TABLE 1

Ligand binding parameters of the wild-type and mutants of the β₁-AR

Site directed mutagenesis of Lys³²¹ and Lys³²⁴ to the amino acids indicated in the table was performed as described under Materials and Methods. [¹²⁵I]ICYP binding was determined as described under Materials and Methods on membranes derived from HEK-293 cells expressing the wild-type β₁-AR or the different β₁-AR mutants. Saturation binding experiments were analyzed by nonlinear regression to determine the equilibrium dissociation constant (K_D) of ICYP to each receptor and the receptor density in each membrane preparation. In HEK-293 cells, cell receptor densities for each β₁-AR construct were comparable and ranged from 850 to 1100 fmol of receptor/mg of protein. Competition binding isotherms were analyzed using the two-site competition algorithms in the Prism 4.0 program. When the binding data were best described by two affinity states, K_IH and K_IL indicate the dissociation constants for the high- and low-affinity state of the receptor, respectively. The results are the mean of four to six independent determinations each in triplicate.