Natriuretic Peptide Receptor A Activation Stabilizes a Membrane-distal Dimer Interface*

We have shown previously (Rondeau, J.-J., McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130–2136) that atrial natriuretic peptide (ANP) stabilizes a dimeric form of the natriuretic peptide receptor A (NPRA) by simultaneously interacting with both receptor subunits. However, the first crystallographic study of unliganded NPRA extracellular domain documented a V-shaped dimer involving a membrane-proximal dimer interface and separate binding sites for ANP on each monomer. We explored the possibility of an alternative A-shaped dimer involving a membrane-distal dimer interface by substituting an unpaired solvent-exposed cysteine for Trp74 in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp74 from both subunits drastically differs, depending on whether the V-shaped (84 Å) or the A-shaped (8 Å) dimer model is considered. In contrast with the expected results for the reported V-shaped dimer, the NPRAW74C mutant was constitutively covalently dimeric. Also, the subunits spontaneously reassociated following transient disulfide reduction by dithiothreitol and reoxidation. However, ANP could neither bind to nor activate NPRAW74C. Permanent disulfide opening by reduction with dithiothreitol and alkylation with N-ethylmaleimide rescued ANP binding to NPRAW74C. The NPRA mutant could be maintained as a covalent dimer while preserving its function by crosslinking with the bifunctional alkylating agent phenylenedimaleimides (PDM), the ortho-substituted oPDM being more efficient than mPDM or pPDM. These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that ANP binding stabilizes the receptor dimer with more stringent spacing at the dimer interface.

The interaction of the natriuretic peptide ANP 1 with the binding domain of its receptor NPRA is a determinant for proper signal transduction leading to cyclic GMP production and cellular response in the cardiovascular system (1). Natriuretic peptides counterbalance the renin-angiotensin system by lowering blood pressure and increasing natriuresis and diuresis. This role is exemplified in knockout mice with abrogated or reduced expression of ANP or its receptor NPRA and who are hypertensive (2,3). Natriuretic peptide receptors are members of the membrane guanylyl cyclase family (4). These receptors are composed of five domains. The glycosylated extracellular domain is required for binding the activating agents, e.g. natriuretic peptides, guanylin, and the sea urchin sperm-activating peptides (4 -6). It is linked through a single transmembrane domain to the intracellular portion, which is composed of three domains. First a membrane-proximal domain, which is homologous to a protein kinase domain and presumably binds ATP but lacks catalytic function. This phosphorylated domain serves as a regulatory component for signal transduction (4,7,8). It is also a target for the intracellular guanylyl cyclase activating protein (GCAP), which directly activates retinal and olfactory guanylyl cyclases (9). The kinase homology domain (KHD) is connected through a coiled-coil (10,11) to the guanylyl cyclase domain, effecting the activation process by producing cyclic GMP (11). Initial studies have shown that natriuretic peptide receptors could be documented as constitutively noncovalently dimeric through the interaction of both the extracellular (13) and intracellular domains (10). Photoaffinity derivatives of ANP, with photosensitive substitutions at both ends of the peptide, could specifically crosslink the receptor dimer, indicating that the peptide must be interacting with both subunits (14).
