Site‐Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.G protein-coupled receptors (GPCRs) 2 are the largest class of membrane-bound receptors, with more than 850 members in the human genome (1). The majority of GPCRs is grouped in the rhodopsin family by the presence of a small number of conserved amino acids in the transmembrane (TM) bundle, such as the DRY motif at the cytoplasmic end of TM3 and the NPXXY motif in TM7 (2). These receptors share a similar molecular architecture within the seven-helix bundle, which was originally represented by the ␣-carbon template of the Baldwin model (3) and later confirmed by the high resolution structure of bovine rhodopsin (4). The extracellular loops, in contrast, are more divergent, both in length and function (5). Ligands frequently bind to the extracellular loops, and the variability of the loops is not surprising when considering the range of ligands known to interact with GPCRs. For many GPCRs, especially those that bind larger peptide ligands, the mechanisms by which ligand binding stimulates G protein activation and how the extracellular loops can influence receptor activation are poorly understood. Considering that more than half of all prescribed medications target GPCRs, mostly by disrupting or mimicking ligand binding (6), a better understanding of the function of the extracellular loops is extremely important.To elucidate how GPCRs function as molecular switches to transduce signals, we study the complement factor 5a receptor (C5aR), a rhodopsin-like GPCR expressed primarily on the surface of neutrophils and other myeloid cells. C5a, a 74-amino acid peptide released during complement activation, binds to C5aR and directs neutrophil chemotaxis and the release of proteolytic enzymes and superoxide (7). C5a binds to both the transmembrane bundle and the amino terminus of C5aR (8, 9), and other binding sites in the extracellular l...

Section: Journal Of Biological Chemistry 12015mentioning

confidence: 60%

Genetic Analysis of the First and Third Extracellular Loops of the C5a Receptor Reveals an Essential WXFG Motif in the First Loop

Klco

Nikiforovich

Baranski

2006

“…Because the oligosaccharide is not required for function, it is unlikely to interact directly with the receptor (45). Also, the side chain of ligand Gly 73 must be in proximity to receptor Val 286 and Ile 116 (21), whereas the ligand C-terminal carboxylate group must be close to the side chain of Arg 206 of the receptor (22,40). Although in one NMR structural study of C5a, the C terminus was not resolved (44), other structures (39) contain a C-terminal helix that must necessarily unwind in the bound state to make the C-terminal residues available for receptor interaction.…”

Section: Resultsmentioning

confidence: 99%

“…The obtained structure was extended outward at the N terminus by manually adding a fragment corresponding to residues 24 -37 of C5aR in a position presumably allowing contacts with residue 27 of C5a. Using the Swiss PDB Viewer (www.expasy.org/spdbv/) (38), a structure of C5a taken from Protein Data Bank entry 1KJS (39) (22,40), the dihedral angles and of the peptide backbone for ligand residues 63-65 and 67 were manually adjusted within the allowable regions of the Ramachandran plot. The resulting docked model was subjected to energy minimization using the program SYBYL (Tripos, St. Louis, MO) to remove clashes between residues.…”

Section: Yeastmentioning

confidence: 99%

Random Mutagenesis of the Complement Factor 5a (C5a) Receptor N Terminus Provides a Structural Constraint for C5a Docking

Hagemann¹,

Narzinski

Floyd

et al. 2006

The N terminus of G protein-coupled receptors has been implicated in binding to peptide hormones. We have used random saturation mutagenesis to identify essential residues in the N terminus of the human complement factor 5a receptor (C5aR). In a library of N-terminal mutant C5aR molecules screened for activation by C5a, residues 24 -30 of the C5aR showed a marked propensity to mutate to cysteine, most likely indicating that sulfhydryl groups at these positions are appropriately situated to form disulfide interactions with the unpaired Cys 27 of human C5a. This presumptive spatial constraint allowed the ligand to be computationally docked to the receptor to form a model of the C5a/C5aR interaction. When the N-terminal mutant C5aR library was rescreened with C5a C27R, a ligand incapable of disulfide interactions, no individual position in the N terminus was essential for receptor signaling. However, the region 19 -29 was relatively highly conserved in the functional mutants, further demonstrating that this region of the C5aR makes a productive physiologic interaction with the C5a ligand.G protein-coupled receptors (GPCRs) 3 transduce signals from a wide variety of biological stimuli, including small molecules, lipids, peptides, proteins, and environmental cues such as odorants and light (1). As the largest family of cell-surface receptors, they have provided fertile ground for pharmacologic modulation. GPCRs share a secondary structure consisting of seven transmembrane helices (TMs 1-7) joined by three intracellular and three extracellular loops, along with N-and C-terminal domains (Fig. 1). The receptors serve as binary switches that, when activated by ligand, exhibit guanine nucleotide exchange factor activity toward heterotrimeric G proteins (2). Because they are large transmembrane proteins, GPCRs are difficult to study by direct structural methods such as x-ray crystallography. A series of crystal structures of bovine rhodopsin has allowed major progress in the understanding of GPCR structure (3-7) but has not made clear the mechanism by which these receptors serve as switches, because rhodopsin has only been crystallized in its resting state. The mechanism of GPCR activation has, however, been productively explored using a variety of biochemical, biophysical, and computational techniques. Overall, ligand binding by GPCRs appears to induce a rigid-body movement of TM6 away from TM3, exposing new receptor surfaces that allow coupling to downstream effectors (8 -11).Our laboratory has studied the human complement factor 5a receptor (C5aR) (12) as a model system because it, like rhodopsin and many other clinically important receptors, belongs to the large family A of GPCRs (13). The C5aR has 19% amino acid identity with rhodopsin and has loop and transmembrane helix lengths similar to those of rhodopsin. Furthermore, the C5aR can be expressed in Saccharomyces cerevisiae, making it amenable to genetic studies.In addition to its role as a model receptor, the C5aR has intrinsic biological interest. The vertebrate...

“…Since the cloning of the C5a receptor (21,22), work has been focused on defining the structural features of the receptor molecule required for the C5a-receptor interaction (23)(24)(25)(26)(27)(28)(29)(30)(31). The cell-surface receptor for C5a is a member of the G-proteincoupled receptor superfamily, with an extracellular N-terminal region, an integral membrane helical domain, and a C-terminal tail extending into the cytoplasmic space (32).…”

mentioning

confidence: 99%

Residues 21–30 within the Extracellular N-terminal Region of the C5a Receptor Represent a Binding Domain for the C5a Anaphylatoxin

Chen¹,

Zhang²,

Gonnella³

et al. 1998

The functions of the C5a anaphylatoxin are expressed through its interaction with a cell-surface receptor with seven transmembrane helices. The interaction of C5a with the receptor has been explained by a two-site model whereby recognition and effector sites on C5a bind, respectively, to recognition and effector domains on the receptor, leading to receptor activation (Chenoweth, D. E., and Hugli, T. E.