FIGURE 4.

Guanylyl cyclase and ligand-binding properties of wild type and spliced GC-A. HEK 293 cells were transiently transfected with each GC-A isoform. A, cells were incubated with vehicle or various ANP concentrations for 10 min, and intracellular cGMP contents were then measured by radioimmunoassay (n = 4). Inset in A, Western blot analysis showing similar expression levels of wild type or spliced GC-A. B, FRET was used to monitor in real time the kinetics and extent of cGMP formation in single HEK 293 cells cotransfected with GC-A (wild type or splice form) and the cGMP indicator pGES-DE2 (17). Top, ratiometric FRET images of cells prior to and 5 min after stimulation with ANP. Bottom, representative ratiometric recordings of single cell FRET signals. C, HEK 293 cells were incubated with ¹²⁵I-ANP in the presence of various concentrations of unlabeled ANP in serum-free DMEM at 37 °C for 20 min, and ¹²⁵I-ANP bound was quantitated with a γ-counter. Binding to wild type GC-A in the absence of unlabeled ANP was assigned a value of 100%. Specific activity of ¹²⁵I-ANP was 904 Ci/mmol (n = 4).