Modulation of C3a Activity: Internalization of the Human C3a Receptor and its Inhibition by C5a1 | The Journal of Immunology

The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Gα16, a pertussis toxin-resistant G protein α subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.

The anaphylatoxins C3a,3 C5a, and C5a-desArg are generated during complement activation. Through binding to the C3aR and C5aR on neutrophils, monocytes, basophils, mast cells, and eosinophils, they function as potent proinflammatory mediators (1, 2, 3, 4, 5, 6, 7, 8, 9). Agonist binding stimulates a pertussis toxin-sensitive (PTX) increase in free cytosolic [Ca2+]i (10, 11, 12) and initiates a repertoire of host defense actions, from secretory granule release from neutrophils (12, 13) to chemotaxis in mast cells (6; for a review, see 14). Recent studies on both receptors suggests a much broader tissue distribution than previously surmised. C3aR is expressed during inflammation in the brain and on activated B lymphocytes (15, 16, 17, 18); in addition, C5aR is expressed on hepatocytes, lung, smooth muscle, and endothelial cells (19, 20, 21, 22, 23, 24). The complement system is strictly regulated by a variety of positive and negative feedback mechanisms to avoid self-destruction of the organism. Similarly, the signaling mediated by the anaphylatoxins must also be tightly regulated. Serum carboxypeptidase N, acting on the anaphylatoxins’ C-terminal arginine residue, rapidly inactivates newly generated C3a and greatly reduces the biologic activity of C5a (25). An additional receptor control mechanism is homologous desensitization. Details of the negative feedback mechanisms of C5aR have been described. The cytosolic C-terminus of the C5aR is phosphorylated within minutes of C5a addition (26, 27, 28), and the receptor is rapidly internalized (29, 30). Major and minor phosphorylation sites at the C5aR C-terminus, which seem to be important for internalization and receptor recycling, have been identified (29, 31, 32). Less is known about the regulation of the C3aR, but, as with C5aR, homologous desensitization has been noted at least in the guinea pig system (33).

Several groups have used the β2-adrenergic receptor as a model system to study G protein-coupled receptor internalization; C-terminal Ser and Thr residues are phosphorylated by G protein-coupled receptor kinases (for a review, see Refs. 34 and 35). Kinase activation is enhanced by their βγ subunit-dependent trans-location from the cytosol to the membrane (36, 37) and their phosphorylation by protein kinase C (38, 39). A member of the β-arrestin family binds to the phosphorylated receptor, thereby uncoupling it from its G protein, but improving its attachment to the clathrin-coated vesicle-mediated endocytic pathway (40, 41, 42). The receptor is then rapidly internalized, and the cells are desensitized. Receptor class desensitization (43) has also been described after stimulation of transfected RBL-2H3 cells by C5a or FMLP, which results in the cross-phosphorylation and desensitization of IL-8R A (44), whereas nonchemotactic receptors are not affected. These pathways are dependent on protein kinases C or A (45, 46, 47). Simultaneously, negative feedback mechanisms distal from the G protein exist, such as the phosphorylation of phospholipase Cβ3 (48). The internalized receptors are either degraded or dephosphorylated and recycled to the cell surface (29).

The aim of this study was the detailed characterization of human C3aR internalization as one negative feedback mechanism on granulocytes, as the largest leukocyte population naturally bearing the C3aR. A subset of experiments was performed on the human mast cell line HMC-1, human monocytes, and differentiated monocyte-like U937 cells to demonstrate that internalization is not a regulatory mechanism limited to granulocytes. Transiently transfected HEK293 cells were additionally investigated. In particular, the influence of C5a and FMLP on C3aR internalization was analyzed.

Read more here: Source link