C5a receptor oligomerization. I. Disulfide trapping reveals oligomers and potential contact surfaces in a G protein-coupled receptor.
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.
Klco
Jeffery M
JM
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Lassere
Tracey B
TB
Baranski
Thomas J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D006224:Cricetinae; D019281:Dimerization; D004220:Disulfides / Q000032:analysis; D019204:GTP-Binding Proteins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry / Q000502:physiology; D010618:Phenanthrolines / Q000494:pharmacology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
D015703:Antigens, CD; D002614:Chelating Agents; D004220:Disulfides; D046911:Macromolecular Substances; D010618:Phenanthrolines; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; C025205:1,10-phenanthroline