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Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern.

Schröter J, Zahedi RP, Hartmann M, Gassner B, Gazinski A, Waschke J, Sickmann A, Kuhn M - FEBS J. (2010)

Bottom Line: In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513.However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP.The functional relevance of this observation was analysed by site-directed mutagenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology, University of Würzburg, Würzburg, Germany.

ABSTRACT
Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3',5'-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.

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Related in: MedlinePlus

Fragment ion spectra of the phosphopeptides VRWEDLQPSpSLER and WEDLQPSpSLER, both representing the phosphorylated Ser487 of the GC-A receptor.
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fig04: Fragment ion spectra of the phosphopeptides VRWEDLQPSpSLER and WEDLQPSpSLER, both representing the phosphorylated Ser487 of the GC-A receptor.

Mentions: To enhance the sensitivity of our measurements, TiO2 affinity chromatography was used to enrich the putative phosphopeptides. Detection was performed by nano-LC-MS/MS. The combined results of two independent biological experiments (two purifications of GC-A, four enrichments by TiO2) are summarized in Table 1, including only phosphopeptides which were reproducibly detected and manually validated. Further potential phosphopeptides which did not pass this manual validation are not included. The spectra are shown in Fig. S2. As depicted in Table 1, all detected tryptic phosphopeptides were derived from the KH domain of GC-A, and all six phosphorylation sites previously deduced by Potter and Hunter [21,22] from experiments with metabolically labelled HEK293 cells were verified: Ser497, Thr500, Ser502, Ser506, Ser510, Thr513 (Fig. 3). In addition, one novel site of phosphorylation was identified at Ser487 within the KH domain (Table 1; Fig. 3). The mass spectra of the two tryptic peptides (478–490 and 480–490 of GC-A) containing this new phosphorylation site are shown in Fig. 4.


Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern.

Schröter J, Zahedi RP, Hartmann M, Gassner B, Gazinski A, Waschke J, Sickmann A, Kuhn M - FEBS J. (2010)

Fragment ion spectra of the phosphopeptides VRWEDLQPSpSLER and WEDLQPSpSLER, both representing the phosphorylated Ser487 of the GC-A receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2901513&req=5

fig04: Fragment ion spectra of the phosphopeptides VRWEDLQPSpSLER and WEDLQPSpSLER, both representing the phosphorylated Ser487 of the GC-A receptor.
Mentions: To enhance the sensitivity of our measurements, TiO2 affinity chromatography was used to enrich the putative phosphopeptides. Detection was performed by nano-LC-MS/MS. The combined results of two independent biological experiments (two purifications of GC-A, four enrichments by TiO2) are summarized in Table 1, including only phosphopeptides which were reproducibly detected and manually validated. Further potential phosphopeptides which did not pass this manual validation are not included. The spectra are shown in Fig. S2. As depicted in Table 1, all detected tryptic phosphopeptides were derived from the KH domain of GC-A, and all six phosphorylation sites previously deduced by Potter and Hunter [21,22] from experiments with metabolically labelled HEK293 cells were verified: Ser497, Thr500, Ser502, Ser506, Ser510, Thr513 (Fig. 3). In addition, one novel site of phosphorylation was identified at Ser487 within the KH domain (Table 1; Fig. 3). The mass spectra of the two tryptic peptides (478–490 and 480–490 of GC-A) containing this new phosphorylation site are shown in Fig. 4.

Bottom Line: In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513.However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP.The functional relevance of this observation was analysed by site-directed mutagenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology, University of Würzburg, Würzburg, Germany.

ABSTRACT
Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3',5'-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.

Show MeSH
Related in: MedlinePlus