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.

Fig. 1

The N-terminal FLAG epitope does not alter the activity and subcellular localization of the GC-A receptor. (A) HEK293 cells expressing either the wild-type (wt) GC-A or FLAG-tagged GC-A receptor were incubated with ANP (10 pm to 100 nm, 10 min). Intracellular cGMP contents were quantified by RIA. Inset in (A): western blot analysis demonstrated similar expression levels of wt and FLAG-tagged GC-A. (B) FRET was used to monitor the kinetics and extent of cGMP formation in single HEK293 cells cotransfected with either wt GC-A or FLAG-tagged GC-A and cGMP indicator (pGES-DE2 [24]). Left: FRET images of two cells prior to and during incubation with ANP: wt GC-A with vehicle (a) and 10 nm ANP (b); FLAG-tagged GC-A with vehicle (c) and ANP (d). Right: representative ratiometric recordings of single-cell FRET signals. (C) Confocal immunofluorescence images of HEK293 cells transfected with wt or FLAG-tagged GC-A demonstrate the colocalization with PMCA.

Fig. 2

Enrichment and purification of the FLAG-tagged GC-A receptor from stably expressing HEK293 cells. (A) Cell fractionation and western blot analyses demonstrated that FLAG-tagged GC-A is predominantly localized in the plasma membrane of HEK293 cells. ERK1/2 and PMCA were used as markers for the cytosolic and membrane fractions, respectively. GC-A was detected with anti-GC-A serum and anti-FLAG IgG. (B) Western blot analysis demonstrated that immunoprecipitation of FLAG-tagged GC-A from the cell membrane fraction led to a 13-fold enrichment of the protein (2 μg protein per lane). (C) The silver-stained gel illustrates the step-wise purification of the receptor (10 μg protein per lane). (D) The immunoprecipitated GC-A protein was separated by SDS/PAGE. After Coomassie staining, the protein band at 130 kDa was excised and subjected to in-gel digestion with trypsin.

Fig. 4

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

Fig. 3

Scheme illustrating the domains of GC-A and the positions of the phosphorylated amino acids (in bold). The numbers within the sequence depict the positions of these amino acids within mature rat GC-A [7].

Fig. 6

The impact of the phosphorylation of the GC-A receptor at Ser487 on the responsiveness and homologous desensitization of the receptor was characterized by site-directed mutagenesis followed by guanylyl cyclase activity assays. Crude membranes prepared from HEK293 cells expressing wild-type (wt) GC-A or GC-A S487E were incubated with vehicle, ANP or detergent (Triton X-100). cGMP production was measured by RIA [fmol cGMP·(μg protein)⁻¹·min⁻¹]. All values were calculated as X-fold of the maximal (Triton X-100-induced) activity (means ± SEM). The western blots shown in the insets demonstrate similar expression levels of wt and mutated GC-A (all 10 μg protein per lane). (A) ANP evoked concentration-dependent increases in GC-A activity. The GC-A S487E mutant showed significantly reduced responsiveness to ANP (n = 9 from three independent experiments). (B) HEK293 cells were pretreated with ANP (100 nm, 1 h) or vehicle before the preparation of cell membranes. ANP pretreatment decreased significantly the cGMP response of wt GC-A to subsequent stimulation with 10 nm ANP, indicating homologous desensitization. The GC-A S487E mutant showed a significantly diminished cGMP response to 10 nm ANP, which was not further inhibited by ANP pretreatment (n = 6 from three independent experiments).

Fig. 5

Multiple reaction monitoring was used for semiquantitative analysis of the tryptic GC-A phosphopeptides obtained from ANP- relative to vehicle-treated (control) FLAG-tagged GC-A-expressing HEK293 cells. Four transitions obtained from MS/MS spectra of the phosphopeptide WEDLQPSpSLER were chosen to analyse the peptide content in ANP-treated relative to untreated samples. Peak areas, which are depicted under the spectra, showed an approximately nine-fold increase in ANP-treated versus untreated samples, indicating an increase in peptide amount (sum of peak areas without pretreatment, 352 000; sum of peak areas after ANP pretreatment, 3 140 000). (A) Transition 720.3/486.3. (B) Transition 720.3/670.4. (C) Transition 720.3/768.4. (D) Transition 720.3/288.1.