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Cloning and functional expression of the canine anaphylatoxin C5a receptor. Evidence for high interspecies variability.

ABSTRACT: A cDNA clone, DTJP03, encoding an orphan receptor, was isolated from a canine thyroid library, and found to exhibit 68.6% amino-acid identity with the recently described human C5a receptor. This relatively low similarity first suggested that DTJP03 encoded either a C5a receptor subtype, or the presumably related C3a receptor. Binding studies performed on membranes from COS-7 cells expressing the recombinant receptor demonstrated that DTJP03 encoded a high-affinity C5a receptor, with a Kd of 1.2 nM. C3a was unable to compete for C5a binding. Intracellular free calcium concentrations were measured by Quin-2 fluorescence assays in Chinese hamster ovary cells stably transfected with the canine C5a receptor. C5a addition elicited an increase in the intracellular calcium concentration. Extracellular EGTA partially prevented this response, suggesting that activation of the C5a receptor promotes both the release of calcium from intracellular stores, and the influx of extracellular calcium. Genes encoding C5a-receptor subtypes were subsequently searched for by PCR in genomic DNA from human, canine, rat and bovine sources. The result was the amplification of a single gene fragment from each species, with about 70% identity between any two of them. The canine C5a receptor has therefore to be considered as orthologous to the human C5a receptor described previously. The low similarity between C5a receptors from different mammalian species is quite unusual for a G-protein-coupled receptor.

SUBMITTER: Perret JJ

PROVIDER: S-EPMC1131973 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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Project description:Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G+ polymorphonuclear neutrophils, depletion of CD11c+SiglecF+ alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1-/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury.

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Cloning and functional expression of the canine anaphylatoxin C5a receptor. Evidence for high interspecies variability.

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