[3H]-Hh-agonist kinetic, saturation and competition binding analysis with Smo-containing membranes. (a) The association (solid line) and dissociation (broken line) time courses for the binding of [3H]-Hh-Ag 1.5 to membranes from Smo-overexpressing 293T cells. The arrow denotes the time at which 2 μM unlabeled [3H]-Hh-Ag 1.5 was added to initiate dissociation studies. (b) The total (squares), nonspecific (triangles) and specific (circles) binding (in cpm) of [3H]-Hh-Ag 1.5 to membranes from Smo-overexpressing 293T cells. Total and specific binding data were derived in the absence and presence of 2 μM unlabeled Hh-Ag 1.5, respectively. The specific curve (red) represents the difference between these curves. Similar specific curves resulted when control membranes or a no-membrane control plate was used to define the nonspecific binding, or if 10 μM Cur61414 was used as the competitor. A dissociation binding constant (Kd) of 0.37 nM is predicted from this single site binding isotherm. (c) A competition assay of [3H]-Hh-Ag 1.5/Smo binding by a set of agonist derivatives including Hh-Ag 1.5, Hh-Ag 1.3, Hh-Ag 1.2, Hh-Ag 1.1, and an inactive Hh-agonist derivative. (d) A competition binding study showing the properties of the binding of KAAD-cyclopamine, Cur61414 and Hh-Ag 1.5 to wild-type Smo, Smowt, and a constitutively active Smo mutant protein, Smoact, which contains an activating W539L amino-acid substitution. Competition curves on Smowt are shown by broken lines and the competition curves on Smoact by solid lines. Standard deviations (n = 4) are represented by error bars for all data points.