Fig. 5. Point mutations in MsGC-ß3 demonstrate that MsGC-ß3/MsGC-1 and MsGC-ß3/MsGC-ß1 heterodimers are inactive. Two separate point mutations were generated that converted glutamate 469 to lysine (MsGC-ß3E469K) and arginine 537 to glutamine (MsGC-ß3R537Q). These two constructs were transfected into COS-7 cells as shown and cell extracts were assayed for guanylyl cyclase activity. (A) Each point mutation is inactive when transfected individually or in combination with either MsGC-1 or MsGC-ß1. When each is cotransfected with wild-type MsGC-ß3, the level of guanylyl cyclase activity measured was similar or greater than the level of activity measured when wild-type MsGC-ß3 was transfected alone. The results shown are the sum of six separate transfection experiments and because the absolute level of GC activity varied between experiments, all the data are expressed as % activity compared to the activity measured when wild-type MsGC-ß3 was transfected alone. Analysis of variance (ANOVA) showed that the activity measured when either mutant was transfected alone or in combination with MsGC-1 or MsGC-ß1 was not significantly different (P>0.05) from the activity measured in COS-7 cells transfected with vector alone. (B) Coexpression of the two mutants generates an active enzyme. MsGC-ß3E469K and MsGC-ß3R537Q were transiently coexpressed in COS-7 cells (5 µg of each plasmid) and the guanylyl cyclase activity measured and compared to COS-7 cells that had been transfected with 10 µg of wild-type MsGC-ß3. Values are means ± S.E.M. of three determinations. (C) Western blot of representative transfections showing that each mutant generates an equivalent level of MsGC-ß3 immunoreactivity. COS-7 cells were transfected with 10 µg MsGC-ß3, 5 µg MsGC-ß3 + 5 µg MsGC-ß3E469K or 5 µg MsGC-ß3 + 5 µg MsGC-ß3R537Q and cell extracts analyzed by western blot. Each sample was run on six separate lanes and the pixel density in each band quantified. A representative band is shown above each histogram. There was no significant difference (ANOVA; P>0.05) between the pixel density of MsGC-ß3 immunoreactivity for each transfection. (D) Each mutant acts as a dominant negative when coexpressed with the NO-sensitive guanylyl cyclase subunits. COS-7 cells were transiently transfected with the plasmids shown and assayed for guanylyl cyclase activity in the presence of 4 mmol l^-1 MgCl₂ ± 125 µmol l^-1 sodium nitroprusside (SNP). The activity levels were normalized for transfection efficiency and show that both mutants reduce the levels of basal and NO-stimulated guanylyl cyclase activity. Both the basal and NO-stimulated activity was significantly lower (ANOVA; P<0.01) when the mutants were contransfected than in their absence. Values are means ± S.E.M. of three determinations.

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