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Molecular and cellular physiology of the dissociation of atrial natriuretic peptide from guanylyl cyclase a receptors.

Guanylyl cyclase subtype A (GCA) is the main receptor that mediates the effects of atrial natriuretic peptide (ANP) in the regulation of plasma volume and blood pressure. The dynamics of the dissociation of ANP from GCA were investigated in cultured Chinese hamster ovary (CHO) cells stably transfected with wild-type (WT) or mutant GCA receptors. The rate of dissociation of specifically bound (125)I-ANP-(1-28) from intact CHOGCAWT cells at 37 degrees C was extremely rapid (K(off) = 0.49 +/- 0.02 min(-1)), whereas in isolated membranes prepared from these cells, the dissociation at 37 degrees C was >10-fold slower (K(off) = 0.035 +/- 0.006 min(-1)). The dissociation of ANP from CHOGCAWT cells showed remarkable temperature dependence. Between 22 and 37 degrees C, K(off) increased approximately 8 times, whereas between 4 and 22 degrees C, it increased only 1.5 times. Total deletion of the cytoplasmic domain or of the catalytic guanylyl cyclase sequence within this domain abolished ANP-induced increases in cGMP, dramatically slowed receptor-ligand dissociation by at least 10-fold, and abolished the temperature dependence of the dissociation of ANP. Deletion of the kinase-like domain led to maximal constitutive activation of guanylyl cyclase, markedly decreased K(off) to 0.064 +/- 0.006 min(-1), and also abolished the temperature dependence of dissociation. Substitution of Ser(506) by Ala and particularly the double substitution of Gly(505) and Ser(506) by Ala within the kinase-like domain markedly reduced ANP-induced increases in cGMP, whereas K(off) decreased modestly (albeit significantly) to 0.36 +/- 0.03 and 0.24 +/- 0.02 min(-1), respectively. As a whole, the results demonstrate for the first time that temperature per se or ATP alone cannot account for rapid GCA receptor-ligand dissociation under physiological conditions and suggest that ligand dissociation is modulated in part by the interaction of still unidentified cytosolic factors with the cytoplasmic domain of GCA.

Atrial natriuretic peptide (ANP), 1 a member of the natri-uretic peptide family that includes brain natriuretic peptide and C-type natriuretic peptide, plays a fundamental role in the regulation of blood pressure, plasma volume, and renal function (1,2). Two distinct classes of ANP receptors, named clearance and guanylyl cyclase (GC) receptors, have been biochemically and functionally well characterized (1,3).
Clearance receptors of ANP, the most abundant class of the natriuretic peptide receptors, have a single transmembrane domain, a short cytoplasmic tail of 37 amino acids, and an extracellular binding domain that has a significant homology to the extracellular domain of GC receptors (1, 4 -6). An extensive series of physiological, pharmacological, cellular, and genetic studies have shown that clearance receptors are importantly involved in the systemic and local clearance of ANP (7)(8)(9)(10)(11). This clearance function is accomplished by an efficient mechanism of receptor-mediated endocytosis. Endocytosed ANP is delivered to lysosomes, where it is hydrolyzed to its constituent amino acids, and the internalized receptors are recycled to the cell membrane (10,12). The efficiency of this receptor-mediated endocytic mechanism is enhanced by a relatively low rate of dissociation of ANP from cell-surface receptors (12).
Guanylyl cyclase subtype A (GCA) receptors mediate all of the known cardiovascular and renal effects of ANP (2,13). GCA receptors have a single transmembrane domain, an extracellular ligand-binding domain, and a cytoplasmic domain constituted by a catalytic GC sequence and a tyrosine kinase-like (TK) sequence interposed between the transmembrane and the catalytic domains (14). Between the TK and GC sequences there is an amphipathic ␣-helical region that is involved in higher order oligomerization of GCA receptors (15,16). Under basal conditions, the TK domain has an inhibitory effect on GC activity. It has been postulated that upon ANP (or brain natriuretic peptide) binding to the extracellular domain, ATP binds to the TK domain and allosterically activates the catalytic GC domain (14,17).
Previous studies in our laboratory have demonstrated that the native GCA in cultured glomerular mesangial and renomedullary interstitial cells is a constitutive membrane resident protein that does not undergo endocytosis and does not mediate lysosomal hydrolysis of ligand (18). Moreover, the dissociation of ANP from native GCA is very slow at subphysiological temperatures and increases exponentially at near physiological temperatures (18). We postulated that the rapid dissociation of ANP from surface GCA receptors at physiologi-cal temperatures was due to an interaction of a cytoplasmic factor with the cytoplasmic domain of GCA. This mechanism would allow for rapid onset of ANP effects upon increasing plasma levels of the hormone and a rapid termination of effects when plasma levels of ANP fall (2,18).
In this study, we examined the dynamics and some of the molecular mechanisms of dissociation of ANP from transfected wild-type and mutant GCA receptors stably transfected into Chinese hamster ovary (CHO) cells. It will be shown that there is a remarkable temperature dependence of receptor-ligand dissociation that is observed only in intact cells and not in isolated membrane preparations. The very fast dissociation of ANP depends on cell integrity, the intactness of the cytoplasmic domain of GCA, and near physiological temperatures.