Receptor dimerization is essential for the activation of the catalytic domain of retinal guanylyl cyclase, because both GTP substrates must interact with each subunit (12). In that model system, the coiled-coil connecting the intracellular domains appears to maintain apart both guanylyl cyclase moieties in the basal state. Activation then appears to involve the release of the constraint imposed by the coiled-coil on the catalytic domain (12). The membrane-proximal kinase homology domain is also a determinant in signal transduction. In the absence of ANP, the KHD domain maintains the receptor in the basal state (7). The incoming activation stimulus appears to favor ATP binding and relieve this tonic inhibition through a concerted transmembrane allosteric change, resulting in activation of the catalytic moiety (15). The initial activation step of NPRA is likely to involve a conformational change in the extracellular juxtamembrane region. Indeed, it has been reported that ANP activation leads to increased protease sensitivity of the juxtamembrane region (16,17). Site-directed mutagenesis of C423S located in the juxtamembrane region leads both to constitutive activation and receptor covalent dimerization through the exposed and unpaired Cys 432 (18). This activation was attributed by Misono et al. (16) to the conformational change occurring in the juxtamembrane region more than to the covalent dimerization process, because a double mutation, C423S/C432S, was shown to be also constitutively active but not covalently dimeric (16). Although this interpretation is viable, the occurrence of a constitutive disulfide bridge still indicates the proximity of the extracellular juxtamembrane region of the receptor subunits. The contribution of the extracellular juxtamembrane region in mediating ANP-induced activation is also well documented in a D435C mutant exposing a free cysteine three residues further toward the transmembrane region (19). This mutant is not covalently dimeric in the basal state, but ANP induces disulfide bridge formation through Cys 435 upon receptor activation.
When the extracellular domain of NPRA is expressed in truncated soluble form, it behaves in solution as a monomer (20). Agonist binding to the soluble extracellular domain induces noncovalent dimerization (20). Misono et al. proposed that ANP is binding to the soluble receptor dimer according to a 2:2 stoichiometric ratio (20). Their results, however, clearly document that at the midpoint of the dimerization process 1 M ANP can dimerize a 2 M receptor subunit, implying a 1:2 stoichiometric ratio. This 1:2 ratio is more in agreement with the ratio that we previously documented in full-length receptor by comparing ANP binding capacity with immunoassayable receptor subunit density (14).
The NPRA receptor extracellular domain has been crystallized in the unbound form by van den Akker et al., showing that it is composed of a homodimer (21). Each subunit displays a typical bilobed periplasmic protein folding and contains a chloride ion. In this initial report, the receptor structure was presented as a V-shaped dimer with a subunit interface located in the membrane-proximal lobe (Fig. 1). Prediction of the localization of the ANP binding region on this receptor was helped by our previous photoaffinity tagging results (22) and was confirmed by site-directed mutagenesis (21). However the predicted localization of the ANP binding site in the V-shaped dimer was more on the lateral face of each receptor subunit in a position not easily amenable to simultaneous contact of the peptide with both receptor subunits or to any conformational change in the receptor (21). A more recent report on the structure of the CNP-bound NPRC receptor, which is devoid of guanylyl cyclase and mainly serves for peptide clearance, indicated an A-shaped dimeric structure with the dimerization interface in the membrane-distal lobe (23). In that receptor dimer structure a single CNP molecule is binding within the intersubunit cleft, therefore interfacing both receptor subunits (23). The A-shaped dimer structure was also recognized by van den Akker (24) in the original crystal structure of NPRA ( Fig.   1). This dimer conformation would conform to the 1:2 stoichiometric ratio of peptide to receptor subunit and provide, if applicable to the natriuretic peptide A receptor, a more conceivable mechanism for ANP high affinity binding and receptor dimer activation.
In attempting to explore the various conformations of the NPRA dimer we noticed that the Trp 74 residues located in the membrane-distal lobe of the receptor were separated by drastically different ␣-carbon distances in the V-dimer and the A-dimer conformations (82 Å versus 8 Å, Fig. 1). The position of Trp 74 in NPRA is analogous to that of a cysteine involved in disulfide bridging of the eel NPRC dimer (25). We thus explored the ability of a W74C mutant of rat NPRA to form a covalent dimer through either a disulfide bridge or longer spacers provided with bifunctional dimaleimide derivatives. The results indicate that the W74C mutant is constitutively covalently dimeric, confirming the A-shaped dimer and excluding the V-shaped dimer conformation. But the disulfide-bridged dimeric mutant is inactive. However, proper binding of ANP and receptor activation can be achieved by maintaining a slightly wider spacing between residues 74 using bifunctional crosslinkers. Reciprocally, ANP binding hinders disulfide bridge formation in the W74C mutant. Thus, the ANP-bound and activated receptor dimer appears to adopt a more stable conformation than in the unbound state. These results contribute to the understanding of the conformational changes occurring early on during the activation of NPRA.

