Many hormones and sensory stimuli signal through a superfamily of seven transmembrane-spanning receptors to activate heterotrimeric G proteins. How the seven transmembrane segments of the receptors (a molecular architecture of bundled ␣-helices conserved from yeast to man) work as "on/off" switches remains unknown. Previously, we used random saturation mutagenesis coupled with a genetic selection in yeast to determine the relative importance of amino acids in four of the seven transmembrane segments of the human C5a receptor (Baranski, T. J., Herzmark, P., Lichtarge, O., Gerber, B. O., Trueheart, J., Meng, E. C., Iiri, T., Sheikh, S. P., and Bourne, H. R. (1999) J. Biol. Chem. 274, 15757-15765). In this study, we evaluate helices I, II, and IV, thereby furnishing a complete mutational map of the seven transmembrane helices of the human C5a receptor. Our analysis identified 19 amino acid positions resistant to non-conservative substitutions. When combined with the 25 essential residues previously identified in helices III and V-VII, they delineate two distinct components of the receptor switch: a ligand-binding surface at or near the extracellular surface of the helix bundle and a core cluster in the cytoplasmic half of the bundle. In addition, we found critical amino acids in the first and second helices that are predicted to face the lipid membrane. These residues form an extended surface that might mediate interactions with lipids and other membrane proteins or function as an oligomerization domain with other receptors.G protein-coupled receptors, a superfamily of seven transmembrane proteins, act as molecular switches that, upon activation by extracellular stimuli, transmit signals to heterotrimeric G proteins on the cytoplasmic face of the plasma membrane. These receptors then catalyze ligand-dependent exchange of GTP for GDP on the ␣-subunit of the heterotrimer, causing dissociation of ␣⅐GTP from the ␤␥-dimer; ␣⅐GTP and free ␤␥ subsequently activate effector enzymes and ion channels (1, 2). More than 1000 G protein-coupled receptors of mammals share with their counterparts in yeast and plants a conserved three-dimensional architecture, comprising seven ␣-helices in a transmembrane bundle (3-6). The switch mechanism is also conserved, as indicated by the abilities of mammalian receptors to activate G protein trimers in yeast (7-9). The switch clearly resides in the seven-helix bundle: swapping of extra-or intracellular loops preserves the ability of ligands to activate G proteins while transferring specificity of ligand binding or G protein activation, respectively, from one receptor to another (10 -13).Our understanding of receptor mechanisms remains limited by a lack of precise structural information and the general difficulties of characterizing integral membrane proteins. A low-resolution (5 Å) electron cryomicroscopic structure of rhodopsin (14), the retinal light receptor, reveals relative positions and tilts of seven transmembrane helices in the plane of the membrane. Based on many mutations, the rhod...

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 G protein-coupled receptors (GPCRs) 1 are a diverse superfamily of receptors that mediate numerous physiological effects in response to sensory, hormonal, and neurotransmitter stimuli (1). There is mounting evidence that GPCRs can associate as homo-and/or hetero-oligomers in the plasma membrane, perhaps with functional consequences (2, 3). In vitro studies of many GPCRs in all major families (rhodopsin-like, secretin, and metabotropic glutamate) demonstrate that receptors can be co-immunoprecipitated as differentially epitope-tagged homodimers in the case of the opioid, Ig-Hepta, and calciumsensing and metabotropic glutamate-5 receptors (4 -8). Disulfide-trapping studies have mapped potential oligomer interfaces in dopamine D2 receptors (9) and the C5a receptor (46). The availability of variants of the green fluorescent protein has enabled in vivo studies of GPCRs. Fluorescence resonance and bioluminescence resonance energy transfer experiments (FRET and BRET, respectively) provide evidence of oligomerization of many GPCRs; the ␤ 2 -adrenergic receptor and the chemokine receptor CCR5 have been shown to homodimerize with BRET, the ␦-opioid receptor has been shown to dimerize with BRET and time-resolved FRET, and the yeast pheromone receptor has been shown to oligomerize with FRET in live yeast (10 -13). Oligomers of GPCRs have recently been visualized; elegant atomic force microscopy studies of native disc membranes reveal that rhodopsin packs in the membrane as rows of dimers (14).Whereas the physical association of GPCRs into oligomers has been well described, the mechanisms that assemble GPCRs into higher ordered structures and the roles of membrane scaffolds and accessory proteins in regulating receptor oligomerization remain poorly understood. Several studies demonstrate that receptor oligomers may occur early in their biosynthesis; for example, the metabotropic GABA B1 receptor is retained in the endoplasmic reticulum unless the GABA B2 subunit is co-expressed (15). In addition, energy transfer studies demonstrate that the mammalian CCR5 receptor (11) and the yeast pheromone receptors (16) form constitutive oligomers in the endoplasmic reticulum, suggesting that this phenomenon might be essential for proper receptor assembly, both in yeast and mammalian cells. For the majority of GPCRs, it is unclear if the association of receptors into oligomers is a receptorautonomous process or if accessory proteins mediate specific associations between homo-and heterodimers. Proteins that interact with receptors to regulate their internalization and desensitization might also be involved in regulating GPCR oligomerization at the plasma membrane; examples include the heterotrimeric G proteins, arrestins, caveolins, clathrin, and adaptor proteins (17). What roles...