Biomedical subjects

D J Dorahy

Publications and source records attributed to D J Dorahy.

13 recordsLinked to original sources

PubMed2002

Integrin alpha(v)beta6-associated ERK2 mediates MMP-9 secretion in colon cancer cells.

There is general consensus that matrix metalloproteinases are involved in tumour progression. We show herein that inhibition of integrin alpha(v)beta6 expression in colon cancer cells suppresses MMP-9 secretion. This integrin-mediated event is dependent upon direct binding between the beta6 integrin subunit and extracellular signal-regulated kinase 2. Targetting either beta6 or its interaction with extracellular signal-regulated kinase in order to inhibit matrix metalloproteinase activity may offer a useful therapeutic approach in preventing growth and spread of colon cancer.

Antigens, Neoplasm↗

PubMed2000

CD36 is a ditopic glycoprotein with the N-terminal domain implicated in intracellular transport.

The CD36 receptor sequence predicts two hydrophobic domains located at the N- and C-termini of the protein, but there are conflicting reports as to whether the N-terminal uncleaved leader sequence functions as a transmembrane domain. To investigate the topology of CD36, we generated a panel of mutants lacking either one or both hydrophobic regions and analyzed their folding and transport in COS-7 cells. The N- and the C-terminal hydrophobic regions were both sufficient to anchor CD36 in the membrane, and a FLAG epitope inserted at the N-terminus was located intracellularly. These results indicate that CD36 adopts a ditopic configuration. Accordingly, neither N- nor C-terminal truncation mutants were secreted. Analysis with conformation-specific monoclonal antibodies showed that the N-terminal transmembrane domain truncated molecule was slowly transported through the exocytic pathway and largely accumulated intracellularly. Thus, dual membrane insertion dictates the correct topogenesis and seems to be necessary for efficient folding and intracellular transport.

Amino Acid Sequence↗

PubMed2000

Cytoplasmic interactions between decay-accelerating factor and intercellular adhesion molecule-1 are not required for coxsackievirus A21 cell infection.

Coxsackievirus A21 (CAV-21) employs a cell receptor complex of decay-accelerating factor (DAF) and intercellular adhesion molecule-1 (ICAM-1) for cell infectivity. In this study, the nature of potential extra- and/or intracellular interactions between DAF and ICAM-1 involved in picornaviral cell entry was investigated. Firstly, it was shown that intracellular interplay between DAF and ICAM-1 is not required for CAV-21 infection, as CAV-21 lytic infection mediated via the DAF/ICAM-1 receptor complex is not inhibited by replacement of the transmembrane and cytoplasmic domains of ICAM-1 with those from an unrelated cell surface molecule, CD36. By immunoprecipitation, chemical cross-linking and picornaviral binding assays, the existence of a close spatial association between DAF and ICAM-1 on the surface of ICAM-1-transfected RD cells was confirmed. Furthermore, it was shown that potential extracellular DAF/ICAM-1 interactions are likely to occur in an area on or proximal to DAF SCR3 and may influence the route of CAV-21 cell entry.

Animals↗

PubMed1998

Active Lyn protein tyrosine kinase is selectively enriched within membrane microdomains of resting platelets.

Circulating platelets are primed to respond very rapidly to thrombogenic stimuli, but most platelets complete their lifespan without ever becoming activated. Platelet activation is accompanied by waves of sequential tyrosine phosphorylation thought to involve members of the Src family of protein tyrosine kinases (PTKs). We show here that resting platelets contain highly active pp53/56(Lyn) PTK within membrane microdomains (rafts) isolated biochemically with or without the use of detergent. This fraction is also greatly enriched in the transmembrane glycoprotein CD36, known to associate with Lyn PTK, but in transfection studies we could find no evidence to suggest that CD36 affects the distribution or function of Lyn. Upon platelet activation Lyn activity remains constant or diminishes and pp60(c-src) PTK within this fraction becomes highly activated, indicating the dynamic nature of the membrane microdomains. It is suggested that the function of active Lyn PTK in the resting platelet is to allow prolonged survival of this anucleate cell.