EXPERIMENTAL PROCEDURES
Construction of NPRA Mutants-A wild type (WT) rat NPRA clone inserted into pBK-CMV (Stratagene) between sites NheI and KpnI (26) was used for generating the various mutants. NPRA W74C (Fig. 2) was obtained by mutating Trp 74 into Cys according to a Clontech kit using the mutagenic primer 5Ј-GACCTCAAGTGTGAGCACAGCC-3Ј. The mutation was checked by sequencing, and the fragment encompassing the mutation was subcloned into NPRA WT . The ⌬KC C423S,C432S and ⌬KC C423S,C432S,W74C mutants ( Fig. 2) were obtained starting from the deletion mutant ⌬KC WT lacking all cytoplasmic domain (19) by sequentially mutating Cys 423 and Cys 432 to Ser with the QuikChange kit (Stratagene) using first the oligonucleotide pair 5Ј-CCTGACGTCCCTA-AATCTGGCTTTGACAATGAGG-3Ј and its complementary and then the oligonucleotide 5Ј-GACAATGAGGACCCAGCCTCCAACCAA-GACCACTTTTC-3Ј and its complementary sequence. The mutation was checked by sequencing, and the fragment between sites EcoRI and KpnI was subcloned into NPRA WT and NPRA W74C to generate mutants ⌬KC C423S,C432S and ⌬KC C423S,C432S,W74C , respectively.
Cell Culture and Receptor Protein Expression-HEK293 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units of penicillin/streptomycin in a 5% CO 2 incubator at 37°C. Transfection assays were carried out in 100-mm plates (1.2 ϫ 10 6 cells) using the calcium phosphate co-precipitation technique as described previously (27).
Whole Cell Guanylyl Cyclase Stimulation-Cells were replated 48 h post transfection in 24-well cluster plates at 10 5 cells per well and incubated 24 h prior to agonist stimulation. The cells were washed twice with serum-free Dulbecco's modified Eagle's medium and incubated at 37°C in quadruplicate wells with varying concentrations of rANP (Peninsula) in the same medium containing 0.5% bovine serum albumin and 0.5 mM 1-methyl-3-isobutylxanthine. After a 1-h incubation, the medium was collected, and extracellular cyclic GMP was measured in duplicate by radioimmunoassay as described previously (28).
Preparation of Membranes-Membrane preparations of transfected HEK293 were done according to Labrecque et al. (18). Essentially, cells were harvested 72 h post transfection and homogenized in ice-cold buffer (10 mM Tris-HCl, pH 7.4, 1 M aprotinin, 1 M leupeptin, 1 M pepstatin, 10 M pefabloc, and 1 mM EDTA). After centrifugation at 40,000 ϫ g for 30 min, the pellets were washed twice and finally resuspended in freezing buffer containing 50 mM Tris-HCl, pH 7.4, protease inhibitors, 1 mM MgCl 2 , and 250 mM sucrose. Membranes were then frozen in liquid nitrogen and kept at Ϫ80°C until used. The protein concentration was determined using a BCA protein assay kit (Pierce).
