Arginine 206 of the C5a Receptor Is Critical for Ligand Recognition and Receptor Activation by C-terminal Hexapeptide Analogs

DeMartino, Julie A.; Konteatis, Zenon; Siciliano, Salvatore; Riper, Gail Van; Underwood, Dennis; Fischer, Paul; Springer, Martin S.

doi:10.1074/jbc.270.27.15966

Cited by 72 publications

(93 citation statements)

References 22 publications

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“…For small agonistic peptides, an interaction of the C-terminal arginine with Arg 206 in the fifth transmembrane helix of the C5aR has been postulated (36). Also unknown is the molecular basis of the distinct bioactivity of C5a desarg .…”

Section: Discussionmentioning

confidence: 99%

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

Eglite

Plüss

Dahinden

2000

The Journal of Immunology

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Anaphylatoxin derived from the fifth complement component (C5a) in the presence of IL-3 induces continuous leukotriene C4 generation and IL-4 and IL-13 expression in human basophils for a period of 16–18 h. This indicates that the G protein-coupled C5a receptor (C5aR) can induce long-lasting cellular responses. Using anti-N-terminal C5aR Abs, C-terminal C5a hexapeptide analogs, and pertussis toxin, we demonstrate that the putative activation site of the C5aR is both necessary and sufficient for these late cellular responses. Furthermore, continuous pertussis toxin-sensitive G protein-coupled receptor activation and receptor-ligand interaction is ongoing and required during the entire period of product release. However, the late basophil responses have a more stringent requirement for optimal receptor activation. Leukotriene C4 generation appears to be influenced mostly by the way the receptor is activated, because the most active hexapeptide is a superagonist for this response. By contrast, C5adesarg, lacking the C-terminal arginine, induces minimal lipid mediator formation but is fully active to induce IL-4 production and is even a superagonist for IL-13 release. Nevertheless, IL-4/IL-13 synthesis in response to C5adesarg could be blocked by both C-terminal antagonistic peptide as well as anti-N-terminal C5aR Abs, indicating only minor differences of ligand-receptor interactions between C5a and C5adesarg. Taken together, our data demonstrate that long-lasting and continuous signaling occurs through a limited activation domain of the C5aR, which can differentially promote separate basophil functions.

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Section: Discussionmentioning

confidence: 99%

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

Eglite

Plüss

Dahinden

2000

The Journal of Immunology

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“…C5a is a 74-amino acid glycoprotein that is liberated during complement activation and mediates such processes as neutrophil chemotaxis, platelet activation, and vascular permeability (21). Binding of C5a to its receptor involves a two-site binding model with the COOH terminus of the ligand inserting into the receptor interhelical crevice and other portions of the ligand interacting with the NH 2 terminus of C5aR to enhance ligand affinity (22). C5aR serves as a useful model to study GPCR oligomerization for two major reasons.…”

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confidence: 99%

C5a Receptor Oligomerization

Klco

Lassere

Baranski

2003

Journal of Biological Chemistry

109

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G protein-coupled receptors (GPCRs), stimulated by hormones and sensory stimuli, act as molecular switches to relay activation to heterotrimeric G proteins. Recent studies suggest that GPCRs form dimeric or oligomeric structures, a phenomenon that has long been established for growth factor receptors. The elucidation of the domains of GPCRs that mediate receptor association is of critical importance for understanding the function of GPCR oligomers. Using a disulfide-trapping strategy to probe the intermolecular contact surfaces, we demonstrate cross-linking of C5a receptors in membranes prepared from both human neutrophils and stably transfected mammalian cells that is mediated by a cysteine in the second intracellular loop. To explore other surfaces that might be involved in the oligomerization of C5a receptors, we constructed receptors with individual cysteines in other intracellular regions. C5a receptors with a cysteine in the first intracellular loop or the carboxyl terminus displayed the fastest kinetics of dimer formation, whereas an intracellular loop 3 cysteine displayed minimal cross-linking. Since the rate of disulfide trapping reflects the proximity of sulfhydryl groups, assuming similar accessibility and flexibility, these results imply a symmetric dimer interface that may involve either transmembrane helices 1 and 2 or helix 4. However, neither model can account for the ability of the native cysteine in the second intracellular loop to mediate efficient crosslinking. Based on these observations, we propose that C5a receptors form higher order oligomers (i.e. tetramers) or clusters in the membrane.G protein-coupled receptors (GPCRs) 1 are an evolutionarily conserved family of transmembrane receptors that are characterized by seven-transmembrane helixes connected by intracellular loops (ICs) and extracellular loops (1, 2). GPCRs mediate a multitude of physiologic responses and serve as common pharmacological targets; more than half of all pharmacological agents that are currently prescribed target GPCRs (3). Receptor activation is accomplished by a remarkably diverse group of ligands to catalyze the exchange of GTP for GDP on the ␣ subunit of heterotrimeric G proteins. This exchange results in the dissociation of the ␣-GTP subunit from the ␤␥ dimer. Both complexes can then activate downstream targets, such as effector enzymes and ion channels (4, 5).Despite numerous structure-function studies of many different GPCRs, a fundamental question remains: Do GPCRs function as monomers or as oligomers? For the ϳ25 members of class C receptors, the answer is clear. These receptors form dimers through specialized structures, such as disulfidebonded extracellular domains (metabotropic glutamate receptors) (6, 7) or coiled-coil interactions of the carboxyl-terminal tails (GABA B receptors) (8). For the more than 500 members of the rhodopsin family of GPCRs and the 60 members of the secretin family of GPCRs, studies now demonstrate that a rapidly increasing number of these GPCRs form oligomers (9, 10). Early work...

