Mass spectrometric identification of phosphorylation sites in guanylyl cyclase A and B.
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.
Yoder
Andrea R
AR
Department of Pharmacology, University of Minnesota, Minneapolis,Minnesota 55455, United States.
Stone
Matthew D
MD
Griffin
Timothy J
TJ
Potter
Lincoln R
LR
D000595:Amino Acid Sequence; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D000099242:Atrial Natriuretic Factor Receptor B
D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B; C091715:atrial natriuretic factor receptor A