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Mutations in Tyr808 reveal a potential auto-inhibitory mechanism of guanylate cyclase-B regulation.

Katafuchi T - Biosci. Rep. (2013)

Bottom Line: CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B.Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases.This finding provides new insight into the activation mechanism of GCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, TX, USA. Takeshi.Katafuchi@UTSouthwestern.edu

ABSTRACT
In this study, Tyr808 in GC-B (guanylate cyclase-B), a receptor of the CNP (C-type natriuretic peptide), has been shown to be a critical regulator of GC-B activity. In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr808 markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na3VO4 and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na3VO4-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.

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Dose–response elevation of cGMP production by WT Myc-GC-B and Tyr808 mutantsHeLa cells expressing WT Myc-GC-B (A) or Tyr808 mutants, such as Y808F (B), Y808A (C), Y808E (D), Y808S (E) and Y808W (F) were stimulated with indicated concentrations of CNP for 5 min, and cGMP production was measured. Data represent means±S.E.M., n=3. (G) Levels of Myc-tagged WT GC-B and its Tyr808 mutants (GC-B), P-VASP (phosphorylated VASP), t-VASP (total VASP) and βAct (β-Actin) in HeLa cells determined by immunoblotting.
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Figure 3: Dose–response elevation of cGMP production by WT Myc-GC-B and Tyr808 mutantsHeLa cells expressing WT Myc-GC-B (A) or Tyr808 mutants, such as Y808F (B), Y808A (C), Y808E (D), Y808S (E) and Y808W (F) were stimulated with indicated concentrations of CNP for 5 min, and cGMP production was measured. Data represent means±S.E.M., n=3. (G) Levels of Myc-tagged WT GC-B and its Tyr808 mutants (GC-B), P-VASP (phosphorylated VASP), t-VASP (total VASP) and βAct (β-Actin) in HeLa cells determined by immunoblotting.

Mentions: Although my mutational analysis indicated that S1P-mediated suppression of GC-B activity cannot be explained by a change in phosphorylation of any of the candidate phosphorylation sites (at least individually), I found that cells expressing the Y808F mutant produced more than 30-fold higher levels of cGMP than cells expressing WT GC-B upon stimulation with 0.1 μM CNP (Figure 2B), despite lower levels of expression of the mutant cyclase (Figure 2C). Phosphorylation of Tyr808 did not contribute to this effect, as neither WT GC-B nor the Y808F mutant was recognized by the 4G10 anti-phosphotyrosine antibody (not shown). To further analyse the significance of Tyr808 for GC-B activity, I substituted this residue with amino acids having different chemical characteristics. The effect of residue volume was examined by substituting Tyr808 with smaller (alanine) or larger (tryptophan) residues (residue volumes of alanine, phenylalanine, tyrosine and tryptophan are 67, 135, 141 and 186 Å3, respectively [29]). The importance of hydrophilicity was examined by replacing Tyr808 with glutamic acid and serine. I expressed these mutants in HeLa cells to measure CNP-dependent cGMP production. Like the Y808F mutant, the Y808A, Y808E and Y808S mutants exhibited markedly higher GC activity than did WT GC-B (Figure 3). Upon stimulation with 1 μM CNP, cells expressing the Y808A, Y808E and Y808S mutants produced more than 70- (750 pmol/mg protein), 8- (92.9 pmol/mg protein) and 16-fold (178.9 pmol/mg protein) higher levels of cGMP than WT (10.7 pmol/mg protein), respectively (Figures 3A, 3C, 3D and 3E). Moreover, these three mutants were constitutively active, generating cGMP in cells 370-fold (78.3 pmol/mg protein, Y808A), 270-fold (56.8 pmol/mg protein, Y808E) and 370-fold (78.1 pmol/mg protein, Y808S) higher than WT (0.2 pmol/mg protein) (Figures 3A, 3C, 3D and 3E), even without CNP stimulation. Interestingly, the level of VASP (phosphorylated vasodilator-stimulated phosphoprotein), a well-characterized substrate of cGMP-dependent kinase [30], is higher in the cells expressing Y808A, Y808E and Y808S mutants than in cells expressing WT-GC-B even without CNP stimulation (Figure 3G). These data indicate that the cGMP production is elevated in intact cells expressing the mutants. Despite the enhancement of catalytic potential by these Tyr808 mutations, the EC50s of WT and mutant GC-Bs were similar, indicating that the mutations did not alter ligand–receptor interactions. The EC50 of the single mutant that displayed lower than WT activity, Y808W, was also unchanged. In summary, GC-B mutants containing smaller and more hydrophilic residues than Tyr808 expressed remarkably higher GC activity than WT.


