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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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Alignment of amino acid sequences of human GC-B with HNOBA domain of soluble GCs (A), and with membrane bound GCs and ADCYs (B)(A) GC-B (amino acids 799–846) was aligned with a partial fragment of the HNOBA domain of soluble GCs of various species. Asterisks show amino acid residues of GC-B having significant similarity to soluble GCs. Ho, Homo sapiens; Dr, Drosophila melanogaster; Xe, Xenopus laevis, Or, Oryzias latipes; Ra, Rattus norvegicus; Da, Danio rerio; Ca, Caenorhabditis elegans. (B) Partial amino acid sequence of human GC-B including Tyr808 was aligned with those of HNOBA domain of all membrane-bound GCs, ADCY2 and ADCY4, all of which have tyrosine residue at the position equivalent to Tyr808 of GC-B. The alignment was constructed using the NCBI BLASTP amino acid sequence homology search engine. Highlighted residues show Tyr808 of GC-B and its equivalent tyrosine residues in other cyclases. Italic shows Arg838 of GC-E, and mutation of which has been linked to autosomal dominant inherited cone degeneration and cone–rod degeneration.
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Figure 7: Alignment of amino acid sequences of human GC-B with HNOBA domain of soluble GCs (A), and with membrane bound GCs and ADCYs (B)(A) GC-B (amino acids 799–846) was aligned with a partial fragment of the HNOBA domain of soluble GCs of various species. Asterisks show amino acid residues of GC-B having significant similarity to soluble GCs. Ho, Homo sapiens; Dr, Drosophila melanogaster; Xe, Xenopus laevis, Or, Oryzias latipes; Ra, Rattus norvegicus; Da, Danio rerio; Ca, Caenorhabditis elegans. (B) Partial amino acid sequence of human GC-B including Tyr808 was aligned with those of HNOBA domain of all membrane-bound GCs, ADCY2 and ADCY4, all of which have tyrosine residue at the position equivalent to Tyr808 of GC-B. The alignment was constructed using the NCBI BLASTP amino acid sequence homology search engine. Highlighted residues show Tyr808 of GC-B and its equivalent tyrosine residues in other cyclases. Italic shows Arg838 of GC-E, and mutation of which has been linked to autosomal dominant inherited cone degeneration and cone–rod degeneration.

Mentions: Based on NCBI (National Center for Biotechnology Information) Conserved Domain Database [32], Tyr808 is located in a niche domain between the KHD and GC domains and thus would not be expected to affect the structure of these domains directly. Interestingly, the niche domain containing Tyr808 shows sequence similarity to a partial fragment of HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GCs and bacterial haem-binding protein kinases [33,34] (Figure 7A). HNOBAs consist of an N-terminal core subdomain having interspersed α-helices and β-strands, followed by an extended C-terminal α-helix, termed the helical linker region, which has the potential to form coiled-coils and, hence, may be involved in dimerization. Based on the sequence alignment (Figure 6A) and on secondary structure predictions of Iyer et al. [34], Tyr808 is located within the helical linker region. Mutations of Tyr808 may alter the dimerization state of GC-B and thereby control its catalytic activity, which fits our hypothesis shown in Figure 6.


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

Katafuchi T - Biosci. Rep. (2013)

Alignment of amino acid sequences of human GC-B with HNOBA domain of soluble GCs (A), and with membrane bound GCs and ADCYs (B)(A) GC-B (amino acids 799–846) was aligned with a partial fragment of the HNOBA domain of soluble GCs of various species. Asterisks show amino acid residues of GC-B having significant similarity to soluble GCs. Ho, Homo sapiens; Dr, Drosophila melanogaster; Xe, Xenopus laevis, Or, Oryzias latipes; Ra, Rattus norvegicus; Da, Danio rerio; Ca, Caenorhabditis elegans. (B) Partial amino acid sequence of human GC-B including Tyr808 was aligned with those of HNOBA domain of all membrane-bound GCs, ADCY2 and ADCY4, all of which have tyrosine residue at the position equivalent to Tyr808 of GC-B. The alignment was constructed using the NCBI BLASTP amino acid sequence homology search engine. Highlighted residues show Tyr808 of GC-B and its equivalent tyrosine residues in other cyclases. Italic shows Arg838 of GC-E, and mutation of which has been linked to autosomal dominant inherited cone degeneration and cone–rod degeneration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Alignment of amino acid sequences of human GC-B with HNOBA domain of soluble GCs (A), and with membrane bound GCs and ADCYs (B)(A) GC-B (amino acids 799–846) was aligned with a partial fragment of the HNOBA domain of soluble GCs of various species. Asterisks show amino acid residues of GC-B having significant similarity to soluble GCs. Ho, Homo sapiens; Dr, Drosophila melanogaster; Xe, Xenopus laevis, Or, Oryzias latipes; Ra, Rattus norvegicus; Da, Danio rerio; Ca, Caenorhabditis elegans. (B) Partial amino acid sequence of human GC-B including Tyr808 was aligned with those of HNOBA domain of all membrane-bound GCs, ADCY2 and ADCY4, all of which have tyrosine residue at the position equivalent to Tyr808 of GC-B. The alignment was constructed using the NCBI BLASTP amino acid sequence homology search engine. Highlighted residues show Tyr808 of GC-B and its equivalent tyrosine residues in other cyclases. Italic shows Arg838 of GC-E, and mutation of which has been linked to autosomal dominant inherited cone degeneration and cone–rod degeneration.
Mentions: Based on NCBI (National Center for Biotechnology Information) Conserved Domain Database [32], Tyr808 is located in a niche domain between the KHD and GC domains and thus would not be expected to affect the structure of these domains directly. Interestingly, the niche domain containing Tyr808 shows sequence similarity to a partial fragment of HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GCs and bacterial haem-binding protein kinases [33,34] (Figure 7A). HNOBAs consist of an N-terminal core subdomain having interspersed α-helices and β-strands, followed by an extended C-terminal α-helix, termed the helical linker region, which has the potential to form coiled-coils and, hence, may be involved in dimerization. Based on the sequence alignment (Figure 6A) and on secondary structure predictions of Iyer et al. [34], Tyr808 is located within the helical linker region. Mutations of Tyr808 may alter the dimerization state of GC-B and thereby control its catalytic activity, which fits our hypothesis shown in Figure 6.

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