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Agonistic Induction of a Covalent Dimer in a Mutant of Natriuretic Peptide Receptor-A Documents a Juxtamembrane Interaction That Accompanies Receptor Activation

2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês

10.1074/jbc.m005550200

ISSN

1083-351X

Autores

Jean Labrecque, Julie Deschênes, Normand McNicoll, André De Léan,

Tópico(s)

Pharmaceutical studies and practices

Resumo

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-AD435C mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-AD435C dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-AD435C has been truncated (ΔKCD435C) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type ΔKC and ΔKCD435C is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain. The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-AD435C mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-AD435C dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-AD435C has been truncated (ΔKCD435C) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type ΔKC and ΔKCD435C is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain. natriuretic peptide receptor rat NPR atrial natriuretic peptide rat ANP (residues 1–28) or natriuretic peptide A porcine brain natriuretic peptide (residues 1–32) C-type natriuretic peptide (residues 1–22) atriopeptin I (rANP residues 5–25) des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP 4–23-NH2 (rat) polyacrylamide gel electrophoresis bovine serum albumin 1-methyl-3-isobutylxanthine extracellular domain transmembrane domain kinase homology domain guanylyl cyclase domain His-tagged ECD Dulbecco's modified Eagle's medium erythropoietin receptor The natriuretic peptide receptors (NPRs)1 are members of a family of single-transmembrane domain receptors that mediate their effects through the production of cyclic GMP (1Garbers D.L. Methods. 1999; 19: 477-484Crossref PubMed Scopus (54) Google Scholar). Three different NPRs have been identified, and two of these, NPR-A and NPR-B, respond to agonists by the activation of their guanylyl cyclase catalytic domain. The production of intracellular cGMP mediates their effects on diuresis, vasorelaxation, and the inhibition of the renin-angiotensin-aldosterone system (2Chinkers M. Annu. Rev. Biochem. 1991; 60: 553-575Crossref PubMed Scopus (201) Google Scholar). A third receptor, called NPR-C or the clearance receptor, displays only 37 amino acids in its intracellular domain and is devoid of guanylyl cyclase activity. NPR-C is a disulfide-bridged dimer that internalizes through a fast intracellular cycle process (3Cohen D. Young G.Koh Nikonova L.N. Gordon P. orter J. Maack T. J. Biol. Chem. 1996; 271: 9863-9869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and might be involved in signal transduction (4Anand-Shrivastava M.B. Sehl P.D. Lowe D.G. J. Biol. Chem. 1996; 271: 19324-19329Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). NPR-A is stimulated by two peptides, ANP and BNP, whereas CNP is the only known agonist of NPR-B (2Chinkers M. Annu. Rev. Biochem. 1991; 60: 553-575Crossref PubMed Scopus (201) Google Scholar, 5Nakao K. Ogawa Y. Shin-ichi S. Imura H. J. Hypertens. 1992; 10: 907-912Crossref PubMed Scopus (209) Google Scholar). NPR-C has nearly equal binding affinity for all of these natriuretic peptides (6Itakura M. Iwashina M. Mizuno T. Teizo I. Hagiwara H. Hirose S. J. Biol. Chem. 1994; 269: 8314-8318Abstract Full Text PDF PubMed Google Scholar,7Suga S. Nakao K. Hosada K. Mukoyama M. Ogawa Y. Shirakami G. Arai H. Saito Y. Kambayashi Y. Inouye K. Imura H. Endocrinology. 1992; 130: 229-239Crossref PubMed Google Scholar). NPR-A is an ∼130-kDa protein that contains four structural domains: an extracellular domain (ECD) with a ligand binding site, a transmembrane domain (TM), a kinase homology domain (KHD), and a guanylyl cyclase domain (GC) (2Chinkers M. Annu. Rev. Biochem. 1991; 60: 553-575Crossref PubMed Scopus (201) Google Scholar). Several studies have demonstrated that this receptor is spontaneously preassociated in noncovalent dimers or oligomers (8Chinkers M. Wilson E.M. J. Biol. Chem. 1992; 267: 18589-18597Abstract Full Text PDF PubMed Google Scholar, 9Wilson E.M. Chinkers M. Biochemistry. 