Figure 1.
Comparison of the N-terminal region of the kinase homology domains of transmembrane guanylyl cyclase receptors.
Purple RK indicates the beginning of intracellular domains. Red residues are confirmed phosphorylation sites. The green residue is a putative conserved phosphorylation site, which is Ser-489 in GC-B and Ser-473 in GC-A. Sequences were aligned with CLUSTAL version 2.0.1. Abbreviations are: rGC-A, rat GC-A; hGC-A, human GC-A; rGC-B, rat GC-B; hGC-B, human GC-B; hGC-C, human GC-C; A. punGC, GC from sea urchin species A. punctalata; S. purGC, GC from sea urchin species S. purpuratus.
Table 1.
The effect of alanine substitutions for known or potential GC-B phosphorylation sites on guanylyl cyclase activity and indication of conservation of sites in GC-A and sea urchin guanylyl cyclases is shown.
Figure 2.
Mutation of Ser-489 in GC-B to alanine but not glutamate decreases CNP-stimulated guanylyl cyclase activity.
293T cells were transiently transfected with WT-GC-B containing alanine or glutamate substitutions for Ser-489 (left side) or with the same substitutions engineered into GC-B-6E (right side). GC assays were conducted for 5 min in the presence of 1 µM CNP, 1 mM ATP and 1 mM magnesium-GTP or 1% Triton X-100 and 5 mM manganese-GTP. GC activity was expressed as CNP-dependent activity/Triton X-100-dependent activity ×100. The results are the mean ± SEM, where n = 8.
Figure 3.
Mutation of Ser-473 in GC-A to alanine but not glutamate decreases ANP-stimulated guanylyl cyclase activity.
293T cells were transiently transfected with WT-GC-A containing alanine or glutamate substitutions for Ser-473 (left side) or with the same substitutions engineered into GC-A-6E (right side). GC assays were conducted in the presence of 1 µM ANP, 1 mM ATP and 5 mM magnesium-GTP or 1% Triton X-100 and 5 mM manganese-GTP. GC activity was expressed as the ANP-dependent activity/Triton X-100-dependent activity ×100. The results are the mean ± SEM, where n = 14.
Figure 4.
Mutation of Val-472 in GC-A to glutamate or alanine had no effect on ANP-stimulated guanylyl cyclase activity.
GC assays were conducted on membrane preparations from 293 cells transiently expressing WT or mutants of rat GC-A as described in figure 2. The results are expressed as the mean ± SEM, where n = 12.
Figure 5.
Alanine substitutions at Ser-489 in GC-B increase the Michaelis constant.
293 cells were transiently transfected with the indicated form of GC-B and assayed for GC activity in the presence of 1 µM CNP, 1 mM ATP, and increasing concentrations of GTP. (A) Comparison between WT-GC-B, WT-GC-B-S489E, GC-B-6E and GC-B-6E-S489E. (B) Comparison between WT-GC-B-S489E and WT-GC-B-S489A. (C) Comparison between GC-B-6E and GC-B-6E-S489A. Vmax and Km values were calculated using nonlinear regression. * Indicates significantly different from WT-GC-B (A) or WT-GC-B-S489E (B) at p<0.02.
Figure 6.
Glutamate substitution for serine 473 enhances homologous desensitization of GC-A.
293 cells transiently expressing WT-GC-A or the indicated mutant forms of GC-A were incubated ±1 µM ANP for 1 hour at 37°C. Membranes were prepared and assayed for GC activity in the presence of ANP, ATP and magnesium-GTP or 1% Triton X-100 and manganese-GTP. The results are expressed as a ratio of hormone-stimulated/detergent-stimulated activity and were normalized to the percent of the activity ratio determined in membranes from cells not exposed to ANP. Data are means determined from multiple experiments ± SEM, where n≥6. * Indicates significantly different from control (GC-A or GC-A-6E) at p<0.01.
Figure 7.
GC-B-6E-489E is resistant to inhibition by hyperosmotic medium and protein kinase C.
293 cells transiently expressing WT-GC-B or GCB-6E-S489E were incubated ±0.1 M NaCl, 0.2 M NaCl or 1 µM PMA for 30 min at 37°C. Membranes were prepared and assayed for GC activity in the presence of CNP, ATP and magnesium-GTP or 1% Triton X-100 and manganese-GTP. The results were expressed as a ratio of hormone-stimulated/detergent-stimulated activity and were normalized to the activity ratio determined in membranes from control cells not exposed to any inhibitory agent. Data are presented as means ± SEM, where n = 4. ** Indicates a p value of <0.01, *** indicates a p value of <0.0001.
Figure 8.
The GC-A tryptic peptide phosphorylated at Ser-473 is poorly detected by mass spectrometry.
One pmol of synthetic peptides corresponding to tryptic peptides containing phosphorylated versions of Ser-473, Ser-487, or Ser-985 in GC-A were mixed and submitted to nLC-MS-MS. The resulting ion signal intensities are represented above by their individual m/z ion traces on a standard intensity scale. The inset shown for the phosphor-Ser-473 peptide has an expanded y-axis scale.