Review: Cromer and DAF: role in health and disease.

The antigens of the Cromer blood group system are located on the protein decay-accelerating factor (DAF). This system consists of ten high-prevalence and three low-prevalence antigens; the molecular basis for all of these antigens is a single nucleotide polymorphism in the DAF gene. DAF is a 70,000-Da plasma membrane protein that is widely distributed on all blood cells and on endothelial and epithelial tissues. The physiological role of DAF is to inhibit the complement cascade at the level of the critical C3 convertase step. By this mechanism,DAF acts to protect autologous cells and tissues from complement-mediated damage and hence can play a role in preventing or modulating autoimmune disease and inflammation. The use of recombinant DAF as a therapeutic agent in autoimmunity and inflammation, and of DAF transgenic animals in xenotransplantation, is being actively investigated. Additionally, DAF serves as a receptor for certain strains of Escherichia coli and certain types of enteroviruses. The DAF protein that contains the Cromer antigens serves important roles in health and disease.

SERF: a new antigen in the Cromer blood group system.

The Cromer blood group system consists of eight high incidence and three low incidence antigens carried on decay-accelerating factor (DAF). This report describes the identification and characterization of a new Cromer high incidence antigen, named SERF. Sequence analyses of DNA from a Thai female whose serum contained the alloantibody to a high incidence antigen in the Cromer blood group system (anti-SERF) and from her two children were performed. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis on cDNA from the proband and PCR-restriction fragment length polymorphism analysis on DNA from Thais were also performed. To map the epitope, DAF deletion mutants were tested by immunoblotting with anti-SERF. Sequence analysis revealed a substitution of 647C>T in exon 5 DAF in the proband. The proband's two children and two of 100 Thais were heterozygotes 647C/T. Analysis using DAF deletion mutants revealed the antigenic determinant to be within short consensus repeat 3 (SCR3), which is encoded by exon 5. This study describes a novel high incidence antigen (SERF) in the Cromer blood group system characterized by the amino acid proline at position 182 in SCR3 of DAF. The SERF-negative proband has a substitution mutation that predicts for leucine at this position. SERF has been provisionally assigned the International Society of Blood Transfusion number 021.012 (CROM 12).

Palmitoylation of caveolin-1 at a single site (Cys-156) controls its coupling to the c-Src tyrosine kinase: targeting of dually acylated molecules (GPI-linked, transmembrane, or cytoplasmic) to caveolae effectively uncouples c-Src and caveolin-1 (TYR-14).

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.

Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets.

Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav-KKSL. Cav-DeltaN2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed caveolin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets. Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.

Induction of endothelial cell decay-accelerating factor by vascular endothelial growth factor: a mechanism for cytoprotection against complement-mediated injury during inflammatory angiogenesis.

OBJECTIVE: Decay-accelerating factor (DAF) is a widely expressed, multifunctional cell surface protein involved in complement regulation and cell signaling. Previous studies have demonstrated that endothelial cell (EC) DAF is up-regulated by tumor necrosis factor alpha and inhibits complement binding. Because vascular endothelial growth factor (VEGF) is cytoprotective to endothelium and is expressed at sites of chronic inflammation, we hypothesized that VEGF may induce DAF expression during inflammatory angiogenesis. METHODS: Human umbilical vein and dermal microvascular EC were isolated using routine procedures, and the regulation and function of DAF, as well as other complement-regulatory proteins (membrane cofactor protein and CD59), were analyzed following stimulation with VEGF. RESULTS: Incubation of large- or small-vessel EC with VEGF led to increased expression of DAF, with maximal expression after 48-72 hours of stimulation. This effect depended on the activation of protein kinase C (PKC) and required increased steady-state messenger RNA levels and de novo protein synthesis. Although VEGF-induced EC proliferation was inhibited by both p38 and p42/44 mitogen-activated protein kinase (MAPK) antagonists, DAF up-regulation in response to VEGF was only sensitive to inhibition of p38 MAPK. VEGF-stimulated EC showed a 60% reduction in C3 deposition following complement activation, and this resulted in a marked reduction in complement-mediated EC lysis. These protective effects were abolished by anti-DAF monoclonal antibody 1H4. CONCLUSION: This study confirms the importance of PKC for the regulation of DAF expression by EC and reveals VEGF to be a physiologic agonist for this pathway. The up-regulation of DAF expression by VEGF may represent an important mechanism for the protection of EC from complement-mediated injury during angiogenesis in inflammatory rheumatic diseases.

Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: identification of a c-Src/Cav-1/Grb7 signaling cassette.

Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.

Recruitment of CD55 and CD66e brush border-associated glycosylphosphatidylinositol-anchored proteins by members of the Afa/Dr diffusely adhering family of Escherichia coli that infect the human polarized intestinal Caco-2/TC7 cells.

The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

Molecular basis of Cromer blood group antigens.