1995; 34: 4696-4701Crossref PubMed Scopus (153) Google Scholar, 10Thompson D.K. Garbers D.L. J. Biol. Chem. 1995; 270: 425-430Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Taken together, these studies have indicated that both extracellular and intracellular domains are involved in NPR-A dimerization. According to the current model of agonist activation, NPR-A signal transduction includes these five sequential steps (11Jewett J.R.S. Koller K.J. Goeddel D.V. Lowe D.G. EMBO J. 1993; 12: 769-777Crossref PubMed Scopus (71) Google Scholar). 1) The binding of the natriuretic peptide to the ectodomain induces a conformational change. 2) This modification corresponds to a signal that migrates through the TM domain. 3) The KHD responds to this signal by adopting a conformation that allows ATP binding. 4) ATP binding has two major effects in derepressing the guanylyl cyclase activity and increasing the off-rate of ANP from the receptor (12Larose L. McNicoll N. Ong H. De Léan A. Biochemistry. 1991; 30: 8991-8996Crossref Scopus (53) Google Scholar). 5) Subsequent desensitization results from reduction in phosphorylation state of the KHD (13Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, 14Potter L.R. Hunter T. Methods. 1999; 19: 506-520Crossref PubMed Scopus (33) Google Scholar). We have previously brought out the remarkable conservation of spacing between the cysteine residues found in the extracellular domain of nearly all of the guanylyl cyclases (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). More precisely, we took as a basic observation the presence two invariant cysteines, spaced by 6–8 residues, which are present in nearly all of the juxtamembrane domains of NPRs (Fig. 1). These two cysteines have been shown to be linked through an intrachain disulfide bond in rat NPR-A (16Miyagi M. Misono K.S. Biochim. Biophys. Acta. 2000; 1478: 30-38Crossref PubMed Scopus (34) Google Scholar). On the other hand, we also pointed out a noticeable exception found in the NPR-C5 receptor, where the first juxtamembrane cysteine is absent (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) (Fig.1). Consequently, the only juxtamembrane cysteine (Cys469) in NPR-C5 is free to form an interchain disulfide bridge. Hence, this receptor is found as a covalent homodimer (6Itakura M. Iwashina M. Mizuno T. Teizo I. Hagiwara H. Hirose S. J. Biol. Chem. 1994; 269: 8314-8318Abstract Full Text PDF PubMed Google Scholar). By analogy to the cysteine distribution of NPR-C5, we previously designed the mutation C423S in NPR-A, which eliminates its first juxtamembrane cysteine (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The expectation was that, in the absence of this cysteine (equivalent to Cys423 in NPR-A), it would permit interchain linkage of the second cysteine (Cys432 in NPR-A, equivalent to Cys469 of NPR-C5). Indeed, this mutation led to a spontaneously disulfide-bridged NPR-AC423S dimer. This NPR-AC423S mutant was also found to be constitutively activated, and it displayed an important increase in the binding affinity of pBNP, a weak agonist (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Using these observations, we proposed a model where agonists are inducing a dimeric "tightening" in the juxtamembrane region of NPR-A, hence allowing catalytic activation of the guanylyl cyclase. However, we indicated at the time that we could not exclude the contribution of a conformational change induced by the mutation independently of the interchain disulfide linkage (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). For instance, it was not known if the disruption of the Cys432–Cys423 bond might by itself take part in the constitutive activation of NPR-AC423S. In the current study, our objective is to definitively demonstrate that a juxtamembrane dimerization event is associated with NPR-A activation. To limit eventual structural alterations, we chose to avoid the disruption of the Cys423–Cys432 internal bond. For this, we referred to a minor splicing isoform of NPR-C (NPR-C6) that displays a supplementary juxtamembrane cysteine also forming an accessory interchain disulfide bridge (17Mizuno T. Iwashina M. Itakura M. Hagiwara H. Hirose S. J. Biol. Chem. 1993; 268: 5162-5167Abstract Full Text PDF PubMed Google Scholar). By comparing the