Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen

FG human pancreatic carcinoma cells use integrin alpha v beta 5 as their primary vitronectin receptor since they fail to express integrin alpha v beta 3. These cells are unable to form focal contacts, spread, or migrate on vitronectin but readily do so on collagen in a beta 1 integrin-dependent manner. Transfection of FG cells with a cDNA encoding the integrin beta 3 subunit results in the surface expression of a functional integrin alpha v beta 3 heterodimer providing these cells with novel adhesive and biological properties. Specifically, FG cells expressing beta 3 acquire the capacity to attach and spread on vitronectin as well as fibrinogen with beta 3 localization to focal contacts. Moreover, these cells gain the capacity to migrate through a porous membrane in response to either vitronectin or fibrinogen. These results demonstrate that the beta 3 and beta 5 integrin subunits when associated with alpha v, promote distinct cellular responses to a vitronectin extracellular environment.     

A molecular model of RGD ligands. Antibody D gene segments that direct specificity for the integrin alpha IIb beta 3.

Following an ill-defined activation event, the Arg-Gly-Asp (RGD) recognition site of the platelet integrin, glycoprotein IIb-IIIa (alpha IIb beta 3), can bind to fluid-phase, RGD-containing protein ligands, such as fibrinogen, or to the murine monoclonal IgM, PAC-1, which contains the sequence Arg-Tyr-Asp (RYD) within the third complementarity-determining region of its heavy chain (H3). PAC-1 has thus become a widely exploited marker of platelet alpha IIb beta 3 activation. In this report, we compare PAC-1 with two murine IgG, OP-G2 (IgG1 kappa) and LJ-CP3 (IgG1 kappa), that also contain the sequence RYD in H3 but bind to alpha IIb beta 3 without prior activation. Each antibody can inhibit the binding of the other two to intact platelets or to purified IIb-IIIa, the binding of each antibody is completely inhibited by peptides containing RGD, and H3 of each antibody uses the germline D-gene DSP 2.10 (CTATAGGTACGAC) which includes the sequence RYD. Two other murine IgG, HP20 and PCG1-1, cloned and sequenced by other laboratories, also utilize the DSP 2.10 sequence, but neither antibody binds to alpha IIb beta 3. From a comparison of the H3 sequences of these antibodies, we have developed a molecular model of the H3 loop region which can explain these differences in specificity. This model predicts that both the ability to bind to alpha IIb beta 3 and the activation dependence of that binding are a function of the orientation and, therefore, accessibility of the RYD sequence. This model and refinements thereof can be exploited to study the molecular basis for specificity and affinity of RGD-containing ligands for integrins.     

A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.     

Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active

Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. In the present study, we identified clusters of integrin that formed upstream of Rac activation. These clusters contained a Rac-binding protein(s) and appeared to be involved in Rac activation. The integrin clusters contained calpain and calpain-cleaved beta3 integrin, while the focal complexes and focal adhesions that formed once Rac and Rho were activated did not. Moreover, the integrin clusters were dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases were dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurred even when calpain inhibitors were present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s).     

Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor.

The B cell antigen receptor (BCR) is a multimeric protein complex consisting of the ligand binding immunoglobulin molecule and the Ig-alpha/beta heterodimer that mediates intracellular signalling by coupling the receptor to protein tyrosine kinases (PTKs). Transfection of the Ig-alpha deficient myeloma cell line J558L microns with expression vectors coding for mutated Ig-alpha allowed us to test the function of the tyrosines in the cytoplasmic region of Ig-alpha in the context of the BCR. Furthermore we expressed Ig-alpha mutations as chimeric CD8-Ig-alpha molecules on K46 B lymphoma cells and tested their signalling capacity in terms of PTK activation and release of calcium. We show here that the conserved tyrosine residues in the cytoplasmic portion of Ig-alpha have a dual role. First, they are required for efficient activation of PTKs during signal induction and second, one of them is subject to phosphorylation by activated src-related PTKs. Phosphorylation on tyrosine in the cytoplasmic portion of Ig-alpha is discussed as a possible mechanism to couple the BCR to SH2 domain-carrying molecules.     

Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion.

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.     