In Vitro Modification of Cysteine Residues-Membranes (0.6 mg/ml) were treated with 5 mM of DTT (Sigma) in Tris-EDTA buffer (50 mM Tris-HCl, pH 7.4, 0.2 mM EDTA) at 22°C for 50 min in darkness under an argon atmosphere. Tubes containing membranes were then cooled on ice and centrifuged at 12,000 ϫ g for 15 min at 4°C. Membranes were washed with Tris-EDTA buffer alone, and pellets were resuspended in the same buffer at a concentration of 0.5 mg/ml. For control, membranes were incubated as described above, except that DTT was omitted. Following the reduction step, membranes were treated with PDM (30) to covalently bridge the cysteine residues. Membrane proteins (100 g) were incubated in darkness for 60 min at 4°C with 0.1 mM oPDM, mPDM, or pPDM (Aldrich) in a final volume of 0.2 ml of Tris-EDTA buffer. Control was obtained with the monovalent maleimide compound NEM (Fisher) at 0.1 mM. At the end of incubation, 10 mM NEM was added to all tubes to block all free unreacted cysteine residues and prevent spurious disulfide formation, and incubation was continued for 10 min at 4°C. Treated membranes were centrifuged, washed once, resuspended in freezing buffer, and kept at Ϫ80°C. For reoxidation of cysteine pairs, the DTT-reduced membrane proteins were treated with CuSO 4 -orthophenanthroline (Cu(OP) 2 ) (Fisher) according to Majima et al. (29). Membranes at a concentration of 0.5 mg/ml were incubated with or without 25 M Cu(OP) 2 for 10 min at 4°C. The reaction was stopped by the addition of NEM and EDTA, both at 10 mM. After further incubation for 10 min at 4°C, treated membranes were centrifuged, and the pellets were washed once in Tris-EDTA buffer. The final pellets were resuspended in freezing buffer and kept at Ϫ80°C. When ANP was tested for its ability to interfere with the covalent receptor dimerization, 100 nM rANP was added at 4°C 20 min prior to the treatment with Cu(OP) 2 .
Immunoblot Analysis-Membrane protein samples (4 -10 g) were solubilized in Laemmli sample buffer (62 mM Tris-HCl, 2% SDS, 10% glycerol, 0.001% bromphenol blue, pH 6.8) without (non-reducing) or with (reducing) 5% ␤-mercaptoethanol and heated at 100°C for 3 min. Electrophoresis was performed in a 7.5% polyacrylamide gel for the ⌬KC mutants and a 5% polyacrylamide gel for the full-length NPRA mutant. Following electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad) using the liquid Mini Trans-Blot system (Bio-Rad). Detection of receptor was achieved using an affinitypurified antibody from a rabbit polyclonal antiserum raised against the carboxyl terminus sequence of NPRA (18). Specific signals were probed  2. Structure of the NPRA mutants used. NPRA WT was used as control for the NPRA W74C mutant exposing an unpaired cysteine (S) at the top of the A-dimer interface (Fig. 1). The ⌬KC C423S,C432S mutant was obtained by truncating the cytoplasmic domain (INT) containing unpaired cysteines and by mutating both exposed Cys 423 and Cys 432 in the extracellular domain (EXT) close to the transmembrane domain (TM). The ⌬KC C423S,C432S,W74C mutant contains an additional unpaired and exposed Cys 74 .
with a horseradish peroxidase-coupled second antibody according to the ECL Western blotting analysis system (Amersham Biosciences).
Receptor Binding Assays-125 I-rANP was prepared using the lactoperoxidase method as described previously (18). The specific activity of the high pressure liquid chromatography-purified radioligand was at least 2000 Ci/mmol. Membranes from HEK293 expressing rat NPRA WT , NPRA W74C , ⌬KC C423S,C432S , or ⌬KC C423S,C432S,W74C (0.2-1.0 g) were incubated in duplicate with 10 fmol of 125 I-rANP for 20 h at 4°C in 1 ml of 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl 2 , 0.1 mM EDTA and 0.5% bovine serum albumin. Nonspecific binding was defined by the addition of non-radioactive rANP at 100 nM. Bound ligand was separated from free ligand by filtration on GF/C filters pretreated with 1% polyethylenimine. Filters were washed 4 times and counted in an LKB gamma counter.
Data Analysis-Dose-response curves were analyzed with the program AllFit for Windows based on the four-parameter logistic equation (31).

