HEK293 cells stably expressing aequorin (HEK-AEQ) were maintained in above media containing 500 μg/ml Geneticin CB-839 (Invitrogen). Mouse hypothalamic tissue of embryonic day 18 mouse was purchased from BrainBits (Springfield, IL), primary neurons were prepared and the [Ca2+]i mobilization assay performed after cultivating neurons in NbActiv4 neuronal culture medium (BrainBits). Detailed protocol for transient transfections and generation of stable SH-SY5Y cell line expressing GHSR1a can be found in Supplemental Experimental Procedures. The aequorin bioluminescence assay was carried out as described previously (Feighner et al.,

1998 and Howard et al., 1996) and live cell Ca2+ mobilization assays in neuronal SH-SY5Y cells and primary hypothalamic neurons were performed with the Fluo-4 direct assay (Invitrogen), see detailed protocols in Supplemental Experimental Procedures. Labeling of cells was performed after 48 hr of transfection, buy CP-673451 as described previously (Maurel et al., 2008). Cell labeling with terbium cryptate donor and acceptor fluorophore, and time-resolved (Tr) FRET measurements were performed as described in Supplemental Experimental Procedures. Fluorescently-labeled ghrelin (red-ghrelin from Cisbio) binding assays were performed on batch labeled cells with terbium-cryptate. Forty-eight hours after transfection with SNAP-tagged receptors, cells

were labeled with 100 nM of BG-TbK (Cisbio) substrate in 6-well plates by incubating for 1 hr at 37°C (95% air/5% CO2) in DMEM containing 0.5% FBS. Cells were then detached, collected by centrifugation (1,000 × g, 10 min) and washed three times with PBS. Pelleted cells were resuspended in Tris-KREBS buffer and seeded in 96-well plates (50 × 103 cells per well). Saturation binding, nonspecific binding, and completion binding experiments were performed as described in Supplemental Experimental Procedures. Mouse brains were dissected as above and crude membrane preparations

were isolated, labeled with biotin, and added to streptavidin plates for labeling with donor and acceptor fluorophores (see details secondly in Supplemental Experimental Procedures). Red-ghrelin (100 nM) and mouse anti-DRD2 antibody (1:100 dilutions, Santa Cruz Biotechnology) were added and the plates incubated overnight at 4°C in the dark. To measure the background signal, membranes were incubated with 100 nM of red-fluorophore and mouse anti-DRD2 antibody. Plates were washed twice with TMEP and incubated with terbium-kryptate labeled anti-mouse antibody (10 nM/well; Cisbio) for 2 hr at 4°C in the dark. After two final washes, 400 mM KF in TMEP was added to each well and Tr-FRET signal was measured using an EnVision plate reader as described above. Mouse brain sections (20 μm) were processed for FRET studies.