Animals↗

PubMed1998

Antibody binding to individual short consensus repeats of decay-accelerating factor enhances enterovirus cell attachment and infectivity.

Decay-accelerating factor (DAF), a widely expressed membrane complement-regulatory protein, is utilized as a cellular receptor by many human enteric pathogens. We show here that the binding of two enteroviruses to individual short consensus repeats (SCR) of DAF on the cell surface is greatly augmented by mAb binding to an alternate SCR: Coxsackievirus A21 binding to the SCR1 of DAF is increased by Ab binding to SCR3 and, conversely, Echovirus 7 binding to SCR3 is enhanced severalfold by Ab binding to SCR1. These Ab-induced increases in viral binding also resulted in increased viral infectivity. Using purified soluble DAF in a solid phase assay it was found that Ab binding to SCR1 is increased greatly in the presence of an Ab against SCR3 and, reciprocally, Ab against SCR1 greatly increases Ab binding to SCR3. In contrast to the results obtained with the larger viral particles, however, this reciprocal Ab-induced enhancement of binding is not seen when measuring Ab binding to membrane-bound DAF SCR on the cell surface. These findings provide a possible explanation for functional differences between membrane-bound and soluble DAF with implications for a potential role for DAF-binding molecules in regulating DAF function. This is the first demonstration of enhancement of viral infectivity mediated by Ab against the viral receptor.

Animals↗

PubMed1997

CD36 forms covalently associated dimers and multimers in platelets and transfected COS-7 cells.

CD36 is a transmembrane glycoprotein expressed on the surface of a number of cell types. The analysis of CD36 from platelets using immunoblotting, gel filtration, and native PAGE indicated the presence of high molecular complexes exceeding the Mr of monomeric CD36. Experiments using transfected COS-7 cells revealed these complexes were homodimers and -multimers of CD36. The multimers could be dissociated by treatment with a reducing agent, indicating they were formed by intermolecular cysteine-bridging. Mutagenesis of the cDNA for CD36 implicated the cysteines in the extracellular domain of the molecule. The potential physiological roles of CD36 multimerisation are discussed.

Animals↗

PubMed1997

TLiSA1 (PTA1) activation antigen implicated in T cell differentiation and platelet activation is a member of the immunoglobulin superfamily exhibiting distinctive regulation of expression.

T lineage-specific activation antigen 1 (TLiSA1) antigen was initially described as a T lineage-specific activation antigen involved in the differentiation of human cytotoxic T cells. Subsequently, the antigen was identified on platelets and was shown to be involved in platelet activation, hence it was renamed platelet and T cell antigen 1 (PTA1), although identity between the two antigens was not established. In the present study we have cloned the cDNA encoding TLiSA1 from Jurkat cells and show it to be a novel member of the immunoglobulin superfamily with the unusual structure of two V domains only. Identity between TLiSA1 and platelet PTA1 is established by immunological criteria, by internal peptide sequences obtained from the purified platelet glycoprotein and by sequencing the platelet transcript after reverse transcriptase-polymerase chain reaction. In Jurkat cells, TLiSA1/PTA1 mRNA and surface protein expression is greatly stimulated by treatment of the cells with phorbol ester, but the T cell proliferative signal of phorbol ester and ionophore combined greatly reduces or abrogates this response, and this suppressive effect of the ionophore is not reversed by incorporating FK506 to inhibit calcineurin. Together with the known signaling role of PTA1, these data substantiate the notion that this molecule is implicated in T cell differentiation, perhaps by engagement of an adhesive ligand.

Amino Acid Sequence↗

PubMed1997

Stimulation of platelet activation and aggregation by a carboxyl-terminal peptide from thrombospondin binding to the integrin-associated protein receptor.