BACKGROUND: The Cromer blood group system consists of 10 antigens located on decay-accelerating factor (DAF). Previous molecular genetic analysis has determined the basis for four of these antigens. The present study was undertaken to identify the mutations that determine the remaining antigens. STUDY DESIGN AND METHOD: Existing or new data were used to localize each Cromer system antigen to a specific short consensus repeat (SCR) domain of DAF. The exon encoding that SCR domain was amplified by using the polymerase chain reaction (PCR) on genomic DNA obtained from individuals of that Cromer phenotype, and the DNA product was subjected to DNA sequence analysis. RESULTS: The Tc(a)/Tc(c) polymorphism is due to an R18P amino acid substitution in SCR1 of DAF. The Es(a+)/Es(a-) polymorphism is due to an I46N mutation in SCR1 of DAF. The WES(b)/WES(a) polymorphism is due to an L48R mutation in SCR1 of DAF. The UMC+/UMC- polymorphism is due to a T216M substitution in SCR4 of DAF. CONCLUSIONS: With information from previous reports and the findings of this study, the molecular genetic basis of all known alleles of the Cromer blood group system has been elucidated. Single amino acid substitutions are responsible for 9 of the 10 antigens (all except the multiple-epitope antigen IFC).

Role of decay-accelerating factor domains and anchorage in internalization of Dr-fimbriated Escherichia coli.

Dr-fimbriated Escherichia coli capable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor. The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr(+) E. coli was characterized in a cell-cell interaction model. Binding of Dr(+) E. coli to the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur. Deletion of short consensus repeat 3 domain or replacement of Ser(165) by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization. Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively. Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr(+) E. coli through a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-beta-cyclodextrin, a sterol-chelating agent. Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr(+) E. coli were morphologically distinct between the anchor variants of DAF. The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes. This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr(+) E. coli and demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.

The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo.

Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.

Expression and functional analysis of glycosyl-phosphatidyl inositol-linked CD46 in transgenic mice.

BACKGROUND: Complement activation plays a pivotal role in hyperacute xenograft rejection. In humans, activation of complement is regulated by a number of cell surface regulatory proteins. Membrane cofactor protein (CD46) is one such regulator that protects cells by acting as a cofactor for the factor I-mediated cleavage of C3b and C4b. Transgenic animals expressing human CD46 may provide organs that are resistant to complement attack. However, attempts to generate mice expressing human CD46 using cDNA-based constructs have been largely unsuccessful. METHODS: Transgenic mice expressing a glycosylphosphatidyl inositol (GPI)-linked form of CD46 were generated by microinjection of a hybrid CD46/CD55 cDNA under the control of the human intercellular adhesion molecule-2 promoter. Expression of CD46-GPI on the vascular endothelium was determined by immunohistochemistry. The ability of CD46-GPI to protect mouse tissues from human complement attack was determined using an ex vivo isolated perfused heart model. RESULTS: Three founder animals expressing CD46-GPI were identified. Histological analysis showed strong and uniform expression of CD46-GPI on the vascular endothelium of all organs examined. Ex vivo perfusion of transgenic mouse hearts with human plasma showed a reduction in C3c deposition and a slightly prolonged function compared with controls. CONCLUSIONS: High-level expression of CD46-GPI was achieved in transgenic mice by using a modified cDNA-based construct. The CD46-GPI was functional, providing some protection from complement-mediated damage in the ex vivo model, and may be useful in xenotransplantation if expressed in combination with CD55 and CD59.

Short consensus repeat domain 1 of decay-accelerating factor is required for enterovirus 70 binding.

Enterovirus 70 (EV70), like several other human enteroviruses, can utilize decay-accelerating factor (DAF [CD55]) as an attachment protein. Using chimeric molecules composed of different combinations of the short consensus repeat domains (SCRs) of DAF and membrane cofactor protein (CD46), we show that sequences in SCR1 of DAF are essential for EV70 binding. Of the human enteroviruses that can bind to DAF, only EV70 and coxsackievirus A21 require sequences in SCR1 for this interaction.

Survival of FimH-expressing enterobacteria in macrophages relies on glycolipid traffic.

Strains of Escherichia coli persist within the human gut as normal commensals, but are frequent pathogens and can cause recurrent infection. Here we show that, in contrast to E. coli subjected to opsonic interactions stimulated by the host's immune response, E. coli that bind to the macrophage surface exclusively through the bacterial lectin FimH can survive inside the cell following phagocytosis. This viability is largely due to the attenuation of intracellular free-radical release and of phagosome acidification during FimH-mediated internalization, both of which are triggered by antibody-mediated internalization. This different processing of non-opsonized bacteria is supported by morphological evidence of tight-fitting phagosomes compared with looser, antibody-mediated phagosomes. We propose that non-opsonized FimH-expressing E. coli co-opt internalization of lipid-rich microdomains following binding to the FimH receptor, the glycosylphosphatidylinositol-linked protein CD48, because (1) the sterol-binding agents filipin, nystatin and methyl beta-cyclodextrin specifically block FimH-mediated internalization; (2) CD48 and the protein caveolin both accumulate on macrophage membranes surrounding bacteria; and (3) antibodies against CD48 inhibit FimH-mediated internalization. Our findings bring the traditionally extracellular E. coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies.