juxtamembrane regions of NPR-C6 and NPR-A, this supplementary cysteine in NPR-C6 aligns with the aspartate 435 in NPR-A (Fig. 1). We thought that the addition of a cysteine at position 435 might lead again to a covalently dimerized NPR-A. We thus verified if NPR-AD435Cforms a disulfide-linked dimer. We found that although this mutant displays only trace levels of spontaneous covalent dimerization, agonists can induce such a dimeric linkage. This characteristic allowed us to define important constraints involved in receptor activation. rNPR-A mutants were engineered in the expression vector PBK-Neo (Stratagene). The construction of the His-tagged HT-ECD has already been described (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). This construct includes all of the extracellular domain up to Leu440 followed by Arg-Ser-His6. HT-ECDD435C was obtained by mutating the Asp435 in Cys with the mutagenic primer 5′-CCTGCAACCAATGCCACTTTTCGAC-3′ using the Transformer mutagenesis kit from CLONTECH. NPR-AD435C was obtained by mutating Asp435 in Cys using the mutagenic primer 5′-CCTGCAACCAATGCCACTTTTCCAC-3′. A deletion mutant of the entire intracellular domain of NPR-A (ΔKC) was obtained by PCR usingTaq polymerase. The amplification was realized using a sense primer 5′-ATGCCTTCAGGAATCTGATGC-3′ and two antisense primers. The first antisense primer (5-AGAGCCTCTTTCACCCTTCCTGTATATGAAGAAAGA-3′) was limiting (1 pmol), and the other (5′-TTTTGGTACCTTAACCTCTGGTAGAAGAGCCTCTTTCACCCTT-3′) was in excess (100 pmol). The amplified fragment included codons 217–464, followed by the epitope GERGSSTRG, a stop codon, and finally a KpnI site. The polymerase chain reaction product was codigested withEcoRV–KpnI. This fragment (codons 416 to Stop) was inserted in PBK-NPR-A, which had been previously codigested with the same enzymes. The resulting construction included the whole ectodomain, the transmembrane-spanning domain, and the two first intracellular residues (Arg463-Lys464), followed by the epitope and a stop codon. The ΔKCD435Cwas obtained by following the same strategy but using NPR-AD435C as an initial polymerase chain reaction template. The constructions and the mutations were confirmed by sequencing on the two strands using the Sequenase kit from Amersham Pharmacia Biotech. The human embryonal kidney cell line 293 (American Type Culture Collection) was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units of penicillin/streptomycin in a 5% CO2 incubator at 37 °C. For the cyclic GMP stimulation experiments, cells of the NPR-A and NPR-AD435C clones were seeded at 105cells/well onto 24-well cluster plates. Experiments were performed when the cells reached subconfluence. Transient expression of ΔKC, ΔKCD435C, HT-ECD, and HT-ECDD435C was obtained by the transfection of constructs in PBK-Neo using the CaHPO4 precipitation as described elsewhere (18Lia F. Rajotte D. Clark S.C. Hoang T. J. Biol. Chem. 1996; 271: 28287-28293Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). For HT-ECD and HT-ECDD435C, 20 μg of DNA/10-cm plate was transfected. For the truncated receptors (ΔKCs), the quantity of transfected PBK-ΔKC had to be reduced to obtain a level of expression comparable with the full-length receptor. This level was obtained by transfecting 2.5 μg of PBK-ΔKC or PBK-ΔKCD435C mixed with 17.5 μg of the PBK-Neo vector. For the stable expression of the rNPR-A and rNPR-AD435C, the cells were transfected with 20 μg of DNA/10-cm plate, and clones were selected in 600 μg/ml G-418 (Geneticin; Roche Molecular Biochemicals) in culture medium. Stable clones expressing wild type NPR-A and NPR-AD435C were plated in 10-cm plates and were allowed to grow to subconfluence. After the cells were washed twice with serum-free DMEM, 6 ml of the same medium (37 °C) containing 0.5% BSA and varying concentrations (10−12 to 10−6m) of rANP were added to individual plates. The induction was allowed to proceed for 30 min in a 5% CO2 incubator at 37 °C. Following incubation, the cells were washed twice with phosphate-buffered saline (37 °C), and all liquid was removed. The plates were put directly in a freezer at −80 °C until further used. For membrane preparation, the frozen plates were put on ice, and 4 ml of ice-cold homogenization buffer (5 mm Tris, pH 7.4, 0.2 