Thrombospondin, a major secretory product of the alpha-granules of activated platelets, is a large trimeric glycoprotein that plays an important role in platelet aggregation. On resting platelets, thrombospondin binds to a single receptor in a cation-independent manner, but upon platelet activation it binds at least two further, distinct receptors that are both dependent upon divalent cations. Each of these receptors on the platelet surface binds to different regions of the thrombospondin molecule, and such binding may be responsible for the multifunctional role of thrombospondin in aggregation. We show here that a peptide from the carboxyl terminus of thrombospondin, RFYVVMWK, directly and specifically induces the activation and aggregation of washed human platelets from different donors at concentrations of 5-25 microM. At lower concentrations the peptide synergizes with suboptimal concentrations of ADP to induce aggregation. Peptide affinity chromatography and immunoprecipitation with a monoclonal antibody were used to identify the receptor for the carboxyl-terminal peptide as the integrin-associated protein. The integrin-associated protein remained bound to the RFYVVMWK-containing peptide column when washed with a scrambled peptide in the presence of 5 mM EDTA, indicating a divalent cation-independent association. It is suggested that integrin-associated protein is the primary receptor for thrombospondin on the surface of resting platelets and is implicated in potentiating the platelet aggregation response.

Adenosine Diphosphate↗

PubMed1997

Mouse cells expressing human intercellular adhesion molecule-1 are susceptible to infection by coxsackievirus A21.

Competitive viral binding assays have revealed previously that coxsackievirus A21 (CAV21) and human rhinovirus 14 (HRV14) share a common cell surface receptor. More recently, intercellular adhesion molecule-1 (ICAM-1) has been identified as the cellular receptor for HRV-14. Also, anti-ICAM-1 monoclonal antibodies (MAbs) blocked infection by HRV14, CAV13, CAV18, and CAV21, suggesting that these viruses share this receptor; however, this has never been established by more direct methods. In this study we show conclusively that CAV21 binds to ICAM-1 and that MAbs directed against the N-terminal domain of the molecule inhibit this attachment. Furthermore, we show that the specific interaction between ICAM-1 and 160S CAV21 virions induces formation of 135S A particles. Finally, we show transfection of normally nonsusceptible mouse L cells with human ICAM-1 cDNA renders them susceptible to infection by CAV21.

Animals↗

PubMed1997

Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry.

It is becoming increasingly apparent that many viruses employ multiple receptor molecules in their cell entry mechanisms. The human enterovirus coxsackievirus A21 (CAV21) has been reported to bind to the N-terminal domain of intercellular adhesion molecule 1 (ICAM-1) and undergo limited replication in ICAM-1-expressing murine L cells. In this study, we show that in addition to binding to ICAM-1, CAV21 binds to the first short consensus repeat (SCR) of decay-accelerating factor (DAF). Dual antibody blockade using both anti-ICAM-1 (domain 1) and anti-DAF (SCR1) monoclonal antibodies (MAbs) is required to completely abolish binding and replication of high-titered CAV21. However, the binding of CAV21 to DAF, unlike that to ICAM-1, does not initiate a productive cell infection. The capacity of an anti-DAF (SCR3) MAb to block CAV21 infection but not binding, coupled with immunoprecipitation data from chemical cross-linking studies, indicates that DAF and ICAM-1 are closely associated on the cell surface. It is therefore suggested that DAF may function as a low-affinity attachment receptor either enhancing viral presentation or providing a viral sequestration site for subsequent high-affinity binding to ICAM-1.

Animals↗

PubMed1996

Biochemical isolation of a membrane microdomain from resting platelets highly enriched in the plasma membrane glycoprotein CD36.

