A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B

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 32PO4 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

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Prevention of guanylyl cyclase–B dephosphorylation rescues achondroplastic dwarfism

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) or inactivating mutations in guanylyl cyclase–B (GC-B), also known as NPR-B or Npr2, cause short-limbed dwarfism. FGFR3 activation causes dephosphorylation and inactivation of GC-B, but the contribution of GC-B dephosphorylation to achondroplasia (ACH) is unknown. GC-B7E/7E mice that express a glutamate-substituted version of GC-B that cannot be inactivated by dephosphorylation were bred with mice expressing FGFR3-G380R, the most common human ACH mutation, to determine if GC-B dephosphorylation is required for ACH. Crossing GC-B7E/7E mice with FGFR3G380R/G380R mice increased naso-anal and long (tibia and femur), but not cranial, bone length twice as much as crossing GC-B7E/7E mice with FGFR3WT/WT mice from 4 to 16 weeks of age. Consistent with increased GC-B activity rescuing ACH, long bones from the GC-B7E/7E/FGFR3G380R/G380R mice were not shorter than those from GC-BWT/WT/FGFR3WT/WT mice. At 2 weeks of age, male but not female FGFR3G380R/G380R mice had shorter long bones and smaller growth plate hypertrophic zones, whereas female but not male GC-B7E/7E mice had longer bones and larger hypertrophic zones. In 2-week-old males, crossing FGFR3G380R/G380R mice with GC-B7E/7E mice increased long bone length and hypertrophic zone area to levels observed in mice expressing WT versions of both receptors. We conclude that preventing GC-B dephosphorylation rescues reduced axial and appendicular skeleton growth in a mouse model of achondroplasia

Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes.

In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20min, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2h, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte

GC-B-6E-489E is resistant to inhibition by hyperosmotic medium and protein kinase C.

<p>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.</p

Mutation of Ser-489 in GC-B to alanine but not glutamate decreases CNP-stimulated guanylyl cyclase activity.

<p>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.</p

Glutamate substitution for serine 473 enhances homologous desensitization of GC-A.

<p>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.</p

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.

*<p>Indicates alanine substitution reduced guanylyl cyclase activity but receptor was determined to be incompletely glycosylated and phosphorylated.</p

Mutation of Ser-473 in GC-A to alanine but not glutamate decreases ANP-stimulated guanylyl cyclase activity.

<p>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.</p