mm EDTA containing 10−6m aprotinin, 10−6m pepstatin, 10−6m leupeptin, 10−5m pefabloc) was immediately added. The cells were scraped, collected in centrifugation tubes, homogenized twice for 20 s with a Polytron homogenizer, and centrifuged once for 30 min at 35,000 × g. The pellets were immediately resuspended in ice-cold freezing buffer (50 mm Tris, pH 7.4, 0.1 mm EDTA, 250 mm sucrose, 1 mm MgCl2, and the protease inhibitors) and stored at −80 °C. The protein concentration was determined using the BCA protein assay kit (Pierce). The induction of receptor dimerization was assessed by Western blotting after the separation of membrane proteins (25 μg) on a 5% SDS-PAGE in the presence or absence of β-mercaptoethanol in the loading buffer. Membranes used for the binding studies and the in vitro induction of NPR-AD435Cdimerization were prepared as follows. 72 h post-transfection for ΔKC and ΔKCD435C, or at subconfluence for the stable clones expressing NPR-A and NPR-AD435C, the cells were rinsed twice with phosphate-buffered saline and lysed in ice-cold homogenization buffer (5 mm Tris, pH 7.4, 0.2 mm EDTA, and the protease inhibitors). The cells were scraped, collected in centrifuge tubes, homogenized twice for 20 s with a Polytron homogenizer, and centrifuged for 30 min at 35,000 × g. The pellets were resuspended and washed twice in the same buffer. Finally, membranes were resuspended in ice-cold freezing buffer (50 mm Tris, pH 7.4, 0.1 mm EDTA, 250 mm sucrose, 1 mm MgCl2, and the protease inhibitors), frozen in liquid nitrogen, and stored at −80 °C. HT-ECD and HT-ECDD435C were purified from cell culture medium collected 72 h post-transfection. Supernatants were dialyzed three times against 90 volumes of 50 mm sodium phosphate buffer, pH 7.4, containing 0.3 m NaCl. After adding 16% glycerol, the dialysate was aliquoted, frozen in liquid nitrogen, and kept at −80 °C. The His-tagged ectodomains were purified on Ni2+-nitrilotriacetic acid-agarose gel (Qiagen) as described elsewhere (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Ectodomains were eluted from the gel with 500 mm imidazole. The eluates were finally dialyzed in sodium phosphate buffer, pH 7.4, containing 0.3 m NaCl using Slide-A-Lyzer cassettes (molecular weight cut-off of 10,000; Pierce). 25 μg of membrane proteins obtained from stable clones expressing NPR-A or NPR-AD435C were added to 500 μl of cold incubation buffer (50 mm Tris, pH 7.4, 0.1 mm EDTA, 0.5% BSA, and the protease inhibitors) containing 1 μm agonist. For the specificity study, rANP, pBNP, atriopeptin I (API), CNP, or C-ANF were included in the incubation mixture. The agonist induction of disulfide linkage was allowed to proceed for 22 h at 4 °C. Following incubation, the samples were centrifuged in a microcentrifuge at 10,000 × gfor 10 min. The pellets were carefully resuspended in ice-cold deionized water, and 2× SDS-PAGE sample buffer (without β-mercaptoethanol) was immediately added. The samples were immediately boiled for 5 min. The covalent dimerization was assessed by Western blotting after the separation of membrane proteins on a 5% SDS-PAGE. The induction of HT-ECDD435C was tested at 4 °C or at 22 °C, with 1 μm rANP, for 22 h in 0.1 ml of binding buffer (50 mm sodium phosphate buffer, pH 7.4, 0.3 m NaCl, 1 mm EDTA, 0.1% BSA, 0.05% lysozyme). The presence of covalent dimer was assessed by Western blotting after the separation of proteins (nonreducing conditions) on a 7.5% SDS-PAGE. 125I-rANP was prepared using the lactoperoxidase method as described elsewhere (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Binding to membranes was performed at 4 °C for 22 h in 1 ml of binding buffer (50 mm Tris, pH 7.4, 0.1 mm EDTA, 5 mm MnCl2, and 0.5% BSA). Competition experiments were done by incubation of 3–5 μg of HEK 293 membrane expressing rNPR-A, rNPR-AD435C, ΔKC, or ΔKCD435C with 10 fmol of 125I-rANP and increasing concentrations of nonradioactive peptides. Bound125I-rANP was separated from free ligand by filtration on GF/C filters precoated with 1% polyethyleneimine. Cells stably expressing rNPR-A and rNPR-AD435C were allowed to grow to subconfluence on 24-well cluster plates. The wells were washed twice with serum-free DMEM and were incubated in a final volume of 1 ml of the same medium containing 0.5 mm3-isobutyl-1-methylxanthine (IBMX), 0.5% BSA, and varying concentrations (10−12 to 10−7m) of rANP. After 1 h of incubation, the medium was collected, and extracellular cyclic GMP was determined by radioimmunoassay as described elsewhere (19Fethiere J. Meloche S. Nguyen T.T. Ong H. De, Lean A. Mol. Pharmacol. 