C5a Receptor Oligomerization

The hepatitis E virus (HEV) capsid consists of a single structural protein, a portion of which is engaged in isosahedral contact to form a basal shell, and another portion in dimeric contact to form the homodimers protruding from the shell. Previous studies revealed that homodimers of the truncated HEV capsid proteins, E2 (amino acids 394 -606) and p239 (amino acids 368 -606), model dominant antigenic determinants of HEV. Immunization with these proteins protected rhesus monkeys against the virus, and three monoclonal antibodies against the homodimers could neutralize HEV infectivity and/or immune-capture of the virus. Furthermore, homodimers of p239 further interact to form particles of 23 nm diameter, rendering it an efficacious candidate vaccine. In light of this we postulate that the interactions involved in the formation of the homodimers and particles might be similar to those involved in assembly of the virus capsid. Presently, mutational analysis was carried out to identify these sites of interactions. The site of dimeric interactions was located to a cluster of six hydrophobic amino acids residues, Ala 597 , Val 598 , Ala 599 , Leu 601 , and Ala 602 ; furthermore, the site involved in particle formation was located at amino acids 368 -394. The possibility that these sites are also involved in assembly of the virus capsid is supported by the fact that they are located at two major and highly conserved hydrophobic regions of the HEV structural protein.

Section: Discussionmentioning

confidence: 99%

Mutational Analysis of Essential Interactions Involved in the Assembly of Hepatitis E Virus Capsid

Zhang

et al. 2005

“…Two days after transfection, total membranes were prepared as described previously (14). 5 g of protein from the membrane preparations was incubated with 40 pM 125 I-C5a in the presence of increasing amounts of unlabeled C5a for 45 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were treated with 250 units of endo-␤-N-acetylglucosaminidase (EndoH; New England Biolabs) at 37°C for 3 h. Samples were resolved by SDS-PAGE and immunoblotted with rabbit polyclonal anti-C5aR, raised against residues 9 -29 of the amino terminus (14).…”

Section: Methodsmentioning

confidence: 99%

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

Self Cite

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...

“…and/or IV in the complement C5a receptor (39). This range of results is fascinating, and combinations of direct experimentation and bio-informatic approaches (40 -41) are likely to be required to provide understanding.…”

Section: Figmentioning

confidence: 99%

Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins

Carrillo

Pediani

Milligan

2003

106

118

The concept that G protein-coupled receptors (GPCRs) 1 exist as dimers or higher order oligomers has moved rapidly from hypothesis to being widely accepted (1-4). A range of approaches has contributed to this understanding. This includes the ability to co-immunoprecipitate differentially epitopetagged forms of a GPCR from cells in which they are co-expressed, and, in intact cells, the application of a number of resonance energy transfer-based techniques. However, the role of dimerization in function and the mechanisms then responsible for initiation of signal transduction by the dimer have been more recalcitrant to analysis. Significant progress in this area has recently been achieved for the class C GPCRs. These contain both a long extracellular N-terminal domain, to which agonist ligands bind, and the prototypic seven transmembrane (TM) helix bundle architecture that is the common feature of all GPCR families (for review, see Ref. 5). The functional ␥-aminobutyric acid, type b receptor is a hetero-dimer of two distinct gene products in which trafficking to the plasma membrane requires interaction between the partner polypeptides (6 -9). This indicates that a key role of dimerization is achieving appropriate cellular localization. This is also true for the class A rhodopsin-like GPCRs because non-functional, truncated splice variants can restrict plasma membrane delivery of fulllength GPCRs and, thus, limit their function (10 -12). Chimeric class C GPCRs consisting of the extracellular domain of one GPCR and the TM and intracellular elements of a second, closely related GPCR or in which the intracellular loops of dimer partners are exchanged have provided strong evidence that the mechanism of action of the dimer involves transactivation (13)(14); that is, ligand binding to one element of the dimer results in activation of G protein produced by the other GPCR within the dimer. Again, the ␥-aminobutyric acid, type b receptor has been particularly informative in this regard as only one of the two gene products that forms the dimer is able to bind the agonist ␥-aminobutyric acid. Equivalent systems are not available for the majority of class A GPCRs. However, for the luteinizing hormone receptor, which also has a long N-terminal domain that binds the ligand, partial reconstitution of function has been achieved by co-expression of distinct pairs of mutants (15-16). Furthermore, in recent studies co-expression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescued hormone-activated cAMP production (17).