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Identification of a haem domain in human soluble adenylate cyclase.

Middelhaufe S, Leipelt M, Levin LR, Buck J, Steegborn C - Biosci. Rep. (2012)

Bottom Line: The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer.Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide).Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, Bochum, Germany.

ABSTRACT
The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

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Tissue-specific expression of sAC-HD and effect of sAC-HD on the activity of the sAC catalytic domains(A) The agarose gel shows products of PCR with primer pairs specific for sAC-HD, the sAC-C2 domain and for cytochrome c as a control, respectively, applied to a panel of mouse tissue cDNA samples as templates. The sAC catalytic domain C2 could be detected in all tissues, but cDNA coding for sAC-HD was significantly amplified only from skeletal muscle and testis cDNA. (B) In a competitive cAMP formation assay (increasing activity leads to decreasing signals), addition of sAC-HD leads to a concentration-dependent increase of the activity of the catalytic domains. BSA addition served as a control. Adding NO together with the sAC-HD did not modulate the activating effect of sAC-HD. Data represent averages of duplicates and are shown as signals relative to control, and error bars represent relative percentage differences.
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Figure 5: Tissue-specific expression of sAC-HD and effect of sAC-HD on the activity of the sAC catalytic domains(A) The agarose gel shows products of PCR with primer pairs specific for sAC-HD, the sAC-C2 domain and for cytochrome c as a control, respectively, applied to a panel of mouse tissue cDNA samples as templates. The sAC catalytic domain C2 could be detected in all tissues, but cDNA coding for sAC-HD was significantly amplified only from skeletal muscle and testis cDNA. (B) In a competitive cAMP formation assay (increasing activity leads to decreasing signals), addition of sAC-HD leads to a concentration-dependent increase of the activity of the catalytic domains. BSA addition served as a control. Adding NO together with the sAC-HD did not modulate the activating effect of sAC-HD. Data represent averages of duplicates and are shown as signals relative to control, and error bars represent relative percentage differences.

Mentions: sAC expression has been postulated to be regulated via tissue-specific alternative splicing [34,35]. Expression of sACfl, which should contain sAC-HD, was demonstrated in testis by immunoprecipitation [36]. We used PCR analysis on a panel of mouse tissue cDNAs with primers for sAC-HD and sAC-C2 to confirm the presence of sAC-HD in testis and to identify other tissues harbouring HD-containing sAC isoforms (Figure 5A). Amplification of cytochrome c cDNA as a control for the PCR reaction yielded strong signals for all tissues. The sAC-C2 domain could also be amplified from cDNAs in all tissues tested, consistent with previous studies demonstrating the presence of active sAC isoforms in a wide variety of tissues [2,6,34,35]. In contrast, PCR analyses of sAC-HD yielded only a strong signal from testis cDNA and a weaker signal from skeletal muscle cDNA. Thus, while confirming that an sAC-HD-containing isoform is present in testis, these data suggest that sAC isoforms harbouring the HD are restricted to select somatic tissues.


Identification of a haem domain in human soluble adenylate cyclase.

Middelhaufe S, Leipelt M, Levin LR, Buck J, Steegborn C - Biosci. Rep. (2012)

Tissue-specific expression of sAC-HD and effect of sAC-HD on the activity of the sAC catalytic domains(A) The agarose gel shows products of PCR with primer pairs specific for sAC-HD, the sAC-C2 domain and for cytochrome c as a control, respectively, applied to a panel of mouse tissue cDNA samples as templates. The sAC catalytic domain C2 could be detected in all tissues, but cDNA coding for sAC-HD was significantly amplified only from skeletal muscle and testis cDNA. (B) In a competitive cAMP formation assay (increasing activity leads to decreasing signals), addition of sAC-HD leads to a concentration-dependent increase of the activity of the catalytic domains. BSA addition served as a control. Adding NO together with the sAC-HD did not modulate the activating effect of sAC-HD. Data represent averages of duplicates and are shown as signals relative to control, and error bars represent relative percentage differences.
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Related In: Results  -  Collection

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Figure 5: Tissue-specific expression of sAC-HD and effect of sAC-HD on the activity of the sAC catalytic domains(A) The agarose gel shows products of PCR with primer pairs specific for sAC-HD, the sAC-C2 domain and for cytochrome c as a control, respectively, applied to a panel of mouse tissue cDNA samples as templates. The sAC catalytic domain C2 could be detected in all tissues, but cDNA coding for sAC-HD was significantly amplified only from skeletal muscle and testis cDNA. (B) In a competitive cAMP formation assay (increasing activity leads to decreasing signals), addition of sAC-HD leads to a concentration-dependent increase of the activity of the catalytic domains. BSA addition served as a control. Adding NO together with the sAC-HD did not modulate the activating effect of sAC-HD. Data represent averages of duplicates and are shown as signals relative to control, and error bars represent relative percentage differences.
Mentions: sAC expression has been postulated to be regulated via tissue-specific alternative splicing [34,35]. Expression of sACfl, which should contain sAC-HD, was demonstrated in testis by immunoprecipitation [36]. We used PCR analysis on a panel of mouse tissue cDNAs with primers for sAC-HD and sAC-C2 to confirm the presence of sAC-HD in testis and to identify other tissues harbouring HD-containing sAC isoforms (Figure 5A). Amplification of cytochrome c cDNA as a control for the PCR reaction yielded strong signals for all tissues. The sAC-C2 domain could also be amplified from cDNAs in all tissues tested, consistent with previous studies demonstrating the presence of active sAC isoforms in a wide variety of tissues [2,6,34,35]. In contrast, PCR analyses of sAC-HD yielded only a strong signal from testis cDNA and a weaker signal from skeletal muscle cDNA. Thus, while confirming that an sAC-HD-containing isoform is present in testis, these data suggest that sAC isoforms harbouring the HD are restricted to select somatic tissues.

Bottom Line: The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer.Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide).Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, Bochum, Germany.

ABSTRACT
The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.

Show MeSH
Related in: MedlinePlus