Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex.

Abstract

We have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a Kd of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the a and I8 subunits of G., respectively. The 36-and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm our earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.branes (10-12). The results imply that the receptor has a molecular mass of 40-50 kDa. In contrast, gel filtration experiments with active, detergent-solubilized receptor indicate that the functional molecule is substantially larger, suggesting that the receptor itself is oligomeric or is associated with another protein (13). Crosslinking studies with the solubilized receptor revealed the existence of 90-kDa species, as well as the previously reported smaller form, providing additional evidence supporting the latter conclusion (13). These data, as well as the GTP sensitivity of the solubilized receptor, led to the hypothesis that the C5a receptor consisted of 40-to 50-kDa binding subunit that was coupled, in the absence of ligand, to a G protein (13,14).In the present study we report the purification of the C5a receptor from human PMN membranes. The isolation was achieved in a single step using a C5a affinity column in which the ligand was attached to Affi-Gel 10 through its N terminus. The isolated protein consists ofthree polypeptide chains with molecular masses of 42, 40, and 36 kDa, which are the receptor proper and the a and p subunits of the inhibitory G protein G,, respectively.C5a is a 74-amino acid glycopeptide generated by cleavage of complement component C5 upon activation of the complement system. The numerous responses elicited by the molecule imply that it is an important mediator of inflammatory processes (see refs. 1-3 for reviews). The peptide stimulates the contraction of smooth muscle, induces increases in vascular permeability, and promotes the synthesis and release of other mediators, including histamine, leukotrienes, and platelet activating factor. C5a is also a potent chemotaxin and secretagogue for phagocytic cells such as polymorphonuclear leukocytes (PMNs) and macrophag...

Two-site binding of C5a by its receptor: analternative binding paradigm for G protein-coupled receptors.

“…As reported (15), the purified receptor exhibits three bands on SDS/PAGE-a band at 42 kDa, which represents the binding subunit of the receptor, and bands at 41 and 36 kDa, which are the a and 13 subunits of the receptor-associated G proteins, respectively (Fig. 4A, lane 2).…”

“…To test effects on binding and degranulation, neutrophil membranes or intact neutrophils were preincubated with crude venom or purified protease for 20 min at 220C, the binding was initiated by the addition oflabeled ligand, and then the assays were carried out as described above. The ability to cleave the purified CSa receptor (15) was examined by incubating 50 ng of pure protease or buffer with receptor from 1.5 x 109 human neutrophils for 90 min at 40C in a total volume of 440 A. The reactions were stopped by adding 5x Laemmli sample buffer; the mixtures were boiled, concentrated, and subjected to SDS/PAGE.…”

Characterization of C3a and C5a Receptors in Rat Cerebellar Granule Neurons during Maturation

“…Cultures prepared by this method were enriched in granule neurons by more than 95%; the cell population was negative for glial fibrillary acidic protein (Sigma), OX 42 (ATCC, Manassas, VA), and calbindin D28K (Sigma) staining (data not shown). Cells were grown at 37°C in a humidified incubator with an atmosphere of 5% CO 2 .…”

“…C3a and C5a exert their biological effects through specific binding to membrane receptors, named, respectively, C3aR 1 and C5aR (CD88). These two receptors belong to the seven-transmembrane receptor superfamily and are coupled to a G i protein (2,3). C3aR and C5aR are expressed in myeloid cells (4 -8) and, more surprisingly, in non-myeloid cells and tissues (7)(8)(9)(10).…”

A comparison of post‐receptor signal transduction events in Jurkat cells transfected with either IL‐8R1 or IL‐8R2 Chemokine mediated activation of p42/p44 MAP‐kinase (ERK‐2)

“…The G~2~ protein has previously been shown to directly couple to the C5a and f-MLP receptors [24,28]. Co-transfection of COS-7 cells with IL-8 receptors and an accompanying PTx-resistant G~-protein revealed however that IL-8R1 and IL-8R2 may also couple to the Gq subclass of G-proteins, G~4 and G~l 6 [29].…”