A functional screen provides evidence for a conserved, regulatory, juxtamembrane phosphorylation site in guanylyl cyclase a and B

Abstract

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ~20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation--processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200-1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor (32)PO(4) co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.

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.

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.