SEARCH · PubMed Health

Results for “Receptor, Anaphylatoxin C5a”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1998

Cloning and characterization of the guinea pig C5a anaphylatoxin receptor: interspecies diversity among the C5a receptors.

The anaphylatoxin C5a receptor (C5aR, CD88 in man) plays a prominent role in mediating inflammatory and host defense processes. Direct evidence of C5aR involvement in host defense mechanisms was demonstrated recently using C5aR knockout mice. Mice deficient in C5aR were unable to clear intrapulmonary-instilled bacteria. The guinea pig system is perhaps unique for exhibiting cross-reactivity with human complement components and its high sensitivity to anaphylatoxins. Therefore, we cloned the guinea pig C5aR from a megakaryocyte cDNA library. The deduced amino acid sequence of guinea pig C5aR is 67% identical to human, 61.6% to dog, 60.2% to mouse and 63.6% to rat C5aR. Transient expression of guinea pig C5aR in COS-7 cells and stable expression on L cell fibroblasts were confirmed by FACS analysis. Competitive binding studies using [125I]C5a and stimulation of calcium mobilization by C5a proved that functional C5aR was expressed on these stably transfected L cells. The N-terminal extracellular region of guinea pig C5aR was five to seven residues shorter than the same region in C5aR from other species and sequence homology was limited to 11%. Other outer membrane loops were also poorly conserved (8-33%) when compared across five species. Transmembrane segments were highly conserved between these various species (46-86%). Guinea pig C5aR binds human C5a, therefore residues critical for C5a binding have been conserved between these species. Sequence comparison of C5aR from multiple species permits conserved elements of the ligand binding sites to be elucidated.

Amino Acid Sequence↗

PubMed1995

Mitogen-activated protein kinase activation requires two signal inputs from the human anaphylatoxin C5a receptor.

The anaphylatoxin C5a receptor activates the Ras/Raf/mitogen-activated protein (MAP) kinase pathway in human neutrophils. The signal pathways involved in Ras/Raf/MAP kinase activation in response to C5a and other chemoattractant receptors is poorly understood. Stimulation of the C5a receptor expressed in HEK293 cells results in modest MAP kinase activation, which is inhibited by pertussis toxin-catalyzed ADP-ribosylation of G(i). Coexpression of the C5a receptor and the G16 alpha subunit (alpha 16) results in the G16-mediated activation of phospholipase C beta and a robust MAP kinase activation. Pertussis toxin treatment of C5a receptor/alpha 16-cotransfected cells inhibits C5a stimulation of MAP kinase activity approximately 60% relative to the control response. Similarly, the protein kinase C inhibitor, GF109203X inhibits activation of MAP kinase activation in C5a receptor/alpha 16-cotransfected cells by 60%; the protein kinase C inhibitor does not affect the modest C5a receptor response in the absence of alpha 16 expression. These results demonstrate that two independent signals are required for the maximal activation of MAP kinase by G protein-coupled receptors.

Antigens, CD↗

PubMed1992

Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor.

The mouse C5a receptor gene was isolated using the human C5a receptor cDNA probe recently described (Gerard, N. P., and C. Gerard. 1991. Nature 349:614). By analogy with the human gene, the mouse homolog contains two exons with the 5' untranslated region and initiating methionine codon present in exon 1 and the remainder of the molecule in exon 2. Generation of an expressible cDNA for the mouse C5a receptor was accomplished using the polymerase chain reaction and a sense oligodeoxynucleotide primer which included an initiation codon just 5' to the sequence encoding the N-linked glycosylation site. When transfected into human 293 kidney epithelial cells the cloned cDNA directs expression of a binding site for human C5a anaphylatoxin with a binding constant of 2.5 +/- 0.3 nM; the human C5a receptor expressed under identical conditions has a Kd of 1.7 +/- 0.2 nM. Overall, the deduced amino acid sequences of the receptors are 65% identical given the analogous gene structures. Alignment of the sequences as seven transmembrane segment receptors reveals that the greatest structural diversity (approximately 70%) exists in the putative extracellular domains. In contrast, species differences among other members of this family of seven membrane-spanning receptors is generally only 10 to 20%, even for receptors whose ligands are relatively small and not expected to interact with sites on the extracellular surfaces. A high degree of structural identify is observed for the C5a receptors in the transmembrane segments and in all but one of the loops predicted to exist in the cytoplasm. Inasmuch as critical structures responsible for high affinity binding of the 74 amino acid polypeptide to both C5a receptors involve features conserved between species, these data provide the starting point for mutagenesis studies to determine the nature of the binding and activation sites for the chemotactic receptors. Additionally, these data provide a reagent for immunologic and molecular genetic studies on the role of C5a receptors in inflammatory models.