Physical and functional association of the T cell receptor and the T3 molecular complex on cytotoxic T cell clones that are differentially inhibitable by anti-T3 antibodies

To examine the hypothesis that the antigen-specific T cell receptor (TcR) can function independently from the T3 complex on cytolytic T lymphocyte (CTL) clones, the physical and functional association of the T3 molecular complex and the T cell receptor has been examined on CTL clones that are differentially susceptible to inhibition by anti-T3 antibodies. From a panel of nine DPw2-specific CTL clones derived from the same donor, two clones (8.4 and 8.8) that were the most disparate in their susceptibility to inhibition by anti-T3 antibody were chosen for study. No significant differences were found between 8.4 and 8.8 for: (1) the levels of cell surface expression of the T3 complex and the TcR; (2) the ability to modulate T3 cell surface molecules; and (3) the capacity of the TcR to comodulate with the T3 complex. Modulation of the T3 complex from clone 8.4 did not significantly affect cytolytic activity, and incubation of modulated 8.4 with additional anti-T3 antibody did not inhibit cytolytic activity. Although no T3 function for clone 8.4 could be demonstrated by simply blocking cytolytic activity with anti-T3 antibody, addition of limiting quantities of anti-T11 (but not anti-T4, anti-Tac, or anti-LFA-1) antibodies plus anti-T3 produced a marked synergistic inhibition of cytolysis. These results suggest that: (1) CTL clones that are resistant to inhibition by anti-T3 antibodies actually have a physical and functional association between the T3 complex and the TcR; and (2) the ability to demonstrate a functional role for T3 by antibody blocking may, in some cases, require limiting the involvement of the T11 molecule in CTL-target interactions. The most likely explanation for the observed heterogeneity in susceptibility to blocking by anti-T3 antibodies is, therefore, thought to be that individual CTL clones possess TcR with differential avidity for specific targets.     

A short motif in the C-terminus of mouse bestrophin 3 [corrected] inhibits its activation as a Cl channel

Bestrophins are a new family of anion channels. Here, we examined the Cl channel activity of mBest4. Surprisingly, wild type mouse bestrophin-4 (mBest4) did not induce functional Cl channels when over-expressed in HEK293 cells. However, deletion of part of the C-terminus (residues 353-669) produced large Cl currents, suggesting the presence of a C-terminal motif that inhibited Cl channel function. Deletion of a short motif (356-364) or substitution of certain residues in this motif with alanines also resulted in expression of robust Cl currents. The channel activity of the mBest4 protein lacking the C-terminus (residues 353-669) was specifically inhibited by co-expression of C-terminal fragments of mBest4 having the inhibitory motif, suggesting that the C-terminal motif blocked mBest4 channel activity probably by interacting with the channel pore.     

A new concept in the molecular process of muscle contraction: functional role of phosphorylated amino acids in myosin

In this report a summary is given of our experiments concerning the in vivo endogenous phosphate (P) content of myosin. It was found that besides the ester type phosphates of myosin there was a considerable amount of N-P type energy-rich phosphoryl groups bond to the basic amino acids of peptide chains. The endogenous P concentration of myosin depends on the source of the preparation. The concentration of P is much higher in myosin preparations of well-trained animals and human subjects compared to those found in the control muscles. As the P binding sites of fresh purified myosin are only partially saturated, the preparations can incorporate P up to a definite saturation only. The phosphorylating ability of myosins disappears after prolonged storage as a consequence of an alteration in structure of the myosin molecule. The P groups are moving inside the myosin molecule. It is supposed that the inorganic P release promoted by actin is connected with the thin filament movements towards the centre of the sarcomere, furthermore P replenishment, P linking and movement involve N3-trimethyl-lysine, 3-methylhistidine, P-Arg and two conformers of P-His. The two net negative charges of P group form electric monopoles of a minor battery (myosin head). They help to force generation at head rotation (90 degrees-45 degrees angle) and produce free energy changes that can be calculated from the number of N-P bonds.     

A structural model for the alpha-subunit of transducin. Implications of its role as a molecular switch in the visual signal transduction mechanism

Transducin is a GTP-binding protein which mediates the light activation signal from photolyzed rhodopsin to cGMP phosphodiesterase and is pivotal in the visual excitation process. Biochemical studies suggest that the T alpha subunit of transducin is composed of three functional domains, one for rhodopsin/T beta gamma interaction, another for guanine nucleotide binding, and a third for the activation of phosphodiesterase. The integration of the primary sequence of T alpha along with secondary structure, hydropathy and folding topology predictions, and a comparison with homologous proteins have led to the construction of a three-dimensional model of the T alpha subunit. A molecular mechanism which underlies the coupling action of T alpha is suggested on the basis of this model.     