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“…Then, possible orientations of the entire structure of C5a were restored by overlapping the rigid core C5a-(1-62) from PDB entry 1KJS with each of the four selected orientations of C5a-(59 -74) within C5aR. It was found that only one specific orientation of C5a satisfied the requirements of spatial proximity of positions 15,18,19,20,27, and 46 in C5a to specific residues in the N-terminal segment 8 -41 of C5aR, as was suggested by available site-directed mutagenesis data (31,32). Seventeen conformations of NT 8 -41 (out of 185) and 11 conformations of the EC1 ϩ EC2 ϩ EC3 loop package (out of 29) did not show steric clashes with this particular orientation of C5a; all these conformations together with the selected orientation of C5a and the TM region of C5aR make up the final model of the C5a-C5aR complex.…”

Section: Methodsmentioning

confidence: 99%

“…3 The model was successfully utilized for rationalizing available site-directed mutagenesis data in the N-terminal fragment of C5aR (mutations involving Asp 15 , Asp 16 , Asp 21 , and Asp 27 (10,14)) as well as in C5a (mutations involving His 15 , Val 18 , and Arg 46 (32)). The model was also validated by comparison to the data on C5aR mutants E199K, D282R, R206A, and R175D (16, 18, 33-35).…”

Section: Use Of C5a C27r/r40c To Trap Interactions With the C5ar Nt-c5amentioning

confidence: 99%

“…Hexapeptide analogs of the C5a C terminus can serve as full agonists or as antagonists for the receptor, albeit with decreased potency (17), and this activity can be modulated by making mutations in the interhelical pocket (15,18), suggesting that the receptor switch mechanism is actuated by the "message" delivered as a consequence of the first-site interaction providing the correct address (19).…”

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confidence: 99%

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

Hagemann

Miller

Klco

et al. 2008

Journal of Biological Chemistry

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Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand.One of the most biologically important families of receptors is the G protein-coupled receptors (GPCRs), 2 which eukaryotic cells use to sense signals as diverse as light, odorants, small molecules, and polypeptide hormones (1). The GPCRs, which number ϳ1000 in the human genome (2), have a shared topology made up of seven transmembrane domains (TMs) linked by intracellular and extracellular (EC) loops, along with an extracellular N terminus and intracellular C terminus (Fig. 1). These receptors also have a shared mechanism of action, whereby the activated receptor serves as a guanosine exchange factor on the ␣ subunit of a heterotrimeric G protein, thus transmitting the signal to the interior of the cell. Many drugs are directed against GPCRs, including adrenergic, muscarinic, dopaminergic, serotonergic, GABAergic, and histaminergic receptors. Taken together, drugs acting on GPCRs make up an estimated 50% of the current pharmacopoeia (3).The structural basis of receptor-ligand interaction is of great interest in both basic and applied pharmacology. In the case of GPCRs, a central question is how receptors with a single common topology can be activated by such a wide variety of ligands. A related question is how specificity is built into these receptors, so that each is activated only by the appropriate ligands.Many GPCRs are activated by polypeptide ligands, including chemokines such as SDF-1 (CXCL12), physiologic modulators such as angiotensin II, glycoprotein hormones such as luteinizing hormone, and chemotactic factors such as complement factor 5a (C5a). These ligands are too large to fit entirely in an interhelical cleft of a GPCR, so their bulk must serve some adaptive function other than receptor activation per se. In some cases, the ligand is recruited by its affinity for the receptor extracellular domains, and a second discret...

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Arginine 206 of the C5a Receptor Is Critical for Ligand Recognition and Receptor Activation by C-terminal Hexapeptide Analogs

Cited by 72 publications

References 22 publications

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

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

C5a Receptor Oligomerization

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

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