α_s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α_s, but not α_s· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α_s and that dissociation of α_s·GTP from βγ is likely to mediate receptor-induced α_s depalmitoylation and translocation of the protein to cytosol in intact cells.     

Potential roles of osteopontin and αVβ3 integrin in the development of coronary artery restenosis after angioplasty

Angioplasty procedures are increasingly used to reestablish blood flow in blocked atherosclerotic coronary arteries. A serious complication of these procedures is reocclusion (restenosis), which occurs in 30–50% of patients. Migration of coronary artery smooth muscle cells (CASMCs) to the site of injury caused by angioplasty and subsequent proliferation are suggested mechanisms of reocclusion. Using both cultured human CASMCs and coronary atherectomy tissues, we studied the roles of osteopontin (OPN) and one of its receptors, α_vβ₃ integrin, in the pathogenesis of coronary restenosis. We also measured the plasma levels of OPN before and after angioplasty and determined the effect of exogenous OPN on CASMC migration, extracellular matrix invasion, and proliferation. We found that cultured CASMCs during log phase of growth and smooth muscle cell layer of the coronary atherosclerotic tissues of patients express both OPN mRNA and protein at a significantly elevated level compared with controls. Interestingly, whereas the baseline plasma OPN levels in control samples were virtually undetectable, those in patient plasma were remarkably high. We also found that interaction of OPN with α_vβ₃ integrin, expressed on CASMCs, causes migration, extracellular matrix invasion, and proliferation. These effects were abolished when OPN or α_vβ₃ integrin gene expression in CASMCs was inhibited by specific antisense S-oligonucleotide treatment or OPN-α_vβ₃ interaction was blocked by treatment of CASMCs with antibodies against OPN or α_vβ₃ integrin. Our results demonstrate that OPN and α_vβ₃ integrin play critical roles in regulating cellular functions deemed essential for restenosis. In addition, these results raise the possibility that transient inhibition of OPN gene expression or blocking of OPN-α_vβ₃ interaction may provide a therapeutic approach to preventing restenosis.     

Interferon γ induces differential upregulation of α and β chemokine secretion in colonic epithelial cell lines

Background—Production of chemoattractant factorsby the intestinal epithelium may contribute to mucosal infiltration byinflammatory cells in inflammatory bowel disease. Secretion of the α chemokine interleukin 8 (IL-8), a neutrophil chemoattractant, has beenwidely studied, but little is known about epithelial secretion of β chemokines, which are preferentially involved in recruiting monocytes.
Aims—To investigate the profiles of α and β chemokine secretion in colonic cell lines and their differentialmodulation by interferon γ (IFN-γ), a product of activated Tlymphocytes and natural killer cells.
Methods and results—HT29-19A, a model of theCl secretory crypt cell, exhibited a parallel secretionof the α chemokines IL-8 and GROα, which could be markedlyupregulated by tumour necrosis factor α (TNF-α) and IL-1β. Thesecells showed no significant expression of the β chemokines RANTES(regulated upon activation T cell expressed and secreted), MIP-1α(macrophage inflammatory protein 1α), and MCP-1 (monocyte chemotacticprotein 1) under these conditions, but IFN-γ in combination withTNF-α caused a dose dependent induction of RANTES and MCP-1secretion. This was accompanied by a marked increase of RANTES mRNA. Incontrast, IFN-γ had no significant effect on TNF-α stimulated IL-8secretion. Caco-2 cells, with features more typical of villusabsorptive cells, were relatively poor secretors of α chemokines butsecreted high levels of MCP-1 in response to IL-1β. IFN-γ did notinfluence α or β chemokine secretion in these cells.
Conclusions—These studies suggest that intestinalepithelial cells may produce chemokines capable of attracting bothneutrophils and monocytes. The ability of IFN-γ to activate theexpression of β chemokines preferentially could facilitate thedevelopment of chronic inflammatory infiltrates.

Keywords:inflammatory bowel disease; RANTES; interferongamma; chemokine

     

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α₁β₁ and α₂β₁ integrins—through induction of mRNAs encoding the α₁ and α₂ subunits. In contrast, VEGF did not induce increased expression of the α₃β₁ integrin, which also has been implicated in collagen binding. Integrin α₁-blocking and α₂-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α₁-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α₁β₁ and α₂β₁ expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α₁-blocking and α₂-blocking Abs. In vivo, a combination of α₁-blocking and α₂-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α₁β₁ and α₂β₁ expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α₁β₁ and α₂β₁ antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Identification of amino acids essential for the antiangiogenic activity of tumstatin and its use in combination antitumor activity

