Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern.
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.
Schröter
Juliane
J
Institute of Physiology, University of Würzburg, Würzburg, Germany.
Zahedi
René P
RP
Hartmann
Michael
M
Gassner
Birgit
B
Gazinski
Alexandra
A
Waschke
Jens
J
Sickmann
Albert
A
Kuhn
Michaela
M
D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000097:blood; D006332:Cardiomegaly / Q000097:blood; D020134:Catalytic Domain; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000378:metabolism*; D006333:Heart Failure / Q000097:blood; D006801:Humans; D007668:Kidney / Q000196:embryology; D020097:Natriuretic Peptide, Brain / Q000097:blood / Q000494:pharmacology; D009842:Oligopeptides; D010455:Peptides / Q000502:physiology; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*; D015290:Second Messenger Systems / Q000502:physiology
D009842:Oligopeptides; D010455:Peptides; D010748:Phosphopeptides; D020097:Natriuretic Peptide, Brain; D009320:Atrial Natriuretic Factor; C085706:FLAG peptide; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; D006152:Cyclic GMP
Am J Physiol Cell Physiol. 2008 Jan;294(1):C324-32
17977944
Methods. 1999 Dec;19(4):506-20
10581150
Nat Methods. 2006 Jan;3(1):23-5
16369548
Cell. 2009 Oct 16;139(2):428-39
19837041
J Biol Chem. 2008 Oct 17;283(42):28313-20
18713751
Mol Syst Biol. 2008;4:222
18854821
Proteomics. 2009 Jul;9(13):3463-73
19579229
Circulation. 2005 Oct 11;112(15):2307-17
16216978
FEBS J. 2009 Mar;276(5):1347-55
19187227
Nature. 1996 Feb 1;379(6564):466-9
8559255
Biochemistry. 1999 Dec 21;38(51):16941-51
10606529
J Clin Invest. 2003 May;111(9):1399-407
12727932
Mol Pharmacol. 2008 Feb;73(2):431-40
17965196
Biochimie. 2009 Jul;91(7):888-93
19393286
J Biol Chem. 1992 Jul 25;267(21):14531-4
1353076
J Biol Chem. 2004 Jul 2;279(27):28625-31
15117952
Mol Cell Biol. 1998 Apr;18(4):2164-72
9528788
Nature. 1995 Nov 2;378(6552):65-8
7477288
Can J Physiol Pharmacol. 2006 May;84(5):539-46
16902599
Annu Rev Neurosci. 1992;15:193-225
1349465
FASEB J. 2001 Jul;15(9):1562-8
11427488
Mol Pharmacol. 2005 Jan;67(1):174-83
15459247
Endocrinology. 2006 Jun;147(6):2974-85
16527839
Oncogene. 1999 Jun 24;18(25):3788-92
10391688
Circ Res. 2003 Oct 17;93(8):700-9
14563709
Nature. 1989 Mar 2;338(6210):78-83
2563900
J Biol Chem. 2001 Mar 2;276(9):6057-60
11152699
Science. 1989 Sep 22;245(4924):1392-4
2571188
Nat Genet. 2009 Jun;41(6):666-76
19430483
Science. 1995 Feb 3;267(5198):679-81
7839143
Front Biosci. 2005 May 01;10:1205-20
15769619
J Biol Chem. 2002 May 3;277(18):16041-7
11821394
J Proteome Res. 2008 Feb;7(2):526-34
18088087