Amino Acid Sequence↗

PubMed1995

Identification of the major phosphorylation sites in human C5a anaphylatoxin receptor in vivo.

Interaction of human C5a anaphylatoxin with cell surface receptors mediates cell activation and receptor desensitization. Treatment of differentiated HL60 cells or transiently transfected COS-7 cells with C5a or phorbol 12-myristate 12-acetate (PMA) results in rapid hyperphosphorylation of the C5aR. In an attempt to gain more insight into the function of phosphorylation in the desensitization of C5aR, we have initiated experiments to identify phosphoacceptor sites at the amino acid level after stimulation of cells with either C5a or PMA. In this report we show that C5aR is phosphorylated exclusively on serine residues in both differentiated HL60 and transfected COS-7 cells irrespective of the stimulus used. Peptide mapping after cyanogen bromide cleavage of phosphorylated C5aR indicates that despite the presence of a protein kinase C consensus motif the third cytoplasmic loop is not phosphorylated when cells are challenged with either C5a or PMA. Thus, whether the cells are stimulated with C5a or PMA, the phosphorylation sites appear to be restricted to serine residues in the carboxyl tail. Phosphoamino acid analysis of a series of mutants in which an individual serine residue was replaced by a threonine residue indicates that the C5aR undergoes C5a-dependent phosphorylation to the maximal stoichiometry of 6 mol of PO4/mol of receptor at Ser314, Ser317, Ser327, Ser332, Ser334, and Ser338. Simultaneous substitution of serine residues by alanine at positions 332, 334, and 338 affected neither the binding of C5a nor the cell surface expression of the mutant, but resulted in a dramatic reduction (more than 80%) of both C5a- and PMA-mediated phosphorylation as compared to the wild type receptor. This result suggests that phosphorylation on the segment extending from Ser332 to Ser338 is required for the subsequent phosphorylation of the carboxyl-terminal tail of C5aR.

Amino Acid Sequence↗

PubMed1999

Characterization of a polymorphism in the coding region of the human C5a anaphylatoxin receptor.

A polymorphism was identified in the coding region of the human C5a anaphylatoxin receptor gene leading to C to T transition at nucleotide position 450 (a silent substitution in the Ala150 codon, GCC to GCT). Its distribution was studied in a population of healthy volunteers from the Québec city region (prevalence of 2.8%) and among patients with end-stage renal failure who had previously undergone renal graft (prevalence 1.4%, not significantly different from that of the control group). This new marker provides a valuable tool to assess the risk for putative C5a-associated disorders with genetic determinism.

Alleles↗

PubMed1995

Expression of the complement C5a anaphylatoxin receptor (C5aR) on non-myeloid cells.

The expression of the complement C5a anaphylatoxin receptor (C5aR) has traditionally been thought to be limited to myeloid blood cells, including neutrophils, monocytes, macrophages, and eosinophils. Immunohistochemistry and ligand-binding studies reported here demonstrate, however, that C5aR is expressed by parenchymal cells of several solid organs, including human liver hepatocytes, lung bronchial and alveolar epithelial cells, and lung vascular smooth muscle and endothelial cells. In addition to C5aR expression in liver and lung, C5aR-specific message is found in other tissues, including the spleen, heart, kidney, and intestine. Collectively, these data indicate that the C5aR is expressed in several different types of cells in liver and lung and in yet undetermined cell types in spleen, heart, intestine, and kidney. Moreover, these data suggest that the C5aR mediates previously unrecognized functions by binding to tissue cells that express the C5aR.

Animals↗

PubMed2002

Phosphorylation of key serine residues is required for internalization of the complement 5a (C5a) anaphylatoxin receptor via a beta-arrestin, dynamin, and clathrin-dependent pathway.

