TGF-α shedding assay

GPCR G_α activity was assessed by the TGF-α shedding assay as previously described (16). Briefly, HEK 293 cells lacking G_αq, G_α11, G_αs/olf, and G_α12/13 were transiently transfected with receptor, modified TGF-α–containing alkaline phosphatase (AP-TGF-α), and the indicated G_α subunit. Cells were reseeded 24 hours later in HBSS (Gibco, Gaithersburg, MD) supplemented with 5 mM Hepes in a Costar 96-well plate (Corning Inc., Corning, NY). Cells were then stimulated with the indicated concentration of ligand for 1hour. Conditioned medium (CM) containing the shed AP-TGF-α was transferred to a new 96-well plate. Both the cell and CM plates were treated with para-nitrophenylphosphate (p-NPP, 100 mM; Sigma-Aldrich, St. Louis, MO) substrate for 1 hour; p-NPP is converted to para-nitrophenol (p-NP) by AP-TGF-α. This activity was measured at an optical density at 405 nm (OD₄₀₅) in a Synergy Neo2 Hybrid Multi-Mode (BioTek, Winooski, VT) plate reader immediately after p-NPP addition and then after a 1-hour incubation. Gα activity was calculated by first determining p-NP amounts by absorbance using the following equation:100*(ΔOD405, CMΔOD405, CM+ΔOD405, cell)where ΔOD₄₀₅ = OD_{405, 1hr} – OD_{405, 0 hours} and ΔOD_{405, cell} and ΔOD_{405, CM} represent the changes in absorbance after1 hour in the cell and CM plates, respectively. Data were normalized to a single well that produced the maximal signal.