C5a Receptor Oligomerization

Floyd, Desiree H.; Geva, Adi; Bruinsma, Stephen P.; Overton, Mark C.; Blumer, Kendall J.; Baranski, Thomas J.

doi:10.1074/jbc.m305607200

Cited by 64 publications

(23 citation statements)

References 46 publications

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“…The ability of C5aR to cross-link in the absence of peripheral membrane proteins argues that the oligomerization that we observe is a receptorautonomous process. Moreover, we demonstrated that urea treatment had little effect on the ability of C5aR molecules to FRET in yeast (57).…”

Section: Resultsmentioning

confidence: 87%

“…Heterodimerization of opioid receptors and ␦ results in novel ligand binding and function, proving that GPCR oligomerization has physiologic relevance (12). More recently, energy transfer studies, using either fluorescence (FRET) or bioluminescence (BRET), have also shown the proximity of monomers of many members of the rhodopsin family of GPCRs, including ␤ 2 -adrenergic receptor (13), somatostatin receptors (14), opioid receptors (15), and the C5a receptor (57).…”

mentioning

confidence: 99%

“…In the second and fourth transmembrane segments of the C5a receptor, essential residues, identified by random saturation mutagenesis analysis, map onto positions in the rhodopsin structure that could mediate dimer/oligomer interactions. Moreover, FRET studies on human C5a receptors tagged with YFP and cyan fluorescent protein demonstrate that C5a receptors form oligomers when expressed in yeast, which should provide a useful genetic system to begin to characterize the oligomerization of C5a receptors (57).…”

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confidence: 99%

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C5a Receptor Oligomerization

Klco

Lassere

Baranski

2003

Journal of Biological Chemistry

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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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Section: Resultsmentioning

confidence: 87%

mentioning

confidence: 99%

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confidence: 99%

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C5a Receptor Oligomerization

Klco

Lassere

Baranski

2003

Journal of Biological Chemistry

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109

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“…tors when co-expressed in yeast (60). Accordingly, we suggest that GXXXG-like motifs may be able to direct the formation of specific GPCR complexes as follows.…”

Section: ␣-Factor Receptor Oligomerization Biogenesis and Signalingmentioning

confidence: 90%

Oligomerization, Biogenesis, and Signaling Is Promoted by a Glycophorin A-like Dimerization Motif in Transmembrane Domain 1 of a Yeast G Protein-coupled Receptor

Overton

Chinault

Blumer

2003

Journal of Biological Chemistry

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132

136

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G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the ␣-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that ␣-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired ␣-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes ␣-factor receptor signal transduction. Structurefunction studies suggested that the GXXXG motif in TM1 of the ␣-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays.

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“…Heterologous expression systems have provided a variety of answers concerning ligand-dependent regulation of GPCR oligomeric states. Ligand binding, depending on the GPCR studied, can promote (3)(4)(5)(6)(7)(8)(9)(10) or inhibit (11)(12)(13) dimer formation, as well as having no effect on preexisting constitutive homo-or heterodimers (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). The fact that heterodimerization may alter the pharmacological properties of a GPCR along with its internalization and signal transduction behavior is of critical importance (26,27).…”

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confidence: 99%

The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts

Savi

Zachayus

Delesque-Touchard

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

279

221

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P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12͞P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met. mechanism of action ͉ platelet ͉ antiaggregant M any G protein-coupled receptors (GPCRs) have been shown to assemble as homodimers, heterodimers, as well as larger oligomers (1, 2). The existence of such oligomeric entities raises questions as to their functional consequences as well as their physiological relevance. Heterologous expression systems have provided a variety of answers concerning ligand-dependent regulation of GPCR oligomeric states. Ligand binding, depending on the GPCR studied, can promote (3-10) or inhibit (11-13) dimer formation, as well as having no effect on preexisting constitutive homo-or heterodimers (14-25). The fact that heterodimerization may alter the pharmacological properties of a GPCR along with its internalization and signal transduction behavior is of critical importance (26, 27).Clustering, even for nonheptahelical receptors, now appears as a common feature of cell signaling. Specialized structures such as clathrin-coated pits, caveolae, and lipid rafts contain high concentrations of signaling molecules. Rafts represent dynamic assemblies of proteins and lipids, mostly sphingolipids and cholesterol (28,29). Proteins such as glycophosphatidylinositol-anchored proteins, nonreceptor tyrosine kinases, G␣ subunits of heterotrimeric G proteins, and palmitoylated proteins appear to localize to these microdomains (30). In addition, recent studies have shown that partitioning of proteins in and out of rafts can depend on their state of activation or dimerization (31-33). A variety of GPCR have also been identified in caveolae or rafts. These include ␣ and ␤-adrenergic receptors (34, 35), adenosine A1 receptor (36), angiotensin II type 1 receptor (37), muscarinic receptor (38), EDG1 receptor (39), bradykinin B1 and B2 receptor...

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C5a Receptor Oligomerization

Cited by 64 publications

References 46 publications

C5a Receptor Oligomerization

C5a Receptor Oligomerization

Oligomerization, Biogenesis, and Signaling Is Promoted by a Glycophorin A-like Dimerization Motif in Transmembrane Domain 1 of a Yeast G Protein-coupled Receptor

The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts

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