The human complement 5a (C5a) anaphylatoxin receptor (CD88) is a G protein-coupled receptor involved in innate host defense and inflammation. Upon agonist binding, C5a receptor (C5aR) undergoes rapid phosphorylation on the six serine residues present in the C-terminal region followed by desensitization and internalization. Using confocal immunofluorescence microscopy and green fluorescent protein-tagged beta-arrestins (beta-arr 1- and beta-arr 2-EGFP) we show a persistent complex between C5aR and beta-arrestins to endosomal compartments. Serine residues in the C5aR C terminus were identified that control the intracellular trafficking of the C5aR-arrestin complex in response to C5a. Two phosphorylation mutants C5aR-A(314,317,327,332) and C5aR-A(314,317,332,334), which are phosphorylated only on Ser(334)/Ser(338) and Ser(327)/Ser(338), respectively, recruited beta-arr 1 and were internalized. In contrast, the phosphorylation-deficient receptors C5aR-A(334,338) and C5aR-A(332,334,338) were not internalized even though observations by confocal microscopy indicated that beta-arr 1-EGFP and/or beta-arr 2-EGFP could be recruited to the plasma membrane. Altogether the results indicate that C5aR activation is able to promote a loose association with beta-arrestins, but phosphorylation of either Ser(334)/Ser(338) or Ser(327)/Ser(338) is necessary and sufficient for the formation of a persistent complex. In addition, it was observed that C5aR endocytosis was inhibited by the expression of the dominant negative mutants of dynamin (K44E) and beta-arrestin 1 (beta-arr 1-(319-418)-EGFP). Thus, the results suggest that the C5aR is internalized via a pathway dependent on beta-arrestin, clathrin, and dynamin.

Amino Acid Sequence↗

PubMed1997

Internalization and recycling of the C5a anaphylatoxin receptor: evidence that the agonist-mediated internalization is modulated by phosphorylation of the C-terminal domain.

The C5a anaphylatoxin receptor is a member of the G protein-coupled receptor family involved in chemoattraction and activation of myeloid cells, as well as in host defence against infection by Pseudomonas aeruginosa. Upon challenge by C5a, the C5a receptor undergoes a rapid phosphorylation on serine residues in the carboxyl-terminal region. In this study, we used cells stably transfected with either the wild-type C5a receptor, or mutants affected in their capacity to be phosphorylated, to examine the role played by phosphorylation in the intracellular trafficking of the C5a receptor. Upon agonist binding, the wild-type receptor was rapidly internalized into endosomes that cluster near the nucleus after 10 minutes. Internalization of a non-phosphorylable mutant was severely impaired relative to wild-type receptor, whereas a mutant phos-phorylated on serine 327 and/or serine 338, showed a rate of internalization intermediate between that of wild-type receptor and that of the non-phosphorylable mutant. Under continuous exposure to C5a and in the absence of protein synthesis, the C5a receptor was maintained in a highly phosphorylated state but was not degraded. Confocal microscopy and ligand-binding studies indicated that internalized receptors were recycled to the plasma membrane. During this process, receptors were dephosphorylated with kinetics that correlated with the kinetics of receptor recovery on the cell surface. Altogether, our data suggest that phosphorylation plays a key role in the intracellular trafficking of the C5a receptor. Phosphoryl-ated receptors might be recognized by an adaptor protein that interacts with the endocytic machinery.

Amino Acid Sequence↗

PubMed2000

Human complement 5a (C5a) anaphylatoxin receptor (CD88) phosphorylation sites and their specific role in receptor phosphorylation and attenuation of G protein-mediated responses. Desensitization of C5a receptor controls superoxide production but not receptor sequestration in HL-60 cells.

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166-19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser(334) was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser(332) and Ser(334) or Ser(334) and Ser(338), was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser(334) and Ser(338) is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8-2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms.

Animals↗

PubMed1995

Identification and characterization of the complement C5a anaphylatoxin receptor on human astrocytes.

The C fragment C5a exerts its important physiologic and pathologic effects through interaction with a specific C5a receptor (C5aR) which is highly expressed on polymorphonuclear leukocytes and some other leukocytes. The presence of this receptor on epithelia and endothelia has recently been documented, raising the possibility that these other cells might also respond to locally generated C5a. C has been implicated in several brain disorders, notably demyelination and neurodegeneration, and cells within brain can synthesize a complete C system. It is thus of interest to examine the mechanisms by which C damages or activates brain cells. To this end we have examined the expression on human fetal astrocytes and astrocyte-derived cell lines of receptors for C fragments. We here report that human astrocytes and cell lines express a receptor for C5a (48 to 72 x 10(3) copies/cell), which is indistinguishable at the protein or mRNA level from that in leukocytes. The astrocyte C5aR was recognized by five different specific Abs, which revealed by Western blotting a protein of 40 to 45 kDa in primary human astrocytes and astrocyte cell lines. Expression was confirmed by RT-PCR using multiple primers. Neither inflammatory cytokines nor PMA caused up-regulation of the receptor on astrocytes. The receptor was functional in that addition of C5a (1 nM to 100 nM) or, at high doses (100 nM), C5adesArg, triggered a calcium transient in astrocytes. We propose that C5aR expression on astrocytes plays an important role in control of inflammation in brain and may be a central component of C-mediated brain injury.

