Mass spectrometric identification of phosphorylation sites in guanylyl cyclase A and B.

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  • Author(s): Yoder AR;Yoder AR; Stone MD; Griffin TJ; Potter LR
  • Source:
    Biochemistry [Biochemistry] 2010 Nov 30; Vol. 49 (47), pp. 10137-45. Date of Electronic Publication: 2010 Nov 08.
  • Publication Type:
    Journal Article; Research Support, N.I.H., Extramural
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370623 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-4995 (Electronic) Linking ISSN: 00062960 NLM ISO Abbreviation: Biochemistry Subsets: MEDLINE
    • Publication Information:
      Original Publication: Washington, American Chemical Society.
    • Subject Terms:
      Receptors, Atrial Natriuretic Factor/*metabolism; Amino Acid Sequence ; Animals ; HEK293 Cells ; Humans ; Phosphorylation ; Rats ; Receptors, Atrial Natriuretic Factor/chemistry ; Tandem Mass Spectrometry
    • Abstract:
      Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides. Some sites of phosphorylation are known for rat GC-A and GC-B, but no phosphorylation site information is available for the human homologues. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both species. Tryptic digests of receptors purified from HEK293 cells were separated and analyzed by nLC-MS-MS. Seven sites of phosphorylation were identified in rat GC-A (S497, T500, S502, S506, S510, T513, and S487), and all of these sites except S510 and T513 were observed in human GC-A. Six phosphorylation sites were identified in rat GC-B (S513, T516, S518, S523, S526, and T529), and all six sites were also identified in human GC-B. Five sites are identical between GC-A and GC-B. S487 in GC-A and T529 in GC-B are novel, uncharacterized sites. Substitution of alanine for S487 did not affect initial ligand-dependent GC-A activity, but a glutamate substitution reduced activity 20%. Similar levels of ANP-dependent desensitization were observed for the wild-type, S487A, and S487E forms of GC-A. Substitution of glutamate or alanine for T529 increased or decreased ligand-dependent cyclase activity of GC-B, respectively, and T529E increased cyclase activity in a GC-B mutant containing glutamates for all five previously identified sites as well. In conclusion, we identified and characterized new phosphorylation sites in GC-A and GC-B and provide the first evidence of phosphorylation sites within human guanylyl cyclases.
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    • Grant Information:
      R21 HL093402 United States HL NHLBI NIH HHS; R21HL0934402 United States HL NHLBI NIH HHS
    • Accession Number:
      EC 4.6.1.2 (Receptors, Atrial Natriuretic Factor)
      EC 4.6.1.2 (atrial natriuretic factor receptor A)
      EC 4.6.1.2 (atrial natriuretic factor receptor B)
    • Publication Date:
      Date Created: 20101028 Date Completed: 20101230 Latest Revision: 20211020
    • Publication Date:
      20240829
    • Accession Number:
      PMC4495887
    • Accession Number:
      10.1021/bi101700e
    • Accession Number:
      20977274