Figure 3.

Glutamate but not alanine phosphorylation site substitutions yield hormonally responsive receptors. (A) Glutamate-substituted receptors are activated by ANP and ATP. Human 293 cells were transiently transfected with the indicated NPR-A expression constructs; 48 h later, crude membranes were prepared and assayed for guanylyl cyclase activity in the presence of Mg²⁺-GTP only (basal) or ANP, ATP, and Mg²⁺-GTP (ANP/ATP). (B) The hormone-dependent activities of the glutamate-substituted receptors are not explained by increased protein levels. Crude membranes were prepared as described above and assayed for guanylyl cyclase activity in the presence of Triton X-100 and Mn²⁺-GTP. (C) The glutamate substitutions are not equivalent to the wild-type receptor. The activity ratio of the wild-type and mutant receptors was determined by dividing the hormone-dependent activity by the detergent activity and multiplying this number by 100. The vertical bars centered above the columns represent the range of values obtained from four separate determinations, which were determined in two separate experiments.