Amino Acid Sequence↗

PubMed1997

Characterization of rat C5a anaphylatoxin receptor (C5aR): cloning of rat C5aR cDNA and study of C5aR expression by rat astrocytes.

Complement system activation within the central nervous system (CNS) is involved in demyelinating and neurodegenerative disorders, but the role of complement in the pathogenic process or in the repair remains unclear. Besides the direct lytic effects of complement on target cells (oligodendrocytes or neurons), complement can exert other functions through interaction of complement fragments with specific receptors. The C5a anaphylatoxin, an inflammatory peptide which is formed during complement activation, might play a role in the CNS pathogenesis, and activation and recruitment of glial cells by binding to its receptor (C5aR) on CNS cells. Using degenerate primers corresponding to homologous regions between human and mouse C5aR cDNAs, we have cloned a rat C5aR cDNA probe from rat monocytes RNAs after RT-PCR experiment. The rat C5aR probe isolated by this procedure allowed us to clone the rat C5aR cDNA-coding sequence using a library screening cloning strategy. This probe was also used to study the expression of the C5aR mRNA in the rat CNS. Northern blotting and RT-PCR experiments demonstrated the constitutive expression of C5aR mRNA in brain, spleen, kidney and lung. This transcript was also observed in primary culture of rat astrocytes. Microfluorimetry experiments demonstrated that C5aR expressed by astrocytes in culture is functional since the addition of C5a induced a dose-dependent increase of intracellular calcium concentration. The expression of the C5aR by astrocytes suggests new roles for the C5a anaphylatoxin in reactive astrogliosis to CNS injuries.

Amino Acid Sequence↗

PubMed1997

cDNA cloning and characterization of rat C5a anaphylatoxin receptor.

Activation of the complement cascade plays an essential role in the early stages of inflammation. C5a and its receptor are particularly active in anaphylaxis. To determine the pathological roles played by C5a and C5a receptor (C5aR) in rats, we cloned C5aR cDNA and analyzed distribution of its mRNA in various organs including lung from an LPS-stimulated rat. Furthermore, we generated a polyclonal antiserum which specifically recognizes rat C5aR, as confirmed by its specific interaction with cells transfected with rat C5aR cDNA.

Animals↗

PubMed1995

Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung.

The small-complement C5 activation fragment, C5a, is a potent phlogistic molecule that, on binding to the C5a Receptor (C5aR), mediates contraction of smooth muscle, enhances vascular permeability, and promotes leukocyte functions such as directed chemotaxis, degranulation, mediator release, and production of superoxide anions. Although C5aR expression has traditionally been thought to be limited primarily to myeloid blood cells, including neutrophils, monocytes, macrophages, and eosinophils, we report here that C5aR is expressed by liver and lung cells as well as by cells in several other tissues. By Northern blot analysis, it was determined that mouse liver, baboon liver, human liver, and the human hepatoma-derived cell line HepG2 express a normal size (2.3 kb) C5aR mRNA; in HepG2 cells, the quantity of C5aR mRNA was comparable to that contained in dbcAMP-differentiated U937 cells. HepG2 cells were demonstrated to express the C5aR on their cell surface by flow cytometric and immunofluorescence analyses as well as by 125I-C5a binding assays. The binding data indicated that HepG2 cells express a single class of C5aR with a Kd of 1.18 nM and approximately 28,000 receptors per cell. In vivo expression of C5aR in human liver cells was demonstrated by in situ hybridization and immunohistochemistry analyses. Northern blot analysis of murine and baboon organs shows that, in addition to the liver, other tissues express C5aR mRNA in significant quantities, including the spleen, lung, heart, kidney, and intestine. Moreover, mice treated with LPS show a large increase in C5aR mRNA in all these tissues except the intestine. Immunostaining of human lung tissue demonstrated that bronchial and alveolar epithelial cells, as well as vascular smooth muscle and endothelial cells, also express the C5aR. Collectively, these data indicate that the C5aR is expressed in several different types of cells in liver and lung, and in yet undetermined cell types in spleen, heart, intestine, and kidney. Furthermore, these data suggest that the C5a anaphylatoxin mediates previously unrecognized functions by binding to tissue cells that express the C5aR.