RESULTS
Covalent Dimeric State of the NPRA W74C Mutant-The two proposed arrangements of the NPRA extracellular domain as a V-shaped and an A-shaped dimer mainly differ in terms of the localization of the dimer interface in the membrane-proximal and the membrane-distal lobes of the receptor subunits (Fig. 1). The surface-exposed residues Trp 74 are also widely spaced in the V-dimer (82 Å), whereas these residues are juxtaposed (8 Å) at the membrane-distal end of the dimer interface in the Ashaped dimer. Therefore, substitution of Trp 74 with Cys should yield a disulfide-bridged dimer only in the case of the A-shaped dimer conformation. When the full-length NPRA W74C mutant transiently expressed in HEK293 cells is studied in SDS-PAGE under non-reducing conditions, we observe that the mutant is present almost exclusively as a dimer (Fig. 3, right panel). This dimer can be reduced by treatment with DTT followed by alkylation of free sulfydryls with NEM (Fig. 3, right panel). However, if the receptor mutant is reoxidized in the presence of Cu(OP) 2 following reduction with DTT, then the receptor mutant reassociates as a disulfide-bridged dimer (Fig. 3, right  panel). Thus, the disulfide bridge between the two exposed FIG. 4. Cys 74 disulfide reduction restores high affinity binding of ANP to NPRA W74C . ANP high affinity binding was measured in NPRA WT (open bars) and NPRA W74C mutant (hatched bars) following reduction and alkylation as described in the Fig. 3 legend. The membranes (1 g) were then incubated with 10 pM 125 I-ANP with (nonspecific binding) or without (total binding) an excess of unlabeled ANP for 20h at 4°C. Specific binding was calculated by subtracting nonspecific from total binding, and values were normalized to those obtained with DTT plus NEM and expressed as mean Ϯ S.E. of duplicates. The experiment was repeated twice with similar results. Cys 74 residues is constitutive and is formed spontaneously when reducing conditions are replaced by mild oxidizing conditions. Therefore, residues 74 from both receptor subunits should be spontaneously adjacent, in accordance with the Ashaped conformation and in drastic contrast with the prediction of the V-shaped dimer originally proposed (Fig. 1). In addition, the disulfide bridge, which is likely to occur early on during biosynthesis of the NPRA W74C mutant, does not appear to result from an imposed constraint, because it can be formed again following reduction of the mature and unstimulated receptor protein.
The NPRA W74C Mutant Is Inactive in a Disulfide-bridged Dimeric State-Because the inter ␣-carbon distance (8 Å) of the two Trp 74 residues is slightly longer than the expected distance for disulfides (5-7 Å), we wondered if bridging the two Cys 74 might interfere with the high affinity binding and activation processes of ANP on NPRA W74C . Indeed the receptor mutant is almost completely devoid of high affinity for ANP in membrane preparations from HEK293 cells transiently expressing NPRA W74C (Fig. 4). The receptor mutant is also almost completely insensitive to ANP-activation with marginal response (Fig. 5). This loss of function is not due to an irreversible alteration by the mutation of receptor folding, because reduc-tion and alkylation of the dimer leading to a monomer restores high affinity binding for ANP (Fig. 4). Moreover, reoxidation of the Cys 74 disulfide following reduction leads again to the loss of high affinity binding for ANP (Fig. 4). Thus, although Cys 74 disulfide bridge formation is spontaneous for the basal state of the receptor, this tight dimer conformation prevents ANP high affinity binding and functional activation. These results suggest that, unlike the case for the basal state of the receptor, the ANP-bound and active state requires more stringent proximity conditions, which the Cys 74 disulfide prevents by slightly constraining the inter ␣-carbon distance of residues 74 to Ͻ8 Å.
