The guanine nucleotide-binding proteincoupled receptor superfamily binds a vast array of biological messengers including lipids, odorants, catecholamines, peptides, and proteins. While some small molecules bind to these receptors at a single interhelical site, we find that the binding domain on the receptor for the inflammatory protein C5a is more complex and consists of two distinct subsites. This more elaborate motif appears to be an evolutionary adaptation of the simpler paradigm to which a second interaction site has been added in the receptor N terminus. Surprisingly, occupation of only one of the subsites is required for receptor activation. The two-site motif is not unique to the C5a receptor but appears to be widely used by the superfamily to accommodate macromolecular ligands.The 74-aa glycoprotein C5a evokes a variety of responses in vivo and in vitro, implying that it is a principal mediator of inflammatory responses (1, 2). C5a is a potent chemotaxin and secretagogue for granulocytes and macrophages; it activates the respiratory burst in these cells and modulates their adhesive properties. The effects of C5a are amplified by its ability to stimulate the release of other mediators including histamine, prostaglandins, leukotrienes, interleukin (IL) 1, and IL-6 (1-3).All of the effects of C5a are initiated when it binds to its cell surface receptor, a member ofthe guanine nucleotide-binding protein (G protein)-coupled receptor superfamily (4, 5). The superfamily consists of over 100 members and binds a variety of ligands ranging in complexity from small molecules to moderately sized proteins. Despite this biologic diversity, a general model for the structure of these receptors has emerged: an extracellular N terminus, seven membranespanning helices connected by alternating intracellular and extracellular loops, and an intracellular C terminus (6, 7). The amino acid sequence of the C5a receptor is consistent with this model and like most members of the family has a short N terminus of about 30 residues in length (4, 5).Family members such as rhodopsin and the ,3adrenergic receptor bind their ligands at a single domain, which lies in the receptor's hydrophobic core, between the helices and below the upper plane of the cellular membrane (6, 8). However, it is unclear whether this binding motif is also used by other members of the superfamily, especially those that interact with more complex ligands like C5a, or whether the motif is altered to accommodate the larger agonists. The little information that exists comes largely from studies with the glycopeptide hormone receptors, a branch ofthe superfamily characterized by a greatly extended extracellular N terminus (9, 10). These receptors, in contrast to rhodopsin and the ,fadrenergic receptor, appear to bind ligands by means of this enlarged N terminus (11,12). We now report that the binding site of the C5a receptor is more complex and consists of two physically separable domains. The first domain is composed of the N terminus and possibly the exter...

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Cells having morphological and histochemical properties of collecting tubules were isolated from rabbit renal papillae. Confluent monolayer cultures of these renal papillary collecting tubule (RPCT) cells formed hemicysts and adhered with morphological asymmetry to Millipore filters. Cultures of 1-day-old RPCT cells synthesized cAMP in response to arginine vasopressin (AVP) (half-maximal response to 10(-10) M), oxytocin, and parathyroid hormone (half-maximal responses at 5 X 10(-9) M) but not to adrenergic agents. After 10 days of growth (fourfold increase in cell number) RPCT cells retained the same pattern of histochemical and hormonal responses as 1-day-old cells. Hormones were tested for their influence on the release of immunoreactive prostaglandins (iPG) by RPCT cells; the major product under both basal and stimulated conditions was iPGE2. At very low concentrations (greater than or equal to 10(-10) M), bradykinin, lysyl-bradykinin, and methionyl-lysyl-bradykinin caused four- to sixfold increases in the rate of iPGE2 formation within 3 min; smaller (less than twofold) increases were observed with relatively high concentrations of epinephrine (10(-5) M), norepinephrine (10(-5) M), and angiotensin II (10(-7) M), but only after longer incubations. Significantly, neither AVP (10(-7) M) nor [deamino]AVP (10(-7) M) caused prostaglandin release by RPCT cells. Our results indicate that kinins can act directly on the collecting tubule to elicit PGE2 formation; furthermore, this effect of kinins may be natriuretic, since PGE2 has been shown to inhibit Na+ resorption by the medullary collecting tubule and thick ascending limb.

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...

The anaphylatoxin C5a is implicated in a number of inflammatory diseases. It is a highly cationic protein with 13 of 74 amino acids being either arginine or lysine. A search focusing on positively charged molecules, particularly amine-containing functionalities, led to the discovery of substituted 4,6-diaminoquinolines 1 [N,N'-bis(4-amino-2-methyl-6-quinolyl)urea] and 7 [6-N-(2-chlorocinnamoyl)-4,6-diamino-2-methylquinoline] as inhibitors of C5a receptor binding. These two compounds inhibited the binding of radiolabeled C5a to its receptor isolated from human neutrophils with IC50's = 3.3 and 12 micrograms/mL, respectively. Our efforts to enhance their potencies by chemical modification revealed a narrow profile of potency for effective C5a receptor binding inhibition.