Amino Acid Sequence↗

PubMed1988

Expression of C5a anaphylatoxin receptor in monoblastic cells involves facilitation of an adenosine 3',5'-monophosphate-dependent process.

We have previously reported a synergistic effect of 1,25-dihydroxyvitamin D [1,25-(OH)2D] and agents that elevate intracellular cAMP to induce the expression of the C5a complement receptor in U937 cells. In this report we examine the mechanism of this synergy, considering the hypothesis that the steroid hormone works by facilitating what is ultimately a cAMP-dependent process. We show that U937 cells cultured with 1,25-(OH)2D alone before culture with prostaglandin (PGE2) alone will express C5a receptor (an average of 55 +/- 4% of the receptors expressed with continuous exposure of cells to both agents; P less than 0.05). The reverse, PGE2 followed by 1,25-(OH)2D, causes very little receptor induction. This demonstrates the ability of 1,25-(OH)2D to induce changes in the state of the cell, such that activation of cAMP-dependent protein kinase has effects that are otherwise not seen, in other words 1,25-(OH)2D can prime the cell for the subsequent action of the cAMP messenger system. Furthermore, we are able to substitute, during the priming period, the protein synthesis inhibitor cycloheximide (CHX) for 1,25-(OH)2D. Cells cultured for 24 h with CHX will express C5a receptor when cultured for a second 2-day period with PGE2 at about 77 +/- 7% of the amount obtained with simultaneous exposure to 1,25-(OH)2D and PGE2. The CHX effect is time dependent and visible after 2 h. CHX is not synergistic with 1,25-(OH)2D. Other agents that can also substitute for 1,25-(OH)2D, but not for cAMP, in facilitating C5a receptor expression include retinoic acid and ionomycin, but with less potency. The 1,25-(OH)2D and PGE2 synergy is sensitive to the presence of isobutylmethylxanthine, implicating its dependence on the maintained elevation of intracellular cAMP levels. The synergy does not appear to be sensitive to changes in extracellular or intracellular calcium. We conclude from these results that 1,25-(OH)2D may promote the expression of C5a receptor in these cells in a fashion similar to that by which CHX potentiates other genes, i.e. that 1,25-(OH)2D increases levels of the mRNA encoding the C5a receptor. The mechanism of cAMP's subsequent and necessary action in the induction of C5a receptor expression is not yet clear.

1-Methyl-3-isobutylxanthine↗

PubMed2002

Distribution of rat C5a anaphylatoxin receptor.

The anaphylatoxin, complement 5a (C5a), plays a key role in mediating various inflammatory reactions following complement activation. Several investigators have reported that C5a receptor (C5aR) is expressed in non-myeloid cells under certain conditions or in different cell lines. In our study, the abundance of C5aR-positive myeloid cells in rats depended on the organs examined. C5aR was usually expressed at the site of exposure to pathogens, such as in salivary gland or lung, and was up-regulated in liver in the inflammatory state induced by lipopolysaccharide (LPS) administration. Furthermore, the increased expression of C5aR antigen was not accompanied by an increase in C5aR mRNA in Kupffer cells following LPS challenge.

Animals↗

PubMed2006

Mycobacteria-primed macrophages and dendritic cells induce an up-regulation of complement C5a anaphylatoxin receptor (CD88) in CD3+ murine T cells.

Complement C5a anaphylatoxin is a potent activator of macrophages, neutrophils, and dendritic cells (DC) and binds the C5a receptor (C5a-R; CD88). Although C5a is chemotactic for T cells, expression of C5a-R on murine T cells has been disputed. We report here that naïve, Con A-activated, and cytokine (IL-12, IL-18)-stimulated murine CD3+ T cells from three strains of mice [C57Bl/6, B10.nSn (C5+/+), B10.on (C5-/-)] lacked C5a-R, as evaluated by immunophenotyping with an anti-C5a-R mAb. Ligation of CD3 induced a modest up-regulation with 3% of CD3+ T cells expressing cell surface C5a-R. T cells primed by APC differentiate into effector T cells. Activation of mycobacteria [bacillus Calmette-Guerin (BCG)]-sensitized T cells through MHC II and TCR interactions via BCG-infected macrophages enhanced the expression of C5a-R with approximately 14% of CD3+ T cells positive for C5a-R. Comparable expression was found in C5+/+ as well as C5-/- strains of mice (14% and 15%, respectively). Furthermore, anti-CD3-activated T cells were primed by BCG-infected DC, and a larger proportion of the primed T cells expressed C5a-R (30-40%). Finally, mice infected with BCG showed significant numbers of CD3+ T cells expressing C5a-R in the spleens during infection. As APC, such as macrophages and DC, can secrete C5 and cleave C5 to C5a and C5b through a peptidase, we suggest that macrophage and DC-T cell interactions can up-regulate C5a-R on T cells through MHC II-TCR and provide a C5a peptide for additional local activation of T cells via C5a-R.