Proper Spacing of Cross-linked Cys 74 in ⌬KC C423S,C432S,W74C Mutant Is Required for High Affinity ANP Binding-To verify that proper spacing of residues 74 from both receptor subunits is required for the active state, we looked for various types of bifunctional cross-linking agents specific for surface-exposed sulfydryls. Such agents had to be reacting with Cys 74 following the disulfide bridge opening with DTT. Initial attempts to use dual methane thiosulfonate reagents (MTS reagents; Toronto Research Company) were fraught with difficulties. We observed that preliminary reduction with DTT left other free and reactive cysteines besides Cys 74 . Thus, although MTS compounds could properly cross-link free Cys 74 , additional spuri- ous disulfide formation occurred, resulting in multiple receptor oligomers. Also, because MTS compounds form a reducible covalent link with cysteines, SDS-PAGE could not be performed under reducing conditions, precluding elimination of spurious disulfides. We thus resorted to dimaleimide crosslinking reagents, which have been used for sizing inter-cysteine distances in proteins (30). oPDM, mPDM, and pPDM can efficiently cross-link neighboring exposed cysteines, and the resulting dimers can be studied in SDS-PAGE under reducing conditions with the advantage of removing spurious disulfides. In addition, the number of potential free and exposed cysteines was reduced by truncating the cytoplasmic domain of NPRA with many potentially exposed free cysteines and by mutating both Cys 423 and Cys 432 , which are exposed in the juxtamembrane region, leaving only two buried and unreactive disulfides (Fig. 2). Thus for the ⌬KC C423S,C432S,W74C mutant, the additional W74C mutation could provide the only exposed free cysteine, avoiding spurious cysteine reactions.
Following transient reduction with DTT, the ⌬KC C423S,C432S,W74C mutant was efficiently crosslinked as a non-reducible dimer by oPDM and mPDM and somewhat less by pPDM (Fig. 6, right panel). The untreated disulfide-bridged ⌬KC C423S,C432S,W74C mutant was devoid of high affinity ANP binding (Fig. 7). As for the full-length NPRA W74C mutant, reduction and alkylation of ⌬KC C423S,C432S,W74C with DTT and NEM could restore ANP binding by cleaving the interchain disulfide (Fig. 7). However crosslinking of the receptor subunits with oPDM completely restored ANP binding (Fig. 7) while maintaining a covalent dimer with a wider spacing of the Cys 74 from both subunits than was obtained with the disulfide (Fig.  6). mPDM and pPDM were much less effective, presumably because the inter Cys 74 spacing imposed by those cross-linking agents was too wide, therefore interfering with optimal positioning of the two subunits at the dimer interface. ANP binding measurements of the cross-linked receptor indicated that those differences were not due to reduction in binding capacity but in binding affinity (data not shown), suggesting that suboptimal spacing of the receptor subunits perturbed the binding interaction of ANP in the binding cleft.
ANP binding hinders Cys 74 Disulfide Bridging of the ⌬KC C423S,C432S,W74C Mutant-Because disulfide bridging of W74C mutants leads to a slightly constrained inactive dimer, presumably because the dimer conformation does not satisfy the more stringent interface spacing required for high affinity ANP binding, we wondered whether, in reciprocal fashion, ANP binding to the ⌬KC C423S,C432S,W74C mutant could prevent Cys 74 disulfide formation. The receptor mutant was first reduced with DTT (Fig. 8) and then incubated with saturating concentrations of ANP before attempting to reoxidize the Cys 74 disulfide in the presence of Cu(OP) 2 . When studied with SDS-PAGE under non-reducing conditions, the truncated W74C mutant was spontaneously dimeric (Fig, 8, right panel). In analogy with the full-length receptor (Fig. 4), this truncated mutant was also devoid of high affinity for ANP (Fig. 9), but peptide binding could be restored by reduction and alkylation. Incubation with ANP inhibited dimer formation following transient reduction with DTT (Fig. 8, right panel). Thus, unlike the basal inactive state of the NPRA receptor, which allows for spontaneous Cys 74 disulfide formation due, presumably, to conformational flexibility and mobility at the dimer interface, the ANP-bound and activated state displays more stringent interface positioning requirements, probably because ANP binding stabilizes the receptor dimer and thus reduces the conformational mobility of the subunits interface. DISCUSSION We have provided documentation that the extracellular domain of the natriuretic peptide receptor adopts an A-shaped dimer conformation with a membrane-distal dimer interface. All experiments were based on a membrane-anchored receptor, therefore ensuring that the conclusions would be representative of native cellular NPRA. The results do not confirm the initial V-shaped dimer conformation proposed for the unbound state of NPRA. According to that originally proposed conformation, the membrane proximal lobes would provide the dimer interface. In addition, the V-shaped conformation would allow for one ANP binding surface located on each side of the dimer, therefore resulting in a 2:2 stoichiometric ratio for ANP binding to NPRA (21). Although the membrane-proximal localization of the dimer interface is analogous to that for growth hormone receptor (32), it provides few hints about the activation process of NPRA, because the ANP molecules would preferentially, if not exclusively, interface with only one dimer subunit, in contrast with many cases documented to date for growth factor and cytokine receptors (32)(33)(34). The ANP-bound NPRA dimer is not likely to adopt the V-shaped conformation, because covalent cross-linking of the W74C mutant with oPDM preserves peptide binding and maintains the membrane-distal lobes in proximity, albeit more apart than with disulfide bridging but still considerably closer than what the V-dimer could accommodate.