Tumstatin is an angiogenesis inhibitor that binds to αvβ3 integrin and suppresses tumor growth. Previous deletion mutagenesis studies identified a 25-aa fragment of tumstatin (tumstatin peptide) with in vitro antiangiogenic activity. Here, we demonstrate that systemic administration of this tumstatin peptide inhibits tumor growth and angiogenesis. Site-directed mutagenesis identified amino acids L, V, and D as essential for the antiangiogenic activity of tumstatin. The tumstatin peptide binds to αvβ3 integrin on proliferating endothelial cells and localizes to select tumor endothelium in vivo. Using 3D molecular modeling, we identify a putative interaction interface for tumstatin peptide on αvβ3 integrin. The antitumor activity of the tumstatin peptide, in combination with bevacizumab (anti-VEGF antibody), displays significant improvement in efficacy against human renal cell carcinoma xenografts when compared with the single-agent use. Collectively, our results demonstrate that tumstatin peptide binds specifically to the tumor endothelium, and its antiangiogenic action is mediated by αvβ3 integrin, and, in combination with an anti-VEGF antibody it exhibits enhanced tumor suppression of renal cell carcinoma.     

Autoregulation of insulin receptor signaling through MFGE8 and the αvβ5 integrin

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvβ5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/β5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvβ5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/β5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvβ5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.

Acute insulin resistance can be viewed as a protective response under specific physiological conditions that necessitate increased insulin secretion. Nevertheless, the increasing prevalence of chronic insulin resistance (1) in the current obesity epidemic hastens the development of type 2 diabetes (T2D) and induces compensatory hyperinsulinemia. Hyperinsulinemia can produce potentially maladaptive consequences at least in part, due to the mitogenic roles of insulin (2–4). As such, there remains a critical need for new therapies to improve insulin sensitivity in order to prevent T2D, avoid the need for insulin treatment in patients with T2D, or reduce the insulin dose required to normalize blood glucose in such individuals.Insulin binding to the alpha subunit of the insulin receptor induces a conformational change that triggers activation of insulin receptor beta subunit (IRβ) tyrosine kinase activity (5–7). The activated insulin receptor phosphorylates target molecules that mediate downstream signaling leading to glucose uptake and other metabolic effects (8, 9). Dephosphorylation of IRβ and insulin receptor substrate-1 (IRS-1) aids in termination of insulin signaling pathways (10, 11) and is the basis of clinical trials targeting putative phosphatases to treat diabetes (12). Despite their potential therapeutic relevance, there is a relative paucity of knowledge regarding molecular mechanisms that lead to termination of insulin receptor signaling.The integrin families of cell surface receptors mediate bidirectional signaling between the cell and its external environment. Previous work has identified interactions between integrin receptors and other growth factor receptor tyrosine kinases (13–16) that lead to modulation of downstream signaling (17–19). For example, the αvβ3 and α6β4 integrins function as coreceptors for insulin-like growth factor-1 and 2 (IGF1 and 2) and potentiate IGF1 receptor (IGF1R)-mediated signaling (19–23). Immunoprecipitation studies have demonstrated a physical association between the αv integrins and IRβ (24, 25). The impact of these associations on glucose homeostasis has not been evaluated. A role for β1 integrins in the regulation of glucose homeostasis is well established. This class of integrins appears to be particularly important in regulating insulin-mediated glucose homeostasis in the obese state. The effect of β1 integrins on glucose homeostasis appears to be primarily due to obesity-associated matrix remodeling (26–30) rather than a direct effect secondary to a physical association between β1 integrins and the insulin receptor.Milk fat globule epidermal growth factor like 8 (MFGE8) is a secreted integrin ligand which binds the αvβ3, αvβ5, and α8β1 integrins (31, 32). Several recent observations suggest a role for MFGE8 in modulating insulin resistance. In humans, serum MFGE8 levels are increased in the context of diabetes and correlate positively with the extent of hemoglobin glycosylation (33, 34). Indeed, serum MFGE8 levels correlate with indices of insulin resistance in two independent cohorts of patients with T2D or gestational diabetes from China (35, 36). A missense variation in the gene encoding MFGE8, present in South Asian Punjabi Sikhs, is associated with increased circulating MFGE8 levels and increased risk of developing T2D (37). Increased circulating levels of MFGE8 in diabetic patients may impact T2D through effects on inflammation and cardiovascular disease. Humans with increased MFGE8 expression have a greater risk of developing coronary artery disease (38). In contrast, in murine models, MFGE8 deficiency exacerbates cardiac hypertrophy and atherosclerosis (39, 40). MFGE8 also improves wound healing responses in diabetic foot ulcers (41, 42) by triggering apoptotic cell clearance and promoting resolution of inflammation (43–45).Despite the notable links between MFGE8, insulin resistance, and T2D pathology, the biology underlying these associations has not been investigated. We therefore evaluated the effect of acute antibody-mediated disruption of the MFGE8/β5 pathway on glucose homeostasis in wild-type (WT) mice. We report here that MFGE8 markedly attenuates the effect of insulin on skeletal muscle glucose uptake. Antibody-mediated blockade of MFGE8 or αvβ5 enhances while recombinant MFGE8 (rMFGE8) reduces insulin-stimulated glucose uptake in vitro and in vivo. Mechanistically, insulin acts to promotes cell-surface enrichment of skeletal muscle MFGE8, which then binds to cell surface αvβ5 and increases the interaction between the integrin and the insulin receptor. This interaction subsequently aids in terminating insulin receptor signaling.     