1989; 35: 584-592PubMed Google Scholar). After the assay, 1× SDS-PAGE sample buffer (95 °C) was added to several wells. The wild type and mutant receptor levels were estimated by Western blotting, which was used to normalize their relative cGMP production. Stable clones expressing NPR-A and NPR-AD435C were allowed to grow to subconfluence on 10-cm plates. After having washed the cells with serum-free DMEM, 6 ml of DMEM (37 °C) containing 0.5% BSA and 10−7m rANP was added to the plates. The incubation was allowed to proceed for 30 min in a 5% CO2 incubator at 37 °C. The cells were then carefully washed twice with DMEM, 0.5% BSA (37 °C) and incubated for another 30 min. After this postincubation, the cells were washed again twice with phosphate-buffered saline (37 °C). Membrane preparation was done as described above except that homogenization, washings (three times), and freezing were realized in 50 mm HEPES, pH 7.4, containing 20% glycerol, 50 mm NaCl, 10 mm NaPO4, 0.1m NaF, 1 mm Na3VO4, and the protease inhibitors. The protein concentration was determined, and these membranes were used for guanylyl cyclase assays as described in other studies (13Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, 20Potter L.R. Hunter T. Mol. Cell. Biol. 1998; 18: 2164-2172Crossref PubMed Scopus (125) Google Scholar). 5 μg of membrane proteins were incubated during 10 min at 37 °C in 50 mm Tris-HCl, pH 7.6, with 10 mm theophylline, 2 mm IBMX, 10 mm creatine phosphate, 10 units of creatine kinase, 1 mm GTP, and 4 mm MgCl2. Different conditions were tested using GTP alone (basal) or by adding 1 μm rANP, 1 mm ATP, rANP and ATP together or adding 1% Triton X-100 with 4 mm MnCl2instead of MgCl2. Cyclic GMP was separated from GTP by chromatography on alumina and evaluated by radioimmunoassay as previously reported (19Fethiere J. Meloche S. Nguyen T.T. Ong H. De, Lean A. Mol. Pharmacol. 1989; 35: 584-592PubMed Google Scholar). Membrane proteins were separated on SDS-PAGE in the presence or absence of 5% β-mercaptoethanol in the loading buffer. The proteins were transferred to a nitrocellulose membrane (Bio-Rad) using the liquid Mini Tans-Blot System (Bio-Rad). Detection of NPR-A and ΔKC was achieved using a rabbit polyclonal antiserum raised against the sequence YGERGSSTRG and purified by affinity chromatography. This sequence corresponds to human NPR-A carboxyl terminus preceded by a tyrosine for radioiodination purposes. The rat NPR-A differs from this epitope at a single position; however, both receptors are recognized. Specific signal was probed with an HRP-coupled anti-rabbit polyclonal antibody according to the ECL Western Blotting Analysis System (Amersham Pharmacia Biotech). For the His-tagged HT-ECD and HT-ECDD435C, purified aliquots were run on a 7.5% SDS-PAGE without β-mercaptoethanol in the sample buffer. In this case, 6m urea was included in the sample buffer and in the gel, since it was found to improve the immunodetection. Proteins were transferred to a nitrocellulose membrane as described above, and the ectodomains were detected using a commercial mouse anti-tetrahistidine antibody (Qiagen) according to the technique provided by the manufacturer. Specific signal was probed with a horseradish peroxidase-coupled anti-mouse polyclonal antibody using the ECL Western Blotting Analysis System. Dose-response curves were analyzed with the program AllFit for Windows based on the four-parameter logistic equation (21De Léan A. Hancock A.A. Lefkowitz R.J. Mol. Pharmacol. 1982; 21: 5-16PubMed Google Scholar). Radioligand binding data were analyzed with the same program on a model for the law of mass action (22De Léan A. Munson P.J. Rodbard D. Am. J. Physiol. 