Potential role of a new anti-beta3 integrin antibody in the development of intimal hyperplasia after vascular surgery: an in vitro smooth muscle cell model

The occurrence of intimal hyperplasia after vascular surgery is an ongoing concern in current clinical practice. Among the many factors involved in the development of this pathology, platelet adhesion and myointimal proliferation play a major role. Both these processes are mediated by integrins (mainly alphavbeta3 integrins). Over the past years, several substances have been designed to delay or inhibit the cell proliferation that leads to hyperplasia and mainly include monoclonal antibodies directed against integrins. The aim of the present study was to evaluate the effects of an antibody denoted P37 (anti beta3 integrin) on human smooth muscle cells (SMC) and its role in blocking the B3 subunit. To this end, SMC from human umbilical artery were cultured in the presence or absence of the cell substrate vitronectin (VN) and incubated with P37. After 4 days of treatment, determination was made of cell proliferation and migration. Smooth muscle cells grown on VN showed increased proliferation and migration compared to control VN-free cultures. However, the presence of P37 in the culture medium inhibited proliferation and reduced migration. Combined treatment with VN and P37 led to improved proliferation but VN was unable to reverse the effects on migration observed in the former cultures. Results suggest that in vitro, P37 is capable of blocking human SMC beta3 integrins and thus impedes cell proliferation and migration These findings may have clinical implications related to modulation of the development of hyperplasia.     

The role of semaphorin3A in myogenic regeneration and the formation of functional neuromuscular junctions on new fibres

Current research on skeletal muscle injury and regeneration highlights the crucial role of nerve–muscle interaction in the restoration of innervation during that process. Activities of muscle satellite or stem cells, recognized as the ‘currency’ of myogenic repair, have a pivotal role in these events, as shown by ongoing research. More recent investigation of myogenic signalling events reveals intriguing roles for semaphorin3A (Sema3A), secreted by activated satellite cells, in the muscle environment during development and regeneration. For example, Sema3A makes important contributions to regulating the formation of blood vessels, balancing bone formation and bone remodelling, and inflammation, and was recently implicated in the establishment of fibre‐type distribution through effects on myosin heavy chain gene expression. This review highlights the active or potential contributions of satellite‐cell‐derived Sema3A to regulation of the processes of motor neurite ingrowth into a regenerating muscle bed. Successful restoration of functional innervation during muscle repair is essential; this review emphasizes the integrative role of satellite‐cell biology in the progressive coordination of adaptive cellular and tissue responses during the injury‐repair process in voluntary muscle.     

Natural‐host animal models indicate functional interchangeability between the filamentous haemagglutinins of Bordetella pertussis and Bordetella bronchiseptica and reveal a role for the mature C‐terminal domain,but not the RGD motif,during infection

Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human‐adapted (e.g. Bordetella pertussis) strains produce a surface‐exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg‐Gly‐Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural‐host animal models should apply to B. pertussis FHA as well. We also show that the C‐terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage‐like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.     

Identification and molecular cloning of tactile. A novel human T cell activation antigen that is a member of the Ig gene superfamily.

We have identified and cloned cDNA for a novel cell-surface protein that we have named Tactile for T cell activation, increased late expression. It is expressed on normal T cell lines and clones, and some transformed T cells, but no other cultured cell lines tested. It is expressed at low levels on peripheral T cells and is strongly up-regulated after activation, peaking 6 to 9 days after the activating stimulus. It is also up-regulated on NK cells activated in allogeneic cultures. It is not found on peripheral B cells but is expressed at very low levels on activated B cells. Tactile-specific mAb immunoprecipitates a band of 160 kDa when reduced and bands of 240, 180, and 160 kDa nonreduced. Using an antiserum produced with affinity-purified Tactile protein to screen a lambda gt11 library, we have identified Tactile cDNA. Northern blot analysis shows an expression pattern similar to that of the protein and transfection of COS cells with the full-length 5.2-kb cDNA results in cell-surface expression. Comparison with the sequence databanks show that Tactile is a member of the immunoglobulin gene superfamily, with similarity to Drosophila amalgam, the melanoma Ag MUC-18, members of the carcinoembryonic Ag family, the poliovirus receptor, and the neural cell adhesion molecule. The deduced primary sequence encodes a protein with three Ig domains, a long serine/threonine/proline-rich region typical of an extensively O-glycosylated domain, a transmembrane domain, and a 45 residue cytoplasmic domain. These data suggest that Tactile may be involved in adhesive interactions of activated T and NK cells during the late phase of the immune response.