Here we describe the isolation and characterization of a Triton X-100-insoluble fraction isolated from lysates of platelets by flotation in sucrose gradients. Transmission electron microscopy of the insoluble material revealed a heterogeneous population of vesicles ranging in size from 20 to 1000 nm, and Western blot analyses of platelet lysates for the caveolae structural coat protein, caveolin/VIP21, were negative. Biochemical characterization of the Triton X-100-insoluble fraction showed it to be cholesterol-rich, greatly and specifically enriched in the plasma membrane glycoprotein CD36, and also to contain Src and the Src-related kinase, Lyn. CD36 within this fraction is shown to be palmitoylated, but the fraction itself is not generally enriched in palmitoylated platelet proteins. These results suggest that this fraction represents caveolin-negative, CD36-rich microdomains in the resting platelet membrane. CD36 can form associations with certain Src-related kinases and can signal to activate platelets. These results suggest the possibility that such microdomains are implicated in platelet activation.

Blood Platelets↗

PubMed1996

CD36 is spatially associated with glycoprotein IIb-IIIa (alpha IIb beta 3) on the surface of resting platelets.

Platelet activation and aggregation induced by agonists such as thrombin are accompanied by the phosphorylation of several proteins on tyrosine. Such tyrosine phosphorylation is dependent upon activation and ligand engagement of the major integrin receptor on the surface of platelets, glycoprotein (GP) IIb-IIIa (alpha IIb beta 3), but how this is accomplished is not known. The only platelet membrane GP known to associated with non receptor tyrosine kinases is CD36 (GPIV) which forms associations with pp60Fyn, pp62Yes, and pp54/58Lyn, and antibodies directed against CD36 activate platelets in a process dependent upon GPIIb-IIIa. These and other data suggest a physical association between the two membrane GPs, IIb-IIIa and CD36. By the use of immunoprecipitation of lysates of platelets that have been surface labeled and chemically crosslinked we show here that CD36 and GPIIb-IIIa are spatially associated on the surface of resting platelets.

Antibodies, Monoclonal↗

PubMed1995

Capture by chemical crosslinkers provides evidence that integrin alpha IIb beta 3 forms a complex with protein tyrosine kinases in intact platelets.

Platelet activation is accompanied by a cascade of kinase reactions in which numerous specific proteins are phosphorylated on tyrosine. These events are strictly dependent upon functional activation of an integrin receptor, generally alpha IIb beta 3 (also known as glycoprotein IIb-IIIa). It is not known how alpha IIb beta 3 regulates protein tyrosine kinase activation and, in particular, neither this nor any other integrin has been shown to associate with a protein tyrosine kinase. We employed chemical crosslinking of intact platelets with the bifunctional reagents dithiobis(succinimidyl propionate) (DSP) (lipid-soluble) and dithiobis(sulphosuccinimidyl propionate) (DTSSP) (lipid-insoluble) followed by in vitro kinase assays of immunoprecipitated proteins to identify kinase activity associated with alpha IIb beta 3 in intact platelets. It was found that DSP but not DTSSP crosslinked kinase activity to alpha IIb beta 3, suggesting an internal association. In these immunoprecipitates from DSP-crosslinked platelets, the in vitro kinase reaction revealed a complex of several phosphoproteins in association with alpha IIb beta 3. This association was not seen when the resting platelets were lysed before crosslinking, indicating the specificity of the reaction in crosslinking only molecules in preformed spatial association. The beta 3 subunit of alpha IIb beta 3 was identified as one of the phosphoproteins in the complex obtained after subjecting anti-beta 3 immunoprecipitates from lysates of DSP-treated platelets to an in vitro kinase reaction and SDS/PAGE analysis. Phosphorylation of this subunit is shown to be predominantly on tyrosine. Affinity purification of the crosslinked phosphoprotein complex with anti-beta 3 followed by elution and re-precipitation identified pp60c-src and pp54/58c-lyn as two protein tyrosine kinases associating with the integrin. These results suggest that, upon platelet activation, alpha IIb beta 3 may provide a transmembrane focus for proteins involved in signal transduction.

Blood Platelets↗