RESUMO
The receptor for the inflammatory peptide C3a has scarcely been examined on human cells. This work demonstrates that human tumor-derived basophilic granulocytes express C3a receptors, and presents parts of the hitherto unknown C3a-signal transduction. When incubated with IL-3, these cells specifically liberated histamine on C3a stimulation. Independent from IL-3, 240,000 +/- 100,000 receptors per cell with a Kd of 5.6 +/- 0.9 nM were determined. [Ca2+]i increased from 120 +/- 35 nM to 300 +/- 80 nM after a C3a challenge, as measured by digital imaging fluorescence microscopy, and rested at its basal level in the presence of C3a-desArg, the immediate catabolic product of C3a in vivo. This [Ca2+]i increase could be completely desensitized homologously by C3a as well as inhibited by up to 75% by pertussis toxin. Thus, tumor-derived basophils are suitable for cloning of the human C3a receptor.
Assuntos
Anafilatoxinas/metabolismo , Basófilos/metabolismo , Complemento C3a/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Receptores de Complemento/metabolismo , Ligação Competitiva , Cálcio/metabolismo , Complemento C3a/análogos & derivados , Complemento C5a/metabolismo , Liberação de Histamina , Humanos , Interleucina-3/farmacologia , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The anaphylatoxic peptide C3a is part of a basic immunological defense mechanism, the complement system. Research on the human C3a receptor and signal transduction is hampered by the lack of a suitable human cell or cell line. We screened tumor cell lines and human blood cells for a C3a-dependent increase in cytosolic Ca2+ ([Ca2+]i) and analyzed this reaction in a fura-2/AM fluorescence assay for cells in suspension. U937 cells, when differentiated with dibutyryl-cAMP (Bt2cAMP), and purified human neutrophils reacted in a dose-dependent fashion to C3a and a C3a analogue synthetic peptide. We found complete homologous desensitization of this response and no heterologous desensitization to human C5a. Pertussis toxin totally blocked the increase in [Ca2+]i, indicating the possible involvement of a G-protein. Single-cell analysis by digital imaging fluorescence microscopy indicated that neutrophilic granulocytes responded to C3a. In binding studies with Bt2cAMP-differentiated U937 cells and human granulocytes, the 125I-C3a binding was displaced by C3a, yielding one class of C3a binding sites with dissociation constants (Kd) in the low nanomolar range. We identified myo-inositol 1,4,5-trisphosphate (IP3) as the second messenger possibly causing the [Ca2+]i increase and the release of N-acetyl-beta-D-glucosaminidase as one secretory cell response. By functional and binding studies we demonstrated the expression of the C3a receptor on Bt2-cAMP-differentiated U937 cells and human neutrophils and characterized parts of the C3a signal pathway. Our data support a physiological concept in which C3a might be more important than presently thought.
Assuntos
Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Antígeno de Macrófago 1/metabolismo , Neutrófilos/metabolismo , Acetilglucosaminidase/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Células Cultivadas , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Radioisótopos do Iodo , Cinética , Microscopia de Fluorescência , Dados de Sequência Molecular , Toxina Pertussis , Transdução de Sinais , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
In 1980 the C-terminal pentapeptide LGLAR (C3a 73-77) was described (Caporale, L. H. et al. J. Biol. Chem. 1980, 255: 10758) as the minimal sequence inducing a C3a-specific activity. We have synthesized C3a-analogue peptides connected to non-peptidic acyl residues known to potentiate biological activity. Starting from the acylated hexapeptide fluorenylmethoxycarbonyl(Fmoc)-aminohexanoyl(Ahx)-ALGLAR+ ++, a related series of shorter peptides was synthesized. C3a-specific activity was measured as ATP release from guinea pig platelets. Even the tripeptide LAR, acylated with Fmoc-Ahx, exhibited C3a-specific activity. With 0.34% C3a activity, it was even more potent than the native LGLAR sequence which has 0.01% activity. N-terminal extension of the acylated tripeptide LAR by adding one to three alanines increased activity tenfold up to 3.26% (Fmoc-Ahx-AAALAR), while N-terminal addition of three glycine residues (Fmoc-Ahx-GGGLAR) only increased activity to 0.83% of native C3a. Furthermore, a stimulus-specific desensitization could be observed. Fmoc-Ahx-R and Fmoc-Ahx-AR exhibited neither activity nor desensitizing capacity, but the addition of four alanines to the dipeptide AR led to a sequence (Fmoc-Ahx-AAAAAR) with a C3a-specific activity of 0.14%. Even arginine prolonged N-terminally with five glycines (Fmoc-Ahx-GGGGGR) exhibited some C3a-specific activity so that for biological activity only the appropriate presentation of arginine seems to be essential.