Fig. 1.
Galanin 3 receptor (GalR3) expresses poorly at the plasma membrane in recombinant systems. (A) Forty-eight hours after transient transfection with HA-GalR3 or HA-GalR1, HEK293 cells were fixed and permeabilized (or not) as described in Materials and Methods. Fixed cells were incubated with anti-HA primary antibody (rat) (Roche) and then with anti-rat Alexafluor 588 secondary antibody (Invitrogen) before imaging by confocal microscopy. Scale bar=10 μm. (B) HEK293-CNG parental cells (●) or HEK293-CNG cells stably transfected with Gal1R (▲) or Gal3R (▼) were plated at 14,000 cells per well in a 384-well FLIPR plate. After 18-h incubation at 37°C, 5% CO2, cells were loaded with ACT:One membrane potential dye for 2 h at room temperature. cAMP production in response to increasing concentrations of forskolin was determined according to ACT:One manufacturer's instructions and as described in detail in Table 1. Forskolin EC90s were extrapolated from the concentration response curves for each cell line. (C) Cells were plated and dye-loaded as in (B) before treatment with an EC90 of forskolin together with increasing concentrations of porcine galanin. cAMP levels were determined as in (B). The data presented are means±SEM of triplicate wells (n=3). (D) cAMP biosensor assay principle. The cAMP biosensor assay utilizes a stable cell line expressing a modified cyclic nucleotide gated channel (CNG) acting as a biosensor for cAMP. Changes in cAMP can be measured with a combination of membrane potential dyes and a FLIPR. Gi-coupled GPCRs (e.g., GalR3) promote exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) in Gi α-subunits. Activated Gi acts to inhibit cAMP production at the effector adenylate cyclase, thus Gi-coupled GPCR activity is determined by inhibition of forskolin-stimulated cAMP production. Phosphodiesterases (PDEs), which catalyze the conversion of cAMP into adenosine monophosphate (AMP), are inhibited to maximize the assay window. GPCR, G protein–coupled receptor.