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

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.Structured digital abstract•MINT-7713870, MINT-7713887: PMCA (uniprotkb:P20020) and GC-A (uniprotkb:P18910) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Atrial Natriuretic Peptide Locally Counteracts the Deleterious Effects of Cardiomyocyte Mineralocorticoid Receptor Activation

“…1,34,35 However, despite these high NP levels, HF is characterized by a combined deficiency of the active form of processed BNP 36 and resistance to both NPs. 7,14,16,18 Because of desensitization of the GC-A receptor, NP-dependent intracellular cGMP formation and subsequent cardiovascular effects are markedly blunted. [14][15][16] Of course the mice with genetic cardiomyocyte GC-A or cGKI ablation studied herein present an artificial situation of nonexistent myocyte NP signaling.…”

“…As previously described, 15,18 HEK 293 cells (Invitrogen, Karlsruhe, Germany) stably expressing GC-A, FLAG-tagged GC-A, or cyan fluorescent protein-tagged GC-A were transfected with plasmids encoding for enhanced green fluorescent protein-tagged MR and cGKI with Fugene (Roche, Mannheim, Germany). The cells were maintained in charcoal-stripped (steroid-reduced) medium.…”

“…For immunoprecipitation studies, the membrane and cytosolic proteins were extracted 48 hours after transfection and the membrane proteins were incubated with anti-FLAG antibody coupled to agarose beads (M2; Sigma, Taufkirchen, Germany) for 2 hours at 4°C under rotation as described before. 15,18 Aliquots of the cytosolic and membrane fractions and of the immunoprecipitated proteins were subjected to Western blotting with antibodies against MR (DSHB, Iowa), Hsp90 (BD Biosciences, Heidelberg, Germany), cGKI (Cell Signaling), and GC-A.…”

A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide

“…Next we investigated in HEK293 cells how ANP-induced homologous desensitization of the GC-A receptor affects the GC-A-TRPC complex and its modulation by ANP. For GC-A desensitization, transfected HEK cells were treated with ANP (100 nM) for 60 min (10)(11)(12). Western blot demonstrated unaltered membrane expression of GC-A under these conditions (Fig.…”

“…However, GC-A-mediated cGMP formation and endocrine effects of NPs are blunted, indicating desensitization of the receptor (1). NP-induced homologous desensitization of GC-A is due to posttranslational modifications, particularly to dephosphorylation within the KH domain (10,11). Internalization and degradation of GC-A seem to play no major role (12).…”