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MsGC-ß3 forms active homodimers and inactive heterodimers with NO-sensitive soluble guanylyl cyclase subunits

David B. Morton^* and Esther J. Anderson

Department of Biological Structure and Function, Oregon Health and Science University, Portland, Oregon, USA

View larger version (34K): [in a new window]   Fig. 1. Solubility of native MsGC-ß3. Abdominal nerve cords from pre-pupal Manduca sexta were homogenized in 50 mmoll-1 Tris-HCl, pH 7.5 and centrifuged at 100 000 g for 1 h at 4°C. The pellet was resuspended in 50 mmoll-1 Tris-HCl, pH 7.5 and the proteins in both the supernatant (S) and pellet (P) analyzed by western blot using an affinity-purified anti-MsGC-ß3 antiserum (Nighorn et al., 1999). Molecular mass markers on the left are shown in kDa.

View larger version (20K): [in a new window]   Fig. 2. Gel filtration of recombinant and native MsGC-ß3. Supernatants from extracts of COS-7 cells that had been transiently transfected with either MsGC-ß3 (solid line), MsGC-ß3C338 (broken line) or nerve cords (CNS; dotted line) were homogenized in 50 mmoll-1 Tris HCl, pH 7.5 and separated by gel filtration in the presence of 0.2% octyl ß,D-thioglucopyranoside. Fractions from COS-7 cell extracts were assayed for guanylyl cyclase activity and fractions from nerve cord homogenates were assayed by western blot. Molecular mass estimates were generated by comparing with standard proteins run in parallel.

View larger version (31K): [in a new window]   Fig. 3. Heterodimerization of MsGC-ß3 with MsGC-1 and MsGC-ß1. Extracts of COS-7 cells that were transiently cotransfected with MsGC-ß3 and either empty vector (pcDNA3.1), or hexa-histidine (His6)-tagged MsGC-ß3C338, MsGC-1 or MsGC-ß1, were incubated with nickel-chelated agarose and the bound proteins analyzed by western blot. (A) Blot probed with MsGC-ß3 antiserum shows that MsGC-ß3 is pelleted when cotransfected with either tagged MsGC-ß3C338, tagged MsGC-1 or tagged MsGC-ß1, but the intensity of the immunoreactivity is highest when cotransfected with tagged MsGC-ß3C338. (B) Western blot of input extract stained with anti-MsGC-ß3.

View larger version (26K): [in a new window]   Fig. 4. Alignment of regions of the catalytic domain of selected guanylyl cyclases showing residues predicted to contact Mg-GTP. Homology modeling has been used to predict the residues (shaded) of soluble and receptor guanylyl cyclases that bind Mg-GTP (Liu et al., 1997). Sequences shown are the rat soluble guanylyl cyclase subunits 1, ß1 and ß2, the Manduca soluble subunits MsGC-1, MsGC-ß1 and MsGC-ß3, and the rat receptor guanylyl cyclase, GC-A. Light shading shows the residues from the chain and dark shading those from the ß chain. Note that the homodimeric guanylyl cyclases, rat ß2, GC-A and MsGC-ß3, possess all the residues from both and ß chains. *Residues mutated in MsGC-ß3E469K and MsGC-ß3R573Q.

View larger version (34K): [in a new window]   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.

View larger version (22K): [in a new window]   Fig. 6. Enzymatic properties of MsGC-ß3 compared to MsGC-ß3C338. COS-7 cells were transiently transfected with the plasmids indicated and assayed for guanylyl cyclase activity with GTP concentrations of 0.1 mmol l-1 to 5 mmol l-1 in the presence of 4 mmol l-1 MgCl2 (A,B) or 4 mmol l-1 MnCl2 (C,D). For each concentration the reaction was allowed to proceed for 5, 15 and 30 min and linear regression of these values used to calculate a rate of reaction. The values were also plotted as double reciprocal plots (B,D), which demonstrate that both MsGC-ß3 and MsGC-ß3C338 yield Hill coefficients of 1.0. Values for the Vmax and Km were calculated from the untransformed data using the following equation: Rate=(Vmaxx[GTP])/(Km+[GTP]) using GraphPad Prism 3.0 and are shown in Table 3.