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

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.

Figure 1. Tandem mass spectrometric spectra depicting phosphorylated residues in rat GC-A and human GC-A

Receptors were purified and digested with trypsin prior to analysis by tandem mass spectrometry. Spectra for each phosphopeptide are color coded for matched b- and y-ions in red and blue, respectively. Other matches to the theoretical fragmentation are shown in green. For clarity, only selected species are labeled within the spectra. The primary amino acid sequence for each phosphorylated peptide is denoted on each spectrum and all b- and y- ions matched from the spectrum are marked. Lowercase letters directly preceding amino acids indicate modifications (p = phosphorylated; m = methylated; o = oxidized). Precursor ion m/z value and charge state are listed for the spectrum. SEQUEST discriminant score (XCorr) and mass error (ΔM) values reported for the spectrum were determined by the Proteome Discoverer software. Asterisks indicate ions that support the phosphorylation of specific serines or threonines. (A) Tandem mass spectrum identifying pS497, pT500, and pS502 within rat GC-A. (B) Tandem mass spectrum identifying pS506, pS510, and pT513 within rat GC-A. (C) Tandem mass spectrum identifying pS497 and pT500 within human GC-A. (D) Tandem mass spectrum identifying pS497 and pS502 within human GC-A. (E) Tandem mass spectrum identifying pS506 within human GC-A.

Figure 2. Tandem mass spectrometric spectra depicting phosphorylated residues in rat and human GC-B

Spectra were generated and are labeled as described in Figure 1. (A) Tandem mass spectrum identifying pS513 and pT516 within rat GC-B. (B) Tandem mass spectrum identifying pS513 and pT516 within human GC-B. (C) Tandem mass spectrum identifying pS518 within rat GC-B. (D) Tandem mass spectrum identifying pS518 within human GC-B. (E) Tandem mass spectrum identifying pS523 and pT526 within rat GC-B. (F) Tandem mass spectrum identifying pS523 and pT526 within human GC-B.

Figure 3. Mass spectroscopic identification of phosphorylated Ser-487 in rat and human GC-A and functional characterization of Ser-487 in rat GC-A

Spectra were generated and are labeled as described in Figure 1. (A) Tandem mass spectrum identifying pS487 in rat GC-A. (B) Tandem mass spectrum identifying pS487 in human GC-A. (C) Guanylyl cyclase activities in membranes of 293 cells expressing wild type or mutant forms rat GC-A where n = 25. Assays were conducted in the presence of either 1 µM ANP or 1% Triton X-100. Results are normalized to the maximum cGMP concentrations obtained in the presence of Triton X-100. (D) Homologous desensitization assessed by guanylyl cyclase activities in 293 cells expressing wild type or mutant forms of rat GC-A preincubated with 1 µM ANP for one hour. Assays were conducted in the presence of either 1 µM ANP or 1% Triton X-100. Results are expressed as the activation ratio of ligand-stimulated activity divided by activity determined in the presence of 1 % Triton-X 100 and Mn²⁺ GTP, which were then normalized to the non-pretreated control for each receptor where n = 8. (E) Homologous desensitization assessed by guanylyl cyclase activities in 293 cells expressing wild type or mutant forms of rat GC-A preincubated with 100 nM ANP for one hour. Assays were conducted in the presence of either 10 nM ANP, 1 µM ANP or 1% Triton X-100. Results are expressed as the activation ratio of ligand-stimulated activity divided by activity determined in the presence of 1 % Triton-X 100 and Mn²⁺ GTP, which were then normalized to the non-pretreated control for each receptor where n = 6.

Figure 4. Mass spectroscopic identification of phosphorylated Thr-529 in rat and human GC-B

Spectra were generated and are labeled as described in Figure 1. (A) Tandem mass spectrum identifying pT529 in rat GC-B generated from a tryptic digest of rat GC-B. (B) Tandem mass spectrum identifying pS523 and pT529 in human GC-B. (C) Guanylyl cyclase activities from membranes obtained form 293 cells expressing rat wild type GC-B with T529 mutated to either alanine or glutamic acid where n = 8. (D) Guanylyl cyclase activities from membranes obtained from 293 cells expressing either rat GC-B containing glutamic acid substitutions at all five previously known phosphorylated residues (GC-B-5E) or rat GC-B with a glutamic acid residue substitution at T529 in addition to previously known sites (GC-B-6E) where n = 4. Cyclase activities were determined in the presence or 1 µM CNP or 1% Triton X-100. Results are normalized to the maximum cGMP concentrations obtained in the presence of Triton X-100.

Figure 5. Identified phosphorylation sites in mammalian particulate guanylyl cyclases

(A) Schematic of the rat and human membrane-bound natriuretic peptide receptors illustrating known phosphorylated residues. Phosphorylation sites are identified both by residue number and identification procedure. BLUE residues were identified both by ³²P-phosphopeptide mapping and mass spectrometry. YELLOW residues were identified by mass spectrometry only. GREY residues are predicted to be phosphorylated by sequence homology to known phosphorylated residues. (B) Sequence alignment of phosphorylated regions from mouse (m), rat (r), and human (h) homologs of GC-A, GC-B, and retinal guanylyl cyclase (GC-1). Positions where structurally similar residues are conserved in two or more proteins are shaded light grey. Residues conserved in all proteins are shaded in dark grey. Red residues indicate identified phosphorylation sites. Numbers on top line refer to rat GC-A sites. The alignment was performed with ClustalW.