Animals↗

PubMed2003

Expression and induction of anaphylatoxin C5a receptors in the rat liver.

The C5a-anaphylatoxin which is generated by limited proteolysis upon activation of the fifth component of complement may be induced by the classical, the alternative or the lectin pathway. C5a has been shown, under normal conditions, to induce the release of prostanoids from Kupffer cells (KC) and hepatic stellate cells (HSC) and thereby indirectly to increase glucose output from hepatocytes (HC). A direct action of C5a on HC would require the expression of the specific C5a receptor (C5aR). In studies using quantitative RT-PCR it was shown that non-stimulated HC lack C5aR, in contrast to KC, HSC and sinusoidal endothelial cells (SEC) all of which contained mRNA for the C5aR in decreasing amounts. FACS analyses, immunohisto- and immunocytochemistry as well as functional analyses confirmed the results of the RT-PCR assays. Under inflammatory situations the C5aR was found to be upregulated in various organs and tissues which included the liver. Interleukin-6 (IL-6) as a main inflammatory mediator in the liver induced a de novo expression of functional C5aR in HC in-vitro and in-vivo. In contrast, LPS failed to induce C5aR directly in cultured HC in-vitro but induced C5aR in HC in vivo and in co-cultures of HC and KC which release IL-6 upon stimulation with LPS. So far, the only known effector function of C5a on HSC was the induction of prostanoid release. In an approach to reveal new functions of C5aR in HSC, the cells responsible for liver fibrosis, it could be shown that C5a upregulated fibronectin-specific mRNA five-fold whereas entactin, collagen IV and the structure protein smooth muscle actin were not affected. In addition, C5a did not upregulate specific mRNA for the profibrotic cytokine TGF-beta1 in either isolated KC or HSC. Thus, C5a alone appears to have only a limited role in the induction of liver fibrosis.

Animals↗

PubMed2003

Direct binding of a fragment of the Wiskott-Aldrich syndrome protein to the C-terminal end of the anaphylatoxin C5a receptor.

Migration of myeloid cells towards a source of chemoattractant, such as the C5a anaphylatoxin, is triggered by the activation of a G-protein-coupled receptor. In the present study, we have used a yeast two-hybrid approach to find unknown partners of the C5a receptor (C5aR). Using the cytosolic C-terminal region of C5aR as bait to screen a human leucocyte cDNA library, we identified the Wiskott-Aldrich syndrome protein (WASP) as a potential partner of C5aR. WASP is known to have an essential function in regulating actin dynamics at the cell leading edge. The interaction was detected with both the fragment of WASP containing amino acids 1-321 (WASP.321) and WASP with its actin-nucleation-promoting domain [verprolin-like, central and acidic (VCA) domain] deleted. The interaction between C5aR and the WASP.321 was supported further by an in vitro binding assay between a radiolabelled WASP.321 fragment and a receptor C-terminus glutathione S-transferase (GST) fusion protein, as well as by GST pull-down, co-immunoprecipitation and immunofluorescence experiments. In the yeast two-hybrid assay, full-length WASP showed no ability to interact with the C-terminal domain of C5aR. This is most probably due to an auto-inhibited conformation imposed by the VCA domain. In HEK-293T cells co-transfected with full-length WASP and C5aR, only a small amount of WASP was co-precipitated with the receptor. However, in the presence of the active form of the GTPase Cdc42 (Cdc42V12), which is thought to switch WASP to an active 'open conformation', the amount of WASP associated with the receptor was markedly increased. We hypothesize that a transient interaction between C5aR and WASP occurs following the stimulation of C5aR and Cdc42 activation. This might be one mechanism by which WASP is targeted to the plasma membrane and by which actin assembly is spatially controlled in cells moving in a gradient of C5a.

Actins↗