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

Abstract

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...

Mechanism of Neutrophil Dysfunction: Neutrophil Serine Proteases Cleave and Inactivate the C5a Receptor

“…NSPs have been shown to proteolytically reduce the expression of several cell-surface molecules involved in immunity, including CXCR1 (IL-8RA), CR1, CD16, CD43, and TNFRII, but their impact on C5aR expression has not been investigated. Proteolytic cleavage and inactivation of C5aR has been demonstrated by a variety of endogenous and exogenous proteases, including an endogenous metalloprotease, activated by the action of Loxosceles spider venom sphingomyelinase D, a serine protease from Porphyromonas gingivalis, and a metalloprotease from venom of the spider Plectreurys tristes (22)(23)(24). Direct proteolysis and inactivation of C5aR by an endogenous protease has not been demonstrated so far; however, a reduced chemotactic response to C5a after NE and CG treatment has been reported (25).…”

Requirements for C5a Receptor-Mediated IL-4 and IL-13 Production and Leukotriene C4 Generation in Human Basophils

“…It has two major regions important for ligand binding (15). The first, interacting with the core of C5a, is in the extracellular N-terminal domain, and the second, interacting with the C terminus of C5a, is poorly characterized and lies within the transmembrane helices (possibly helix V) of the receptor.…”

“…The first, interacting with the core of C5a, is in the extracellular N-terminal domain, and the second, interacting with the C terminus of C5a, is poorly characterized and lies within the transmembrane helices (possibly helix V) of the receptor. Several studies have indicated that ligand interaction with the putative transmembrane binding site but not the N terminus of the receptor is responsible for the induction of cellular responses, like chemotaxis and enzyme release from neutrophils (15)(16)(17)(18). Subsequently, a number of C5a C-terminal analogs interacting with the second, "effector" site of the receptor and possessing different agonistic properties were generated (19 -21).…”

Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling

“…As evidence for the role of the first-site interaction, truncation of the N terminus reduces the affinity of the receptor for full-length ligand, but preserves the efficacy of hexapeptide analogs of the C5a C-terminal tail (8). Conversely, the small molecule L-584,020 competes with full-length C5a but not with hexapeptides (8), suggesting that L-584,020 inhibits binding but not activation.…”

“…Conversely, the small molecule L-584,020 competes with full-length C5a but not with hexapeptides (8), suggesting that L-584,020 inhibits binding but not activation. Furthermore, NMR spectroscopy has shown that the structure of the isolated C5aR N terminus is altered by incubation with C5a, suggesting that the N terminus of the receptor interacts with the ligand (11).…”