C5a receptor oligomerization. II. Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast.
Recent studies demonstrate that members of the superfamily of G protein-coupled receptors (GPCRs) form oligomers both in vitro and in vivo. The mechanisms by which GPCRs oligomerize and the roles of accessory proteins in this process are not well understood. We used disulfide-trapping experiments to show that C5a receptors, expressed in mammalian cells, reside in membranes as oligomers (Klco, J. M., Lassere, T. B., and Baranski, T. J. (2003) J. Biol. Chem. 278, 35345-35353). To begin to address how C5a receptors form oligomers, we now use fluorescence resonance energy transfer experiments on human C5a receptors expressed in the lower eukaryote Saccharomyces cerevisiae. C5a receptors tagged with variants of the green fluorescent protein display energy transfer in intact yeast, demonstrating that mammalian accessory proteins are not required for C5a receptor oligomerization. In both intact yeast cells and membrane preparations, agonist does not affect FRET efficiency, and little energy transfer is observed between the C5a receptor and a co-expressed yeast pheromone receptor (encoded by STE2), indicating that C5a receptor oligomerization is both receptor-specific and constitutive. FRET studies performed on fractionated membranes demonstrate similar levels of energy transfer between tagged C5a receptors in endoplasmic reticulum compared with plasma membrane, and urea washing of membranes has little effect on the extent of energy transfer. The oligomerization of C5a receptors expressed in yeast displays characteristics similar to those observed for other GPCRs studied in mammalian cells. This model system should prove useful for further studies to define mechanisms of oligomerization of mammalian GPCRs.
Floyd
Desiree H
DH
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, 63110, USA.
Geva
Adi
A
Bruinsma
Stephen P
SP
Overton
Mark C
MC
Blumer
Kendall J
KJ
Baranski
Thomas J
TJ
D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D046911:Macromolecular Substances; D016133:Polymerase Chain Reaction; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D015703:Antigens, CD; D017931:DNA Primers; D046911:Macromolecular Substances; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase