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Identification and Characterization of the Major Phosphorylation Sites of the B-type Natriuretic Peptide Receptor

1998; Elsevier BV; Volume: 273; Issue: 25 Linguagem: Inglês

10.1074/jbc.273.25.15533

1083-351X

Lincoln R. Potter, Tony Hunter,

Phosphodiesterase function and regulation

C-type natriuretic peptide (CNP) is a newly discovered factor that stimulates vasorelaxation and inhibits cell proliferation. Natriuretic peptide receptor-B (NPR-B) is the primary signaling molecule for CNP. Recently, the guanylyl cyclase activity of NPR-B was shown to correlate with its phosphorylation state, and it was suggested that receptor dephosphorylation is a mechanism of desensitization. We now report the identification and characterization of the major NPR-B phosphorylation sites. Mutagenesis and comigration studies using synthetic phosphopeptides were employed to identify five residues (Ser-513, Thr-516, Ser-518, Ser-523, and Ser-526) within the kinase homology domain that are phosphorylated when NPR-B is expressed in human 293 cells. Mutation of any of these residues to alanine reduced the receptor's phosphorylation state and CNP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in receptor protein level as indicated by immunoblot analysis and determinations of cyclase activity in the absence of CNP or in the presence of detergent. Elimination of all of the phosphorylation sites resulted in a completely dephosphorylated receptor whose CNP-dependent cyclase activity was decreased by >90%. However, unlike NPR-A, the dephosphorylated receptor was not completely unresponsive to hormone. Finally, two additional residues (Gly-521 and Ser-522) were identified that when mutated to alanine reduced the overall phosphorylation state and hormone responsiveness of the receptor without abolishing the phosphorylation of a specific site. These data indicate that phosphorylation of the kinase homology domain is a critical event in the regulation of NPR-B. C-type natriuretic peptide (CNP) is a newly discovered factor that stimulates vasorelaxation and inhibits cell proliferation. Natriuretic peptide receptor-B (NPR-B) is the primary signaling molecule for CNP. Recently, the guanylyl cyclase activity of NPR-B was shown to correlate with its phosphorylation state, and it was suggested that receptor dephosphorylation is a mechanism of desensitization. We now report the identification and characterization of the major NPR-B phosphorylation sites. Mutagenesis and comigration studies using synthetic phosphopeptides were employed to identify five residues (Ser-513, Thr-516, Ser-518, Ser-523, and Ser-526) within the kinase homology domain that are phosphorylated when NPR-B is expressed in human 293 cells. Mutation of any of these residues to alanine reduced the receptor's phosphorylation state and CNP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in receptor protein level as indicated by immunoblot analysis and determinations of cyclase activity in the absence of CNP or in the presence of detergent. Elimination of all of the phosphorylation sites resulted in a completely dephosphorylated receptor whose CNP-dependent cyclase activity was decreased by >90%. However, unlike NPR-A, the dephosphorylated receptor was not completely unresponsive to hormone. Finally, two additional residues (Gly-521 and Ser-522) were identified that when mutated to alanine reduced the overall phosphorylation state and hormone responsiveness of the receptor without abolishing the phosphorylation of a specific site. These data indicate that phosphorylation of the kinase homology domain is a critical event in the regulation of NPR-B. The natriuretic peptide family consists of atrial natriuretic peptide (ANP), 1The abbreviations used are: ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; BNP, B-type natriuretic peptide; HEK, human embryonic kidney; KHD, kinase homology domain; NPR-A, natriuretic peptide receptor A; NPR-B, natriuretic peptide receptor B; PAGE, polyacrylamide gel electrophoresis; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; HPLC, high pressure liquid chromatography. 1The abbreviations used are: ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; BNP, B-type natriuretic peptide; HEK, human embryonic kidney; KHD, kinase homology domain; NPR-A, natriuretic peptide receptor A; NPR-B, natriuretic peptide receptor B; PAGE, polyacrylamide gel electrophoresis; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; HPLC, high pressure liquid chromatography. B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (1Brenner B.M. Ballermann B.J. Gunning M.E. Zeidel M.L. Physiol. Rev. 1990; 70: 665-699Crossref PubMed Scopus (846) Google Scholar, 2Ruskoaho H. Pharmacol. Rev. 1992; 44: 479-602PubMed Google Scholar, 3McDowell G. Shaw C. Buchanan K.D. Nicholls D.P. Eur. J. Clin. Invest. 1995; 25: 291-298Crossref PubMed Scopus (28) Google Scholar). ANP and BNP are stored primarily in the cardiac atria and ventricles, respectively, and are released into the circulation upon an increase in cardiac volume (1Brenner B.M. Ballermann B.J. Gunning M.E. Zeidel M.L. Physiol. Rev. 1990; 70: 665-699Crossref PubMed Scopus (846) Google Scholar, 2Ruskoaho H. Pharmacol. Rev. 1992; 44: 479-602PubMed Google Scholar, 3McDowell G. Shaw C. Buchanan K.D. Nicholls D.P. Eur. J. Clin. Invest. 1995; 25: 291-298Crossref PubMed Scopus (28) Google Scholar). CNP on the other hand, is found primarily in vascular endothelial cells (4Suga S. Nakao K. Itoh H. Komatsu Y. Ogawa Y. Hama N. Imura H. J. Clin. Invest. 1992; 90: 1145-1149Crossref PubMed Scopus (489) Google Scholar), seminal plasma (5Chrisman T.D. Schulz S. Potter L.R. Garbers D.L. J. Biol. Chem. 1993; 268: 3698-3703Abstract Full Text PDF PubMed Google Scholar), and brain tissue (6Sudoh T. Minamino N. Kangawa K. Matsuo H. Biochem. Biophys. Res. Commun. 1990; 168: 863-870Crossref PubMed Scopus (959) Google Scholar). It does not seem to be stored in significant quantities. Instead, it appears to be regulated at the level of transcription by various cytokines, such as TGF-β (4Suga S. Nakao K. Itoh H. Komatsu Y. Ogawa Y. Hama N. Imura H. J. Clin. Invest. 1992; 90: 1145-1149Crossref PubMed Scopus (489) Google Scholar). The physiological responses elicited by these natriuretic peptides are similar but not identical. For instance, all three have been shown to cause vascular relaxation, but although both ANP and BNP have been shown to cause diuresis and natriuresis, CNP's role in these processes is dubious. Likewise, both ANP and CNP have been shown to inhibit the proliferation of various cell types (7Appel R.G. Am. J. Physiol. 1992; 262: F911-F918PubMed Google Scholar, 8Furuya M. Yoshida M. Hayashi Y. Ohnuma N. Minamino N. Kangawa K. Matsuo H. Biochem. Biophys. Res. Commun. 1991; 177: 927-931Crossref PubMed Scopus (174) Google Scholar, 9Hagiwara H. Sakaguchi H. Itakura M. Yoshimoto T. Furuya M. Tanaka S. Hirose S. J Biol Chem. 1994; 269(14): 10729-10733Google Scholar) but only CNP has thus far been shown to be a potent inhibitor of intimal thickening after vascular injury (10Furuya M. Miyazaki T. Honbou N. Kawashima K. Ohno T. Tanaka S. Kangawa K. Matsuo H. Ann N Y Acad Sci. 1995; 748: 517-523Crossref PubMed Scopus (23) Google Scholar, 11Brown J. Chen Q. Hong G. Am J Physiol. 1997; 272(6 Pt 2): H2919-31Google Scholar, 12Ueno H. Haruno A. Morisaki N. Furuya M. Kangawa K. Takeshita A. Saito Y. Circulation. 1997; 96: 2272-2279Crossref PubMed Scopus (85) Google Scholar).The cognate signaling receptor for ANP and BNP is the natriuretic peptide receptor A (NPR-A), which is also known as guanylyl cyclase A (13Chinkers M. Garbers D.L. Chang M.S. Lowe D.G. Chin H.M. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (882) Google Scholar, 14Lowe D.G. Chang M.S. Hellmiss R. Chen E. Singh S. Garbers D.L. Goeddel D.V. EMBO J. 1989; 8: 1377-1384Crossref PubMed Scopus (313) Google Scholar). The primary signaling receptor for CNP is the natriuretic peptide receptor B (NPR-B), which is also known as guanylyl cyclase B (15Chang M.S. Lowe D.G. Lewis M. Hellmiss R. Chen E. Goeddel D.V. Nature. 1989; 341: 68-72Crossref PubMed Scopus (499) Google Scholar, 16Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (488) Google Scholar). NPR-A and NPR-B are approximately 40 and 78% identical within their extracellular and intracellular regions, respectively (16Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (488) Google Scholar). Motifs present in the primary amino acid sequence of both NPR-A and NPR-B suggest that they contain five primary functional domains: an extracellular ligand-binding domain, a single membrane-spanning region, a juxtamembrane protein kinase homology domain (KHD), an α-helical hinge region involved in oligomerization (17Wilson E.M. Chinkers M. Biochemistry. 1995; 34: 4696-4701Crossref PubMed Scopus (151) Google Scholar), and a carboxyl-terminal guanylyl cyclase catalytic domain (18Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar, 19Garbers D.L. Koesling D. Schultz G. Mol. Biol. Cell. 1994; 5: 1-5Crossref PubMed Scopus (79) Google Scholar). Maximal activation of these receptors is thought to require ligand binding extracellularly and ATP binding intracellularly (20Kurose H. Inagami T. Ui M. FEBS Lett. 1987; 219: 375-379Crossref PubMed Scopus (137) Google Scholar, 21Chang C.H. Kohse K.P. Chang B. Hirata M. Jiang B. Douglas J.E. Murad F. Biochim. Biophys. Acta. 1990; 1052: 159-165Crossref PubMed Scopus (87) Google Scholar, 22Chinkers M. Singh S. Garbers D.L. J. Biol. Chem. 1991; 266: 4088-4093Abstract Full Text PDF PubMed Google Scholar, 23Duda T. Sharma R.K. Mol. Cell. Biochem. 1995; 152: 179-183Crossref PubMed Scopus (15) Google Scholar). The effect of ATP is presumably mediated allosterically by the KHD, because nonhydrolyzable adenine nucleotide analogs effectively substitute for ATP (20Kurose H. Inagami T. Ui M. FEBS Lett. 1987; 219: 375-379Crossref PubMed Scopus (137) Google Scholar, 21Chang C.H. Kohse K.P. Chang B. Hirata M. Jiang B. Douglas J.E. Murad F. Biochim. Biophys. Acta. 1990; 1052: 159-165Crossref PubMed Scopus (87) Google Scholar, 22Chinkers M. Singh S. Garbers D.L. J. Biol. Chem. 1991; 266: 4088-4093Abstract Full Text PDF PubMed Google Scholar), and deletion mutants lacking the KHD are unresponsive to hormone (24Chinkers M. Garbers D.L. Science. 1989; 245: 1392-1394Crossref PubMed Scopus (268) Google Scholar, 25Koller K.J. de Sauvage F.J. Lowe D.G. Goeddel D.V. Mol. Cell. Biol. 1992; 12(6): 2581-2590Crossref Scopus (93) Google Scholar). Within the KHD of NPR-A is the sequence GXGXXXG, which is similar to the known ATP-binding motif GXGXXG found in many protein kinases (26Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3782) Google Scholar). Sharma and colleagues (27Goraczniak R.M. Duda T. Sharma R.K. Biochem. J. 1992; 282: 533-537Crossref PubMed Scopus (73) Google Scholar, 28Duda T. Goraczniak R.M. Sharma R.K. FEBS Lett. 1993; 315: 143-148Crossref PubMed Scopus (49) Google Scholar) have coined this corresponding region in natriuretic peptide receptors the ATP-regulatory module. They have reported that the mutation of some but not all of the residues within the ATP-regulatory module region dramatically reduces hormone-dependent activation of these receptors. The mechanism for this effect has not been demonstrated, but it has been speculated that it is a result of the disruption of the putative ATP-binding domain.Both NPR-A and NPR-B are known to be phosphorylated on serine and threonine residues in unstimulated cells (29Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, 30Potter L.R. Garbers D.L. J. Biol. Chem. 1994; 269: 14636-14642Abstract Full Text PDF PubMed Google Scholar, 31Potter L.R. Biochemistry. 1998; 37: 2422-2429Crossref PubMed Scopus (85) Google Scholar, 32Koller K.J. Lipari M.T. Goeddel D.V. J. Biol. Chem. 1993; 268: 5997-6003Abstract Full Text PDF PubMed Google Scholar). Upon binding to their cognate hormone, the guanylyl cyclase activity of these receptors is markedly enhanced, resulting in dramatic elevations in intracellular cGMP concentrations. After the initial stimulation, the activity of these enzymes decreases with kinetics that are coincident with receptor dephosphorylation (29Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, 31Potter L.R. Biochemistry. 1998; 37: 2422-2429Crossref PubMed Scopus (85) Google Scholar). Thus, it has been suggested that dephosphorylation mediates their desensitization. We recently, identified six phosphorylation sites within the KHD of NPR-A (33Potter L.R. Hunter T. Mol. Cell. Biol. 1998; 18: 2164-2172Crossref PubMed Scopus (122) Google Scholar). As was predicted from the desensitization studies, the mutation of these sites to alanine dramatically decreased the ANP-dependent activity of NPR-A. Furthermore, the conversion of four or more of the phosphorylation sites to alanine resulted in an hormonally unresponsive receptor. In this report, we describe the identification of fivein vivo phosphorylation sites for NPR-B. The evidence for these sites is based primarily on deletion and site-specific mutagenesis studies. However, for two of the sites we were able to show comigration with synthetic phosphopeptides. Four of the five sites correspond to residues present in NPR-A, and the additional site is only one amino acid carboxyl-terminal to a known NPR-A site. All of the single phosphorylation site mutations decreased CNP-dependent activity, and a completely dephosphorylated form of NPR-B retained less than 10% of its wild type activity. Finally, we show that a previously described inactivating mutation within the so called ATP-regulatory module of NPR-B (G521A) also results in receptor dephosphorylation, suggesting that dephosphorylation may explain the functional consequence of this mutation as well.DISCUSSIONIn this report, we describe the identification and characterization of five phosphorylated residues located in the putative ATP-binding portion of the KHD of NPR-B. The data for these conclusions are based on a number of experiments including: deletion mutagenesis (33Potter L.R. Hunter T. Mol. Cell. Biol. 1998; 18: 2164-2172Crossref PubMed Scopus (122) Google Scholar), single site mutagenesis, and comigration studies involving synthetic phosphopeptides. The experiments involving the comigration of synthetic phosphopeptides with tryptic phosphopeptides of NPR-B isolated from 32P-labeled cells provide strong evidence for the phosphorylation of Thr-516 and Ser-518. Furthermore, the high correlation of the mutagenesis data with the predicted phosphopeptide maps together with the corroborating phosphoamino acid analysis, make a very strong case for the correct identification all five sites.Although most of the NPR-A phosphorylation sites were conserved in NPR-B, we were unable to find conclusive evidence for the phosphorylation of Thr-529. Mutation of the corresponding residue in NPR-A, Thr-513, was shown to decrease the phosphorylation state and change the tryptic phosphopeptide maps of this receptor, whereas mutation of Thr-529 to alanine in NPR-B had little or no effect. However, because phosphoamino acid analysis of peptides that are predicted to contain this residue revealed the presence of low levels of phosphothreonine, we cannot rule out the possibility that Thr-529 may be a minor phosphorylation site. Furthermore, the replacement of this residue with alanine resulted in CNP-dependent guanylyl cyclase activities that were similar to those observed for the S513A, T516A, S518A, and S522A mutations. Whether the reduction associated with the T529A mutant is because of the loss of a specific phosphorylation site or is simply because of structural differences between the threonine and alanine residues is not known. It is also interesting that Ser-522 does not appear to be phosphorylated in NPR-B, because the corresponding residue in NPR-A (Ser-506) has been shown to be a major site. It is possible that the function of this residue is subserved by the adjacent residue, Ser-523, which lacks a counterpart in NPR-A. Regardless of its phosphorylation state, the structural characteristics of this residue appear to be important for maximal phosphorylation of the known sites, because the conservative mutation of this residue to alanine reduced the overall phosphorylation state of the receptor (Fig. 4). In a like manner, the mutation of glycine 521 to alanine also resulted in a decrease in the phosphorylation state of NPR-B without abolishing the phosphorylation of a specific site (Fig. 7). The structural characteristics of these amino acids are likely to be critical for the function of this region, because the sequence RGSSYGSL is absolutely conserved in all the NPR-B molecules identified to date, including humans, rats, cows, and eels (15Chang M.S. Lowe D.G. Lewis M. Hellmiss R. Chen E. Goeddel D.V. Nature. 1989; 341: 68-72Crossref PubMed Scopus (499) Google Scholar, 16Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (488) Google Scholar, 38Fenrick R. Babinski K. McNicoll N. Therrien M. Drouin J. De Lean A. Mol. Cell. Biochem. 1994; 137: 173-182Crossref PubMed Scopus (23) Google Scholar, 39Katafuchi T. Takashima A. Kashiwagi M. Hagiwara H. Takei Y. Hirose S. Eur. J. Biochem. 1994; 222: 835-842Crossref PubMed Scopus (62) Google Scholar).It has been suggested by Sharma and colleagues (23Duda T. Sharma R.K. Mol. Cell. Biochem. 1995; 152: 179-183Crossref PubMed Scopus (15) Google Scholar, 27Goraczniak R.M. Duda T. Sharma R.K. Biochem. J. 1992; 282: 533-537Crossref PubMed Scopus (73) Google Scholar, 28Duda T. Goraczniak R.M. Sharma R.K. FEBS Lett. 1993; 315: 143-148Crossref PubMed Scopus (49) Google Scholar, 40Duda T. Sharma R.K. Biochem. Biophys. Res. Commun. 1995; 209: 286-292Crossref PubMed Scopus (26) Google Scholar) that the GXGXXXG region in NPR-A and the corresponding region in NPR-B function as ATP-binding modules. The primary evidence for these claims is: (i) A similar motif (GXGXXG) is found in many, but not all, known protein kinases; (ii) ATP is required for maximal activation of these enzymes; (iii) ATP appears to exert its effects on highly purified preparations of NPR-A; and (iv) mutagenesis of the conserved glycines within this region reduce the ability of these receptors to be stimulated by ligands. Specifically, the mutation of glycine 505 and serine 506 to alanine and valine, respectively, in NPR-A, and the mutation of glycine 521 to alanine in NPR-B were found to reduce hormone-stimulated activity. In contrast, when Koller et al. (32Koller K.J. Lipari M.T. Goeddel D.V. J. Biol. Chem. 1993; 268: 5997-6003Abstract Full Text PDF PubMed Google Scholar) mutated all three of the glycines in the putative ATP-binding domain to alanine in NPR-A, they found very little or no effect on the ability of this mutant to be phosphorylated or hormonally activated. This suggests that the conversion of glycine 505 to alanine is not sufficient to mediate this inhibition. Because we now know that Ser-506 is a major phosphorylation site of NPR-A, it is more likely that the removal of this site is responsible for the inactivity. With respect to the glycine 521 to alanine mutation in NPR-B, the effect of this mutation may also be related to the receptor dephosphorylation. But, instead of inhibiting the phosphorylation of a specific site, it appears to causes a global dephosphorylation of the receptor, possibly by making the KHD of NPR-B a less desirable substrate for its protein kinase. Finally, it should be noted that although we have identified five phosphorylation sites within NPR-B, we have not determined the exact stoichiometry of phosphorylation. It is likely that this receptor is multiply phosphorylated in these cells as evidenced by the presence of peptides containing more than one phosphate group. However, the fraction of the total cellular NPR-B pool that is phosphorylated has not yet been determined.In conclusion, we have presented evidence for the correct identification and characterization of the major phosphorylation sites of NPR-B. These data are consistent with our previous report, which suggested that NPR-B is desensitized by dephosphorylation (31Potter L.R. Biochemistry. 1998; 37: 2422-2429Crossref PubMed Scopus (85) Google Scholar). We have now extended our molecular understanding of this process by determining the exact location of the phosphorylated residues. Because the selective removal of these sites via site-directed mutagenesis mimics the effect of phosphatase treatment on NPR-B in crude membranes, it appears likely that dephosphorylation of only NPR-B is sufficient to inhibit its ability to respond to hormone. Now that these sites are known, the role of phosphorylation/dephosphorylation in the regulation of this receptor can be further addressed. It will be interesting to determine whether or not the removal of specific sites can abrogate the protein kinase C-mediated heterologous desensitization response. Likewise, the effect of phosphorylation/dephosphorylation on hormone binding and receptor trafficking and can now be effectively investigated using receptor mutants. The natriuretic peptide family consists of atrial natriuretic peptide (ANP), 1The abbreviations used are: ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; BNP, B-type natriuretic peptide; HEK, human embryonic kidney; KHD, kinase homology domain; NPR-A, natriuretic peptide receptor A; NPR-B, natriuretic peptide receptor B; PAGE, polyacrylamide gel electrophoresis; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; HPLC, high pressure liquid chromatography. 1The abbreviations used are: ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; BNP, B-type natriuretic peptide; HEK, human embryonic kidney; KHD, kinase homology domain; NPR-A, natriuretic peptide receptor A; NPR-B, natriuretic peptide receptor B; PAGE, polyacrylamide gel electrophoresis; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; HPLC, high pressure liquid chromatography. B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (1Brenner B.M. Ballermann B.J. Gunning M.E. Zeidel M.L. Physiol. Rev. 1990; 70: 665-699Crossref PubMed Scopus (846) Google Scholar, 2Ruskoaho H. Pharmacol. Rev. 1992; 44: 479-602PubMed Google Scholar, 3McDowell G. Shaw C. Buchanan K.D. Nicholls D.P. Eur. J. Clin. Invest. 1995; 25: 291-298Crossref PubMed Scopus (28) Google Scholar). ANP and BNP are stored primarily in the cardiac atria and ventricles, respectively, and are released into the circulation upon an increase in cardiac volume (1Brenner B.M. Ballermann B.J. Gunning M.E. Zeidel M.L. Physiol. Rev. 1990; 70: 665-699Crossref PubMed Scopus (846) Google Scholar, 2Ruskoaho H. Pharmacol. Rev. 1992; 44: 479-602PubMed Google Scholar, 3McDowell G. Shaw C. Buchanan K.D. Nicholls D.P. Eur. J. Clin. Invest. 1995; 25: 291-298Crossref PubMed Scopus (28) Google Scholar). CNP on the other hand, is found primarily in vascular endothelial cells (4Suga S. Nakao K. Itoh H. Komatsu Y. Ogawa Y. Hama N. Imura H. J. Clin. Invest. 1992; 90: 1145-1149Crossref PubMed Scopus (489) Google Scholar), seminal plasma (5Chrisman T.D. Schulz S. Potter L.R. Garbers D.L. J. Biol. Chem. 1993; 268: 3698-3703Abstract Full Text PDF PubMed Google Scholar), and brain tissue (6Sudoh T. Minamino N. Kangawa K. Matsuo H. Biochem. Biophys. Res. Commun. 1990; 168: 863-870Crossref PubMed Scopus (959) Google Scholar). It does not seem to be stored in significant quantities. Instead, it appears to be regulated at the level of transcription by various cytokines, such as TGF-β (4Suga S. Nakao K. Itoh H. Komatsu Y. Ogawa Y. Hama N. Imura H. J. Clin. Invest. 1992; 90: 1145-1149Crossref PubMed Scopus (489) Google Scholar). The physiological responses elicited by these natriuretic peptides are similar but not identical. For instance, all three have been shown to cause vascular relaxation, but although both ANP and BNP have been shown to cause diuresis and natriuresis, CNP's role in these processes is dubious. Likewise, both ANP and CNP have been shown to inhibit the proliferation of various cell types (7Appel R.G. Am. J. Physiol. 1992; 262: F911-F918PubMed Google Scholar, 8Furuya M. Yoshida M. Hayashi Y. Ohnuma N. Minamino N. Kangawa K. Matsuo H. Biochem. Biophys. Res. Commun. 1991; 177: 927-931Crossref PubMed Scopus (174) Google Scholar, 9Hagiwara H. Sakaguchi H. Itakura M. Yoshimoto T. Furuya M. Tanaka S. Hirose S. J Biol Chem. 1994; 269(14): 10729-10733Google Scholar) but only CNP has thus far been shown to be a potent inhibitor of intimal thickening after vascular injury (10Furuya M. Miyazaki T. Honbou N. Kawashima K. Ohno T. Tanaka S. Kangawa K. Matsuo H. Ann N Y Acad Sci. 1995; 748: 517-523Crossref PubMed Scopus (23) Google Scholar, 11Brown J. Chen Q. Hong G. Am J Physiol. 1997; 272(6 Pt 2): H2919-31Google Scholar, 12Ueno H. Haruno A. Morisaki N. Furuya M. Kangawa K. Takeshita A. Saito Y. Circulation. 1997; 96: 2272-2279Crossref PubMed Scopus (85) Google Scholar). The cognate signaling receptor for ANP and BNP is the natriuretic peptide receptor A (NPR-A), which is also known as guanylyl cyclase A (13Chinkers M. Garbers D.L. Chang M.S. Lowe D.G. Chin H.M. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (882) Google Scholar, 14Lowe D.G. Chang M.S. Hellmiss R. Chen E. Singh S. Garbers D.L. Goeddel D.V. EMBO J. 1989; 8: 1377-1384Crossref PubMed Scopus (313) Google Scholar). The primary signaling receptor for CNP is the natriuretic peptide receptor B (NPR-B), which is also known as guanylyl cyclase B (15Chang M.S. Lowe D.G. Lewis M. Hellmiss R. Chen E. Goeddel D.V. Nature. 1989; 341: 68-72Crossref PubMed Scopus (499) Google Scholar, 16Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (488) Google Scholar). NPR-A and NPR-B are approximately 40 and 78% identical within their extracellular and intracellular regions, respectively (16Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (488) Google Scholar). Motifs present in the primary amino acid sequence of both NPR-A and NPR-B suggest that they contain five primary functional domains: an extracellular ligand-binding domain, a single membrane-spanning region, a juxtamembrane protein kinase homology domain (KHD), an α-helical hinge region involved in oligomerization (17Wilson E.M. Chinkers M. Biochemistry. 1995; 34: 4696-4701Crossref PubMed Scopus (151) Google Scholar), and a carboxyl-terminal guanylyl cyclase catalytic domain (18Garbers D.L. Lowe D.G. J. Biol. Chem. 1994; 269: 30741-30744Abstract Full Text PDF PubMed Google Scholar, 19Garbers D.L. Koesling D. Schultz G. Mol. Biol. Cell. 1994; 5: 1-5Crossref PubMed Scopus (79) Google Scholar). Maximal activation of these receptors is thought to require ligand binding extracellularly and ATP binding intracellularly (20Kurose H. Inagami T. Ui M. FEBS Lett. 1987; 219: 375-379Crossref PubMed Scopus (137) Google Scholar, 21Chang C.H. Kohse K.P. Chang B. Hirata M. Jiang B. Douglas J.E. Murad F. Biochim. Biophys. Acta. 1990; 1052: 159-165Crossref PubMed Scopus (87) Google Scholar, 22Chinkers M. Singh S. Garbers D.L. J. Biol. Chem. 1991; 266: 4088-4093Abstract Full Text PDF PubMed Google Scholar, 23Duda T. Sharma R.K. Mol. Cell. Biochem. 1995; 152: 179-183Crossref PubMed Scopus (15) Google Scholar). The effect of ATP is presumably mediated allosterically by the KHD, because nonhydrolyzable adenine nucleotide analogs effectively substitute for ATP (20Kurose H. Inagami T. Ui M. FEBS Lett. 1987; 219: 375-379Crossref PubMed Scopus (137) Google Scholar, 21Chang C.H. Kohse K.P. Chang B. Hirata M. Jiang B. Douglas J.E. Murad F. Biochim. Biophys. Acta. 1990; 1052: 159-165Crossref PubMed Scopus (87) Google Scholar, 22Chinkers M. Singh S. Garbers D.L. J. Biol. Chem. 1991; 266: 4088-4093Abstract Full Text PDF PubMed Google Scholar), and deletion mutants lacking the KHD are unresponsive to hormone (24Chinkers M. Garbers D.L. Science. 1989; 245: 1392-1394Crossref PubMed Scopus (268) Google Scholar, 25Koller K.J. de Sauvage F.J. Lowe D.G. Goeddel D.V. Mol. Cell. Biol. 1992; 12(6): 2581-2590Crossref Scopus (93) Google Scholar). Within the KHD of NPR-A is the sequence GXGXXXG, which is similar to the known ATP-binding motif GXGXXG found in many protein kinases (26Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3782) Google Scholar). Sharma and colleagues (27Goraczniak R.M. Duda T. Sharma R.K. Biochem. J. 1992; 282: 533-537Crossref PubMed Scopus (73) Google Scholar, 28Duda T. Goraczniak R.M. Sharma R.K. FEBS Lett. 1993; 315: 143-148Crossref PubMed Scopus (49) Google Scholar) have coined this corresponding region in natriuretic peptide receptors the ATP-regulatory module. They have reported that the mutation of some but not all of the residues within the ATP-regulatory module region dramatically reduces hormone-dependent activation of these receptors. The mechanism for this effect has not been demonstrated, but it has been speculated that it is a result of the disruption of the putative ATP-binding domain. Both NPR-A and NPR-B are known to be phosphorylated on serine and threonine residues in unstimulated cells (29Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, 30Potter L.R. Garbers D.L. J. Biol. Chem. 1994; 269: 14636-14642Abstract Full Text PDF PubMed Google Scholar, 31Potter L.R. Biochemistry. 1998; 37: 2422-2429Crossref PubMed Scopus (85) Google Scholar, 32Koller K.J. Lipari M.T. Goeddel D.V. J. Biol. Chem. 1993; 268: 5997-6003Abstract Full Text PDF PubMed Google Scholar). Upon binding to their cognate hormone, the guanylyl cyclase activity of these receptors is markedly enhanced, resulting in dramatic elevations in intracellular cGMP concentrations. After the initial stimulation, the activity of these enzymes decreases with kinetics that are coincident with receptor dephosphorylation (29Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, 31Potter L.R. Biochemistry. 1998; 37: 2422-2429Crossref PubMed Scopus (85) Google Scholar). Thus, it has been suggested that dephosphorylation mediates their desensitization. We recently, identified six phosphorylation sites within the KHD of NPR-A (33Potter L.R. Hunter T. Mol. Cell. Biol. 1998; 18: 2164-2172Crossref PubMed Scopus (122) Google Scholar). As was predicted from the desensitization studies, the mutation of these sites to alanine dramatically decreased the ANP-dependent activity of NPR-A. Furthermore, the conversion of four or more of the phosphorylation sites to alanine resulted in an hormonally unresponsive receptor. In this report, we describe the identification of fivein vivo phosphorylation sites for NPR-B. The evidence for these sites is based primarily on deletion and site-specific mutagenesis studies. However, for two of the sites we were able to show comigration with synthetic phosphopeptides. Four of the five sites correspond to residues present in NPR-A, and the additional site is only one amino acid carboxyl-terminal to a known NPR-A site. All of the single phosphorylation site mutations decreased CNP-dependent activity, and a completely dephosphorylated form of NPR-B retained less than 10% of its wild type activity. Finally, we show that a previously described inactivating mutation within the so called ATP-regulatory module of NPR-B (G521A) also results in receptor dephosphorylation, suggesting that dephosphorylation may explain the functional consequence of this mutation as well. DISCUSSIONIn this report, we describe the identification and characterization of five phosphorylated residues located in the putative ATP-binding portion of the KHD of NPR-B. The data for these conclusions are based on a number of experiments including: deletion mutagenesis (33Potter L.R. Hunter T. Mol. Cell. Biol. 1998; 18: 2164-2172Crossref PubMed Scopus (122) Google Scholar), single site mutagenesis, and comigration studies involving synthetic phosphopeptides. The experiments involving the comigration of synthetic phosphopeptides with tryptic phosphopeptides of NPR-B isolated from 32P-labeled cells provide strong evidence for the phosphorylation of Thr-516 and Ser-518. Furthermore, the high correlation of the mutagenesis data with the predicted phosphopeptide maps together with the corroborating phosphoamino acid analysis, make a very strong case for the correct identification all five sites.Although most of the NPR-A phosphorylation sites were conserved in NPR-B, we were unable to find conclusive evidence for the phosphorylation of Thr-529. Mutation of the corresponding residue in NPR-A, Thr-513, was shown to decrease the phosphorylation state and change the tryptic phosphopeptide maps of this receptor, whereas mutation of Thr-529 to alanine in NPR-B had little or no effect. However, because phosphoamino acid analysis of peptides that are predicted to contain this residue revealed the presence of low levels of phosphothreonine, we cannot rule out the possibility that Thr-529 may be a minor phosphorylation site. Furthermore, the replacement of this residue with alanine resulted in CNP-dependent guanylyl cyclase activities that were similar to those observed for the S513A, T516A, S518A, and S522A mutations. Whether the reduction associated with the T529A mutant is because of the loss of a specific phosphorylation site or is simply because of structural differences between the threonine and alanine residues is not known. It is also interesting that Ser-522 does not appear to be phosphorylated in NPR-B, because the corresponding residue in NPR-A (Ser-506) has been shown to be a major site. It is possible that the function of this residue is subserved by the adjacent residue, Ser-523, which lacks a counterpart in NPR-A. Regardless of its phosphorylation state, the structural characteristics of this residue appear to be important for maximal phosphorylation of the known sites, because the conservative mutation of this residue to alanine reduced the overall phosphorylation state of the receptor (Fig. 4). In a like manner, the mutation of glycine 521 to alanine also resulted in a decrease in the phosphorylation state of NPR-B without abolishing the phosphorylation of a specific site (Fig. 7). The structural characteristics of these amino acids are likely to be critical for the function of this region, because the sequence RGSSYGSL is absolutely conserved in all the NPR-B molecules identified to date, including humans, rats, cows, and eels (15Chang M.S. Lowe D.G. Lewis M. Hellmiss R. Chen E. Goeddel D.V. Nature. 1989; 341: 68-72Crossref PubMed Scopus (499) Google Scholar, 16Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (488) Google Scholar, 38Fenrick R. Babinski K. McNicoll N. Therrien M. Drouin J. De Lean A. Mol. Cell. Biochem. 1994; 137: 173-182Crossref PubMed Scopus (23) Google Scholar, 39Katafuchi T. Takashima A. Kashiwagi M. Hagiwara H. Takei Y. Hirose S. Eur. J. Biochem. 1994; 222: 835-842Crossref PubMed Scopus (62) Google Scholar).It has been suggested by Sharma and colleagues (23Duda T. Sharma R.K. Mol. Cell. Biochem. 1995; 152: 179-183Crossref PubMed Scopus (15) Google Scholar, 27Goraczniak R.M. Duda T. Sharma R.K. Biochem. J. 1992; 282: 533-537Crossref PubMed Scopus (73) Google Scholar, 28Duda T. Goraczniak R.M. Sharma R.K. FEBS Lett. 1993; 315: 143-148Crossref PubMed Scopus (49) Google Scholar, 40Duda T. Sharma R.K. Biochem. Biophys. Res. Commun. 1995; 209: 286-292Crossref PubMed Scopus (26) Google Scholar) that the GXGXXXG region in NPR-A and the corresponding region in NPR-B function as ATP-binding modules. The primary evidence for these claims is: (i) A similar motif (GXGXXG) is found in many, but not all, known protein kinases; (ii) ATP is required for maximal activation of these enzymes; (iii) ATP appears to exert its effects on highly purified preparations of NPR-A; and (iv) mutagenesis of the conserved glycines within this region reduce the ability of these receptors to be stimulated by ligands. Specifically, the mutation of glycine 505 and serine 506 to alanine and valine, respectively, in NPR-A, and the mutation of glycine 521 to alanine in NPR-B were found to reduce hormone-stimulated activity. In contrast, when Koller et al. (32Koller K.J. Lipari M.T. Goeddel D.V. J. Biol. Chem. 1993; 268: 5997-6003Abstract Full Text PDF PubMed Google Scholar) mutated all three of the glycines in the putative ATP-binding domain to alanine in NPR-A, they found very little or no effect on the ability of this mutant to be phosphorylated or hormonally activated. This suggests that the conversion of glycine 505 to alanine is not sufficient to mediate this inhibition. Because we now know that Ser-506 is a major phosphorylation site of NPR-A, it is more likely that the removal of this site is responsible for the inactivity. With respect to the glycine 521 to alanine mutation in NPR-B, the effect of this mutation may also be related to the receptor dephosphorylation. But, instead of inhibiting the phosphorylation of a specific site, it appears to causes a global dephosphorylation of the receptor, possibly by making the KHD of NPR-B a less desirable substrate for its protein kinase. Finally, it should be noted that although we have identified five phosphorylation sites within NPR-B, we have not determined the exact stoichiometry of phosphorylation. It is likely that this receptor is multiply phosphorylated in these cells as evidenced by the presence of peptides containing more than one phosphate group. However, the fraction of the total cellular NPR-B pool that is phosphorylated has not yet been determined.In conclusion, we have presented evidence for the correct identification and characterization of the major phosphorylation sites of NPR-B. These data are consistent with our previous report, which suggested that NPR-B is desensitized by dephosphorylation (31Potter L.R. Biochemistry. 1998; 37: 2422-2429Crossref PubMed Scopus (85) Google Scholar). We have now extended our molecular understanding of this process by determining the exact location of the phosphorylated residues. Because the selective removal of these sites via site-directed mutagenesis mimics the effect of phosphatase treatment on NPR-B in crude membranes, it appears likely that dephosphorylation of only NPR-B is sufficient to inhibit its ability to respond to hormone. Now that these sites are known, the role of phosphorylation/dephosphorylation in the regulation of this receptor can be further addressed. It will be interesting to determine whether or not the removal of specific sites can abrogate the protein kinase C-mediated heterologous desensitization response. Likewise, the effect of phosphorylation/dephosphorylation on hormone binding and receptor trafficking and can now be effectively investigated using receptor mutants. In this report, we describe the identification and characterization of five phosphorylated residues located in the putative ATP-binding portion of the KHD of NPR-B. The data for these conclusions are based on a number of experiments including: deletion mutagenesis (33Potter L.R. Hunter T. Mol. Cell. Biol. 1998; 18: 2164-2172Crossref PubMed Scopus (122) Google Scholar), single site mutagenesis, and comigration studies involving synthetic phosphopeptides. The experiments involving the comigration of synthetic phosphopeptides with tryptic phosphopeptides of NPR-B isolated from 32P-labeled cells provide strong evidence for the phosphorylation of Thr-516 and Ser-518. Furthermore, the high correlation of the mutagenesis data with the predicted phosphopeptide maps together with the corroborating phosphoamino acid analysis, make a very strong case for the correct identification all five sites. Although most of the NPR-A phosphorylation sites were conserved in NPR-B, we were unable to find conclusive evidence for the phosphorylation of Thr-529. Mutation of the corresponding residue in NPR-A, Thr-513, was shown to decrease the phosphorylation state and change the tryptic phosphopeptide maps of this receptor, whereas mutation of Thr-529 to alanine in NPR-B had little or no effect. However, because phosphoamino acid analysis of peptides that are predicted to contain this residue revealed the presence of low levels of phosphothreonine, we cannot rule out the possibility that Thr-529 may be a minor phosphorylation site. Furthermore, the replacement of this residue with alanine resulted in CNP-dependent guanylyl cyclase activities that were similar to those observed for the S513A, T516A, S518A, and S522A mutations. Whether the reduction associated with the T529A mutant is because of the loss of a specific phosphorylation site or is simply because of structural differences between the threonine and alanine residues is not known. It is also interesting that Ser-522 does not appear to be phosphorylated in NPR-B, because the corresponding residue in NPR-A (Ser-506) has been shown to be a major site. It is possible that the function of this residue is subserved by the adjacent residue, Ser-523, which lacks a counterpart in NPR-A. Regardless of its phosphorylation state, the structural characteristics of this residue appear to be important for maximal phosphorylation of the known sites, because the conservative mutation of this residue to alanine reduced the overall phosphorylation state of the receptor (Fig. 4). In a like manner, the mutation of glycine 521 to alanine also resulted in a decrease in the phosphorylation state of NPR-B without abolishing the phosphorylation of a specific site (Fig. 7). The structural characteristics of these amino acids are likely to be critical for the function of this region, because the sequence RGSSYGSL is absolutely conserved in all the NPR-B molecules identified to date, including humans, rats, cows, and eels (15Chang M.S. Lowe D.G. Lewis M. Hellmiss R. Chen E. Goeddel D.V. Nature. 1989; 341: 68-72Crossref PubMed Scopus (499) Google Scholar, 16Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (488) Google Scholar, 38Fenrick R. Babinski K. McNicoll N. Therrien M. Drouin J. De Lean A. Mol. Cell. Biochem. 1994; 137: 173-182Crossref PubMed Scopus (23) Google Scholar, 39Katafuchi T. Takashima A. Kashiwagi M. Hagiwara H. Takei Y. Hirose S. Eur. J. Biochem. 1994; 222: 835-842Crossref PubMed Scopus (62) Google Scholar). It has been suggested by Sharma and colleagues (23Duda T. Sharma R.K. Mol. Cell. Biochem. 1995; 152: 179-183Crossref PubMed Scopus (15) Google Scholar, 27Goraczniak R.M. Duda T. Sharma R.K. Biochem. J. 1992; 282: 533-537Crossref PubMed Scopus (73) Google Scholar, 28Duda T. Goraczniak R.M. Sharma R.K. FEBS Lett. 1993; 315: 143-148Crossref PubMed Scopus (49) Google Scholar, 40Duda T. Sharma R.K. Biochem. Biophys. Res. Commun. 1995; 209: 286-292Crossref PubMed Scopus (26) Google Scholar) that the GXGXXXG region in NPR-A and the corresponding region in NPR-B function as ATP-binding modules. The primary evidence for these claims is: (i) A similar motif (GXGXXG) is found in many, but not all, known protein kinases; (ii) ATP is required for maximal activation of these enzymes; (iii) ATP appears to exert its effects on highly purified preparations of NPR-A; and (iv) mutagenesis of the conserved glycines within this region reduce the ability of these receptors to be stimulated by ligands. Specifically, the mutation of glycine 505 and serine 506 to alanine and valine, respectively, in NPR-A, and the mutation of glycine 521 to alanine in NPR-B were found to reduce hormone-stimulated activity. In contrast, when Koller et al. (32Koller K.J. Lipari M.T. Goeddel D.V. J. Biol. Chem. 1993; 268: 5997-6003Abstract Full Text PDF PubMed Google Scholar) mutated all three of the glycines in the putative ATP-binding domain to alanine in NPR-A, they found very little or no effect on the ability of this mutant to be phosphorylated or hormonally activated. This suggests that the conversion of glycine 505 to alanine is not sufficient to mediate this inhibition. Because we now know that Ser-506 is a major phosphorylation site of NPR-A, it is more likely that the removal of this site is responsible for the inactivity. With respect to the glycine 521 to alanine mutation in NPR-B, the effect of this mutation may also be related to the receptor dephosphorylation. But, instead of inhibiting the phosphorylation of a specific site, it appears to causes a global dephosphorylation of the receptor, possibly by making the KHD of NPR-B a less desirable substrate for its protein kinase. Finally, it should be noted that although we have identified five phosphorylation sites within NPR-B, we have not determined the exact stoichiometry of phosphorylation. It is likely that this receptor is multiply phosphorylated in these cells as evidenced by the presence of peptides containing more than one phosphate group. However, the fraction of the total cellular NPR-B pool that is phosphorylated has not yet been determined. In conclusion, we have presented evidence for the correct identification and characterization of the major phosphorylation sites of NPR-B. These data are consistent with our previous report, which suggested that NPR-B is desensitized by dephosphorylation (31Potter L.R. Biochemistry. 1998; 37: 2422-2429Crossref PubMed Scopus (85) Google Scholar). We have now extended our molecular understanding of this process by determining the exact location of the phosphorylated residues. Because the selective removal of these sites via site-directed mutagenesis mimics the effect of phosphatase treatment on NPR-B in crude membranes, it appears likely that dephosphorylation of only NPR-B is sufficient to inhibit its ability to respond to hormone. Now that these sites are known, the role of phosphorylation/dephosphorylation in the regulation of this receptor can be further addressed. It will be interesting to determine whether or not the removal of specific sites can abrogate the protein kinase C-mediated heterologous desensitization response. Likewise, the effect of phosphorylation/dephosphorylation on hormone binding and receptor trafficking and can now be effectively investigated using receptor mutants. We thank Jill Meisenhelder for peptide synthesis, Nigel Carter for HPLC purification of the synthetic phosphopeptides, and Anthony Craig for mass spectroscopy. We are also grateful to Dr. David L. Garbers for the generous donation of antiserums Z658 and R1215.