Membrane cofactor protein (CD46) is a basolateral protein that is not endocytosed. Importance of the tetrapeptide FTSL at the carboxyl terminus.

Membrane cofactor protein (MCP) is a widely distributed complement regulatory protein that is expressed on the basolateral surface of polarized epithelial cells. The basolateral targeting of the BC1 isoform of MCP was analyzed by generating deletion mutants and point mutants within the cytoplasmic tail of 16 amino acids. A sequence of four amino acids, FTSL, was found to be indispensable for the basolateral transport of MCP. This tetrapeptide has two unique features compared with the targeting motifs of other basolateral proteins: (i) it contains a phenylalanine rather than a tyrosine at position 1; (ii) it is located at the very COOH-terminal end. Replacement of the phenylalanine or the leucine by an alanine resulted in a nonpolarized delivery to the cell surface. On the other hand, substitution of a tyrosine for the phenylalanine did not affect the basolateral transport of MCP. The latter mutant, however, was efficiently internalized, whereas the wild type protein was not subject to endocytosis. Our results indicate that the targeting signal YXX-large aliphatic that is involved in various sorting events has been modulated in MCP in such a way that it allows basolateral transport but not endocytosis.

Identification of a heparin binding domain in the seventh short consensus repeat of complement factor H.

Surface polyanions such as sialic acid and heparin are thought to enhance the binding of complement factor H (fH) to C3b deposited on particles and cell surfaces, thereby reducing complement activation. fH contains 20 short consensus repeat (SCR) domains, and it has been proposed that SCR 13 contains a heparin binding site. We used recombinant proteins to determine the heparin binding site on fH. Full-length fH (H20) and truncated and SCR deletion mutant proteins were cloned and expressed in Chinese hamster ovary cells. Supernatants were applied to heparin-agarose affinity columns to determine their binding and elution profiles. Deletion of SCR 13 from H20 did not prevent heparin binding nor alter its salt elution profile, indicating that SCR 13 does not contain an essential heparin binding site. We found that SCR 7 contains a heparin binding site, as SCRs 1 through 7 were the smallest truncated proteins to bind heparin (89 +/- 3%). Furthermore, deletion of SCR 7 from a protein containing SCRs 1 through 9 reduced heparin binding, whereas deletion of SCR 6 did not (17 +/- 13 vs 81 +/- 13%; p = 0.02). It is likely that other heparin binding sites exist within SCRs 10 through 20; an SCR 7 deletion mutant of H20 eluted earlier than H20, but still showed >99% binding to immobilized heparin. SCR 13 does not contain such a site because a double deletion of SCRs 7 and 13 from H20 showed >97% heparin binding and had an elution profile smilar to that of a single deletion of SCR 7.

CD36 is palmitoylated on both N- and C-terminal cytoplasmic tails.

The membrane protein CD36 has been reported to carry out a wide range of potential functions, including serving as a receptor for thrombospondin, collagen, oxidized low density lipoprotein, fatty acids, anionic phospholipids, and Plasmodium falciparum malaria parasitized erythrocytes. This implicates CD36 in cellular adhesion, human atherosclerotic lesion formation, lipid metabolism, and malaria. A presumed rat homolog of CD36 was previously reported to be palmitoylated. We confirmed that human CD36 is palmitoylated and identified cysteines 3, 7, 464, and 466 as the palmitoylation sites using a mutagenesis approach. This result suggests that both the N- and C-terminal tails of CD36 are cytoplasmic. Published models for the topology of CD36 have the C terminus located in the cytoplasm but differ as to whether the N terminus is cytoplasmic or extracellular. To address this question, a C-terminal truncation mutant of CD36 was made by introducing a stop codon just upstream of the C-terminal transmembrane domain. This mutant was found membrane-bound when expressed in human embryonic kidney 293 cells, indicating that the N-terminal hydrophobic domain serves as a transmembrane anchor, and thus supporting a CD36 topology with two transmembrane domains.

The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55).

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.

Activation of the gene encoding decay accelerating factor following nerve growth factor treatment of sensory neurons is mediated by promoter sequences within 206 bases of the transcriptional start site.

Using two independent differential screening procedures designed to identify novel mRNAs induced by nerve growth factor (NGF) treatment of adult dorsal root ganglion (DRG) neurons, we have isolated cDNA clones derived from the gene encoding decay accelerating factor (DAF). Hybridization analysis and semi-quantitative polymerase chain reaction confirmed that the DAF mRNA was indeed induced in NGF-treated adult DRG neurons. Moreover, the DAF gene promoter is NGF inducible (approximately two- to threefold) when transfected into DRG neurons, and this effect is primarily dependent on sequences between -206 and -77 relative to the transcriptional start site. Hence, the DAF gene constitutes a novel NGF-inducible gene whose mRNA is elevated in response to NGF treatment of DRG neurons. The potential significance of this effect is discussed in terms of the role of NGF in modulating the transcriptional activity and function of adult DRG neurons.