1978; 235: E97-E102Crossref PubMed Google Scholar). For simplicity, the same binding data were also analyzed as dose-response curves, and ED50 values are mentioned in the discussion instead ofK d values. We considered the possibility that the introduction of a cysteine at position 435 could cause a covalent dimerization of rNPR-A subunits. This site was chosen because of its linear alignment with an accessory cysteine involved in the linkage of NPRC-C6 dimers (Fig. 1). Initial studies with rNPR-AD435C expressed in HEK293 showed, however, only low levels of spontaneous dimerization. At this point, we proceeded to further testing and explored the possibility that some dimeric linkage might be induced by rANP, the main agonist of NPR-A. This hypothesis turned out to be right, since dimers of rNPR-AD435C could be detected after a cellular incubation with 1 μm rANP (30 min, 37 °C). We further detailed this induction through a dose-effect study using a clone stably expressing NPR-AD435C (Fig.2). A clear signal of ∼260 kDa corresponding to NPR-AD435C dimers is detected on nonreducing SDS-PAGE. It is noteworthy that such an induction is not seen in cells expressing wild type NPR-A. A densitometric analysis of the dimeric induction gave an approximate ED50 of ∼900 pm, which is higher than the ED50 obtained in cellular guanylyl cyclase stimulation (87 pm). The exact explanation for this difference is not known. However, the reaction between cysteines leading to disulfide formation could be a limiting element of the process and might affect the apparent dose-effect curve. Therefore, considering this supplementary element, an ED50of ∼900 pm constitutes an acceptable value. These results are showing that ANP induces a particular dimeric contact in the juxtamembrane domain of NPR-AD435C. This conclusion is in accordance with our previous hypothesis based on the observation of constitutively dimerized NPR-AC423S mutant (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; 274: 9752-9759Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, the ANP-inducible dimerization properties of NPR-AD435C directly proves this mechanism. Furthermore, the near absence of NPR-AD435C covalent dimerization at basal state reveals the existence of a constraint that represses this contact in absence of ANP. To assess if the D435C mutation altered the NPR-A function, we studied cellular guanylyl cyclase activation by rANP. As shown in Fig. 3, the ED50 obtained for the wild type (71 ± 12.8 pm) and the mutant (87 ± 4.6 pm) are essentially comparable, and their maximal levels of stimulation are similar. It should be noted that we reproducibly observed a very slight increase in the basal activity of NPR-AD435C as compared with that of NPR-AWT (Fig. 3). This almost negligible increase might indicate that the D435C mutation has very slightly modified the interactions in the juxtamembrane region. Alternatively, it may be attributed to a trace level of NPR-AD435C dimer sometimes detectable on Western blot through signal overexposure (not shown). From this result, it can be reasonably concluded that NPR-AD435C displays a response to rANP that is very close to that for wild type NPR-A. Therefore, the conclusions based on the results obtained with this mutant are likely to be applicable to the wild type receptor. We investigated if the induction of NPR-AD435C covalent dimerization goes along with persistent activation. Guanylate cyclase activity was tested in vitrowith membrane preparations obtained from ANP-treated cells. Stable clones expressing NPR-AWT and NPR-AD435C were treated for 30 min (37 °C) with 10−7m ANP. Removal of the ligand was realized through extensive washing of cells, followed by postincubation without ligand and washings during membrane preparation (see "Experimental Procedures"). The membranes were submitted to several conditions using GTP alone (basal) or together with ATP, ANP, ATP plus ANP, and Triton/Mn2+. The results were expressed as a percentage of the activation obtained with Triton/Mn2+. This condition stimulates NPR-A to its maximal catalytic level and is commonly used as an internal reference of enzyme activity. Several observations can be made from the results. First, the level of costimulation with ATP plus ANP tends to diminish in membranes obtained from ANP-treated cells (NPR-A: nontreated 38.9 ± 4.5%, treated 29.8 ± 5.9%; NPR-AD435C: nontreated 39.8 ± 6.9%, treated 29.6 ± 2.6%). This may be attributed to some

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