Comparative Agonist/Antagonist Responses in Mutant Human C5a Receptors Define the Ligand Binding Site

Higginbottom, Adrian; Cain, Stuart A.; Woodruff, Trent M.; Proctor, Lavinia M.; Madala, Praveen K.; Tyndall, Joel D. A.; Taylor, Stephen M.; Fairlie, David P.; Monk, Peter N.

doi:10.1074/jbc.m410797200

Cited by 49 publications

(87 citation statements)

References 29 publications

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“…In our studies in yeast, C5a stimulated weak signaling in D282A-C5aR, whereas C5a-des-Arg 74 induced potent signaling in D282A-C5aR (Fig. 6), consistent with the earlier studies in mammalian cells (54,55). However, both C5a and C5a-des-Arg 74 failed to stimulate signaling in S272A-C5aR (Fig.…”

Section: Ec3-n Terminus Interaction In 7tm Receptor Functionsupporting

confidence: 81%

“…Mutational studies support that Asp 282 located at the EC3/TM7 junction in the C5a receptor forms a salt bridge interaction with Arg 74 . In support of this model, by removing Arg 74 from C5a (C5a-des-Arg 74 , a natural metabolite of C5a generated by proteolysis), the C5a-des-Arg 74 ligand activated the D282A-C5aR with similar potency compared with the full-length C5a (54). To test if the S272A mutation affects the potential interaction of Arg 74 with EC3, we generated a C5a-des-Arg 74 ligand that could be expressed in yeast.…”

Section: Ec3-n Terminus Interaction In 7tm Receptor Functionmentioning

confidence: 99%

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Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

Rana

Baranski

2010

Journal of Biological Chemistry

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G protein-coupled receptors (GPCRs), 2 often referred to as seven-transmembrane domain receptors, are one of the most biologically important superfamilies of receptors (1). Phylogenetic analysis classifies the superfamily of receptors into glutamate, rhodopsin, adhesive, frizzled, and secretin families (2).The rhodopsin family alone includes ϳ750 GPCRs (3). ϳ60% of GPCRs respond to sensory and olfactory neurons; ϳ30% respond to protein and peptide hormones, amino acids, biogenic amines, and lipids, whereas the remaining GPCRs respond to nutrients and metabolites. In response to ligands, the receptors recruit either G proteins or other intracellular effector proteins (4), which transduce the extracellular chemical signals and further stimulate intracellular signaling cascades (5). This extraordinary ability to sense such a diverse array of chemical signals with selective precision classifies GPCRs as a "druggable proteome" (6) that generates ϳ10% of the global pharma revenue (7).Given their cellular and pharmacological importance, understanding the receptor function at the molecular level is a prerequisite, and indeed the recent advancements in the high resolution crystals of rhodopsin-like GPCRs have been significantly informative (8 -12). Nevertheless, a central question remains how the canonical receptor topology interconverts between several possible conformations in response to diverse chemical stimuli. Traditionally, the GPCR activation mechanism is described as "molecular switches" identified in conserved "microdomains" (13) and recently reviewed elsewhere (14,15). Briefly, ligand/agonist binding or an activating point mutation (CAM) triggers a series of molecular macroswitches, such as the global rotamer toggling (16, 17) on TM6 or the disruption of an ionic lock (18,19), between TM3 and TM6, to unlock the G protein-binding site in the intracellular face of the receptors leading to G protein activation. However, ϳ80% of the rhodopsin family receptors do not have the putative residues to support the described "rotamer toggle" switch, the "ionic lock," or both (20). Although a universal activation mechanism is also suggested by the ability of human receptors to activate evolutionarily distant yeast G proteins, how this is accomplished is difficult to reconcile within a framework of low sequence homology (Յ20%) between receptors (21), the variable length of the peptide loops, and the diversity of N termini in receptors. Indeed, the significant variation observed in pharmacological properties of receptors (22,23) within the same family (24) does not favor a single, unified activation mechanism model.Within the rhodopsin family of GPCRs, the EC3 loop varies in length between 4 and 27 residues and plays a key role in the activation of the neuropeptide class of receptors (25). Minor alterations affecting its folding or structure have been shown to impair both ligand binding and signaling (26 -32). This study * This work was supported, in whole or in part, by National Institutes of Health Grants GM071634 and GM07...

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Section: Ec3-n Terminus Interaction In 7tm Receptor Functionsupporting

confidence: 81%

Section: Ec3-n Terminus Interaction In 7tm Receptor Functionmentioning

confidence: 99%

Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

Rana

Baranski

2010

Journal of Biological Chemistry

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“…Activation of C5aR requires C5a binding at two distinct sites: the main ligand binding site being the extracellular N terminus (aa , and the other signal-transducing site is formed by the extracellular loops formed by a-helices III, VI, and VII (12,(29)(30)(31). Thus, the reduction in responsiveness to C5a but not the C5aR agonist suggested that the loss of binding of the anti-N-terminal Ab and the loss of C5a-induced calcium response was not a consequence of total loss of the C5aR but suggested that only the C5a-binding N terminus was lost but the calcium signaling C terminus was retained.…”

Section: Nsps Cleave and Inactivate The C5armentioning

confidence: 99%

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

Berg

Tambourgi

Clark

et al. 2014

The Journal of Immunology

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Neutrophil dysfunction, resulting in inefficient bacterial clearance, is a feature of several serious medical conditions, including cystic fibrosis (CF) and sepsis. Poorly controlled neutrophil serine protease (NSP) activity and complement activation have been implicated in this phenomenon. The capacity for excess NSP secretion and complement activation to influence the expression and function of the important neutrophil-activating receptor C5aR was investigated. Purified NSPs cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 cleaved C5aR to a 26- to 27-kDa membrane-bound fragment, thereby inactivating its C5a-induced signaling ability. In a supernatant transfer assay, NSPs released from neutrophils in response to C5a induced the cleavage of the C5aR on unstimulated cells. Stimulation of myeolomonocytic U937 cells and purified neutrophils with C5a resulted in downregulation of the C5aR on these cells, which, in the case of U937 cells, was largely caused by NSP-mediated cleavage of C5aR, but in the case of neutrophils, intracellular degradation was likely the main mediator in addition to a small role for NSPs. CG and NE in bronchoalveolar lavage fluid from CF patients both contributed to C5aR cleavage. We propose two converging models for C5a- and NSP-mediated neutrophil dysfunction whereby C5aR cleavage is induced by NSPs, secreted in response to: 1) excess C5a generation or other stimuli; or 2) necrosis. The consequent impairment of C5aR activity contributes to suboptimal local neutrophil priming and bacterial clearance. NSP inhibitors with specificity for both CG and NE may aid the treatment of pathologies associated with neutrophil dysfunction including sepsis and CF.

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“…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%

“…The second site involves the C terminus of C5a and an interhelical pocket of the C5aR (15,16). 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).…”

mentioning

confidence: 99%

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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Comparative Agonist/Antagonist Responses in Mutant Human C5a Receptors Define the Ligand Binding Site

Cited by 49 publications

References 29 publications

Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

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

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

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