Radioreceptor-binding studies of wild-type (square symbol) and E54K (round symbol) mutated receptors using [¹²⁵I] Hcrt-1. HEK-293 cells transfected with wild-type and mutated Hcrtr2 constructs with and without GFP were used (for technical details, see text). Note that wild -type receptors express saturable binding with an approximate Kd of 0.9 nM, while E54K does not bind the ligand at concentrations up to 4 nM. Data obtained with other mutated constructs are not shown, but the cells do not display any specific binding.