Integrin αvβ6 sustains and promotes tumor invasive growth in colon cancer progression

AIM: To detect the mechanism by which colon tumor escapes the growth constraints imposed on normal cells by cell crowding and dense pericellular matrices.METHODS: An immunohistochemical study of integrin αvβ6 and matrix metalloproteinase-9 (MMP-9) was performed on tissue microarrays of 200 spots, including 100 cases of colon tumors.RESULTS: High immunoreactivity for αvβ6 (73.7%; 28/38) and MMP-9 (76.5%; 52/68) was observed in invasive tumor portions. Furthermore, the effects of integrin αvβ6 on tumor invasive growth in nude mice were detected. Tumor invasive growth and high expression of both αvβ6 and MMP-9 were only seen in tumors resulting from WiDr cells expressing αvβ6 in the tumorigenicity assay. Flow cytometry was applied to analyze αvβ6 expression in colon cancer WiDr and SW480 cells. The effects of cell density on αvβ6 expression and MMP-9 secretion were also detected by Biotrak MMP-9 activity assay and gelatin zymography assay. High cell density evidently enhanced αvβ6 expression and promoted MMP-9 secretion compared with low density.CONCLUSION: Integrin αvβ6 sustains and promotes tumor invasive growth in tumor progression via a self-perpetuating mechanism. Integrin ανβ6-mediated MMP-9 secretion facilitates pericellular matrix degradation at high cell density, which provides the basis of invasive growth.     

Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.     

Streaming potential measurements in αβγ-rat epithelial Na+ channel in planar lipid bilayers

Streaming potentials across cloned epithelial Na⁺ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (ΔP) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by ΔP when the Δπ was directed against ΔP. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na⁺] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na⁺ ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by ΔP can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity.     

The α chain of laminin-1 is independently secreted and drives secretion of its β- and γ-chain partners

A mammalian recombinant strategy was established to dissect rules of basement membrane laminin assembly and secretion. The α-, β-, and γ-chain subunits of laminin-1 were expressed in all combinations, transiently and/or stably, in a near-null background. In the absence of its normal partners, the α chain was secreted as intact protein and protein that had been cleaved in the coiled-coil domain. In contrast, the β and γ chains, expressed separately or together, remained intracellular with formation of ββ or βγ, but not γγ, disulfide-linked dimers. Secretion of the β and γ chains required simultaneous expression of all three chains and their assembly into αβγ heterotrimers. Epitope-tagged recombinant α subunit and recombinant laminin were affinity-purified from the conditioned medium of αγ and αβγ clones. Rotary-shadow electron microscopy revealed that the free α subunit is a linear structure containing N-terminal and included globules with a foreshortened long arm, while the trimeric species has the typical four-arm morphology of native laminin. We conclude that the α chain can be delivered to the extracellular environment as a single subunit, whereas the β and γ chains cannot, and that the α chain drives the secretion of the trimeric molecule. Such an α-chain-dependent mechanism could allow for the regulation of laminin export into a nascent basement membrane, and might serve an important role in controlling basement membrane formation.     

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (K_m, 13.6 μM) and pregnenolone (K_m, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-³H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-³H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.     

Multivalent structure of an αβT cell receptor

Whether there is one or multiple αβT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRβ chains incorporate at least two αβTCRs in a single TCR/CD3 complex. Evidence for bispecific αβTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRβ-variable regions. Such (αβ)₂TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (αβ)₂TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.     

Experimental support for a β-propeller domain in integrin α-subunits and a calcium binding site on its lower surface

Integrins are large, heterodimeric surface molecules of wide importance in cell adhesion. The N-terminal half of all integrin α-subunits contains seven weak sequence repeats of ≈60 amino acids that are important in ligand binding and have been predicted to fold cooperatively into a single β-propeller domain with seven β-sheets. We provide evidence supporting this model with a mouse mAb to human Mac-1 (αMβ2, CD11b/CD18). This antibody, CBRM1/20, binds to amino acid residues that are in different repeats and are 94 residues apart in the primary structure in the loop between strands 1 and 2 of β-sheet 5 and in the loop between strands 3 and 4 of β-sheet 6. The 1–2 loops of β-sheets 5–7 in integrins have EF hand-like Ca²⁺-binding motifs. CBRM1/20 binds to Mac-1 in the presence of Ca²⁺ or Sr²⁺ with an EC₅₀ of 0.2 mM. Mg²⁺ or Mn²⁺ cannot substitute. Antibodies to other epitopes on the Mac-1 β-propeller domain bind in the absence of calcium. mAb CBRM1/20 does not block ligand binding. Thus, the region on the lower surface of the β-propeller domain to which mAb CBRM1/20 binds does not bind ligand and, furthermore, cannot bind other integrin domains, such as those of the β-subunit.