Mutations in Tyr808 reveal a potential auto-inhibitory mechanism of guanylate cyclase-B regulation.

Katafuchi T - Biosci. Rep. (2013)

Dose–response elevation of cGMP production by WT Myc-GC-B and Tyr808 mutantsHeLa cells expressing WT Myc-GC-B (A) or Tyr808 mutants, such as Y808F (B), Y808A (C), Y808E (D), Y808S (E) and Y808W (F) were stimulated with indicated concentrations of CNP for 5 min, and cGMP production was measured. Data represent means±S.E.M., n=3. (G) Levels of Myc-tagged WT GC-B and its Tyr808 mutants (GC-B), P-VASP (phosphorylated VASP), t-VASP (total VASP) and βAct (β-Actin) in HeLa cells determined by immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3673034&req=5

Figure 3: Dose–response elevation of cGMP production by WT Myc-GC-B and Tyr808 mutantsHeLa cells expressing WT Myc-GC-B (A) or Tyr808 mutants, such as Y808F (B), Y808A (C), Y808E (D), Y808S (E) and Y808W (F) were stimulated with indicated concentrations of CNP for 5 min, and cGMP production was measured. Data represent means±S.E.M., n=3. (G) Levels of Myc-tagged WT GC-B and its Tyr808 mutants (GC-B), P-VASP (phosphorylated VASP), t-VASP (total VASP) and βAct (β-Actin) in HeLa cells determined by immunoblotting.
Mentions: Although my mutational analysis indicated that S1P-mediated suppression of GC-B activity cannot be explained by a change in phosphorylation of any of the candidate phosphorylation sites (at least individually), I found that cells expressing the Y808F mutant produced more than 30-fold higher levels of cGMP than cells expressing WT GC-B upon stimulation with 0.1 μM CNP (Figure 2B), despite lower levels of expression of the mutant cyclase (Figure 2C). Phosphorylation of Tyr808 did not contribute to this effect, as neither WT GC-B nor the Y808F mutant was recognized by the 4G10 anti-phosphotyrosine antibody (not shown). To further analyse the significance of Tyr808 for GC-B activity, I substituted this residue with amino acids having different chemical characteristics. The effect of residue volume was examined by substituting Tyr808 with smaller (alanine) or larger (tryptophan) residues (residue volumes of alanine, phenylalanine, tyrosine and tryptophan are 67, 135, 141 and 186 Å3, respectively [29]). The importance of hydrophilicity was examined by replacing Tyr808 with glutamic acid and serine. I expressed these mutants in HeLa cells to measure CNP-dependent cGMP production. Like the Y808F mutant, the Y808A, Y808E and Y808S mutants exhibited markedly higher GC activity than did WT GC-B (Figure 3). Upon stimulation with 1 μM CNP, cells expressing the Y808A, Y808E and Y808S mutants produced more than 70- (750 pmol/mg protein), 8- (92.9 pmol/mg protein) and 16-fold (178.9 pmol/mg protein) higher levels of cGMP than WT (10.7 pmol/mg protein), respectively (Figures 3A, 3C, 3D and 3E). Moreover, these three mutants were constitutively active, generating cGMP in cells 370-fold (78.3 pmol/mg protein, Y808A), 270-fold (56.8 pmol/mg protein, Y808E) and 370-fold (78.1 pmol/mg protein, Y808S) higher than WT (0.2 pmol/mg protein) (Figures 3A, 3C, 3D and 3E), even without CNP stimulation. Interestingly, the level of VASP (phosphorylated vasodilator-stimulated phosphoprotein), a well-characterized substrate of cGMP-dependent kinase [30], is higher in the cells expressing Y808A, Y808E and Y808S mutants than in cells expressing WT-GC-B even without CNP stimulation (Figure 3G). These data indicate that the cGMP production is elevated in intact cells expressing the mutants. Despite the enhancement of catalytic potential by these Tyr808 mutations, the EC50s of WT and mutant GC-Bs were similar, indicating that the mutations did not alter ligand–receptor interactions. The EC50 of the single mutant that displayed lower than WT activity, Y808W, was also unchanged. In summary, GC-B mutants containing smaller and more hydrophilic residues than Tyr808 expressed remarkably higher GC activity than WT.

Bottom Line: CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B.Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases.This finding provides new insight into the activation mechanism of GCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, TX, USA. Takeshi.Katafuchi@UTSouthwestern.edu

ABSTRACT
In this study, Tyr808 in GC-B (guanylate cyclase-B), a receptor of the CNP (C-type natriuretic peptide), has been shown to be a critical regulator of GC-B activity. In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr808 markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na3VO4 and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na3VO4-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.

Show MeSH
Related in: MedlinePlus