The A-shaped dimer conformation subsequently proposed by van den Akker (24) for NPRA and documented by Garcia and co-workers for NPRC (23) is more similar to the conformation of other dimeric receptors, e.g. the glutamate metabotropic receptor (35). The constitutively dimeric properties of the native NPRA W74C mutant strongly suggest that this A-shaped conformation is natural and potentially contributes to a loose dimer in the basal inactive state of the membrane-anchored receptor. However, as pointed out by van den Akker (24) the membranedistal dimer interface area is probably insufficient to maintain by itself the A-dimer conformation, therefore explaining the monomeric state of the unbound extracellular domain in soluble truncated form (20). Indeed, ANP binding to an A-shaped NPRA dimer would be expected to significantly contribute to the surface of the dimer interface and therefore stabilize the dimer. This would explain the observation of ANP-induced dimerization of the extracellular domain (20) that would result from a huge increase of the dimerization constant induced by peptide binding.
Although the data presented show that the unbound state of NPRA is characterized by greater flexibility and mobility at the dimer interface, which is required for allowing for Cys 74 disulfide formation, the ANP-bound and active state of NPRA displays more stringent intersubunit distance requirements that are incompatible with Cys 74 disulfide formation but could be satisfied by proper spacing with oPDM. Thus, ANP binding is likely to stabilize the NPRA dimer, presumably by fitting within the inter subunit cleft below the dimer interface, therefore substantially contributing to the buried surfaces of the dimer interface and restraining its mobility. Reciprocally, binding of ANP within this cleft is likely to tightly retain the bound peptide, resulting in a high affinity and slow dissociation rate in agreement with reported observations. Thus, the monitoring of residues 74 at the membrane-distal end of the dimer interface is providing a very sensitive assessment of dimer positioning during receptor activation.
Although these results fully support the A-shaped dimer conformation involving a dimer interface in the membrane distal lobe of the extracellular domain, they do not exclude the existence of another extracellular domain interface in the juxtamembrane domain. Indeed the CNP-bound NPRC dimer used by Garcia and co-workers for crystallography included an interchain disulfide in the juxtamembrane portion of the extracellular domain (23). Also, the constitutive formation of a disulfide bridge at Cys 432 in the NPRA C423S mutant and the observation of an agonist-induced disulfide bridge three residues further in the case of the NPRA D435C mutant both strongly suggest that the juxtamembrane region connecting the bi-lobed periplasmic folded domain with the transmembrane domain is involved in some additional dimer interface, possibly contributing to the signal transduction process from the extracellular to the intracellular domains of the receptor. Crystallographic documentation of the structure of the ANPbound NPRA extracellular domain of the membrane-anchored receptor as well as the kinase homology domain should provide further insight into the signal transduction mechanism of membrane guanylyl cyclases and also contribute to better understanding of other single transmembrane domain receptors.