EXPERIMENTAL PROCEDURES
Materials-CHO-K1 cells were obtained from American Type Culture Collection (Manassas, VA). 125 I-ANP-(1-28) (rat) and the cGMP assay system were purchased from Amersham Pharmacia Biotech. Cell culture media and all supplements were from Life Technologies, Inc. Bovine calf serum was obtained from Hyclone Laboratories (Salt Lake City, UT). The Muta-Gene M13 mutagenesis kit was purchased from Bio-Rad. Oligonucleotides were from Genosys Biotechnologies, Inc. (The Woodlands, TX). The MC1061 bacterial strain and Sequenase DNA sequencing kit were from U. S. Biochemical Corp. The Wizard DNA purification kits, JM109 bacterial strain, and competent cells were from Promega (Madison, WI). M13 bacteriophage vectors, DNA restrictases, and other enzymes were from New England Biolabs Inc. (Beverly, MA). The protein assay reagent kit was from Pierce. The pAXNEO mammalian expression vector and full-length clones of the human kidney GCA receptor (GCAWT) and the cytoplasmic domain-deleted GCA receptor (GCACYT Ϫ ) were a kind gift from Dr. John Lewicki (Scios Inc., Mountain View, CA). Rat ANP-(1-28) was from Peninsula Laboratories, Inc. (Belmont, CA), and GF/C filter membranes were from Whatman (Kent, United Kingdom). HEPES, protease inhibitors, BSA, 3-isobutyl-1-methylxanthine, and all other chemicals were from Sigma.
Site-directed and Deletion Mutagenesis-For mutagenesis constructs, full-length transfected GCAWT (19) was first cloned into SalI-XbaI cloning sites of the M13 bacteriophage vector. The mutagenesis procedures were performed following the method of Kunkel et al. (20) using the Muta-Gene M13 in vitro mutagenesis kit. Point mutations within the TK domain of GCA were performed in Hanks' subdomain I, which is well conserved in the GCA receptor (16,17,21). GCAA505 was constructed by substituting the GGC codon (codon 1653 in full-length GCAWT), coding for Gly 505 , for GCC, coding for alanine. GCAA506 was obtained by substituting the serine codon TCC (codon 1656 in fulllength GCAWT) for GCC, coding for alanine. The double mutant GCAA505/A506 was obtained by the same mutation in codon 1656 using GCAA505 as a template. Deletion of the GC domain was accomplished by introducing the stop codon TGA in place of TCC (codon 2496), coding for Ser 786 . Deletion of the TK domain was accomplished by a small modification of the procedure described by Koller et al. (17). Briefly, two XhoI sites were introduced into M13GCAWT recombinant vector at the positions Leu 467 (codon 1536)-Glu 468 (codon 1539) and Leu 767 (codon 2439)-Thr 768 (codon 2442). Two oligonucleotides (5Ј-AG TTC CTT CTC GAG CTG CAT CTT-3Ј and 5Ј-T GTT AAA TTT GCG CAA CTC GAG GCG GAT CTG CTG GAA T-3Ј) were used for mutagenesis. Positive mutants were selected by SalI-XhoI-XbaI restriction analysis, and deletion of the entire TK domain was performed by digestion with XhoI and subsequent self-ligation. Successfully deleted recombinants were selected by SalI-XhoI-XbaI digestion. Therefore, the total TK domain deletion comprised 300 amino acids, from Leu 469 (codon 1536 of GCAWT) to Thr 768 (codon 2442). All mutations were confirmed by sequencing. GCAWT and mutant GCA receptors were subcloned into the SalI-XbaI cloning sites of the mammalian expression vector pAX-NEO using standard techniques (22).
Culture Medium and Binding Solution-The culture medium consisted of Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 2 g/liter NaHCO 3 , 10% bovine calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 g/ml amphotericin B (pH 7.15). The binding solution consisted of Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 3.6 g/liter HEPES, 3.7 g/liter NaCl, and 2 mg/ml BSA (pH 7). The washing solution was the same as the binding solution, except that it did not contain BSA.

Cell Culture and Stable Transfection of Recombinant Receptors-
Wild-type and transfected CHO-K1 cells were propagated and maintained in the culture medium described above and placed at 37°C in a humidified atmosphere of 95% O 2 and 5% CO 2 . Stable transfections of GCAWT and mutant GCA receptors in CHO cells were accomplished by the calcium phosphate precipitation method, followed by selection with Geneticin according to standard techniques (22). 10 -20 clones were harvested by cloning cylinders and grown in 75-cm 2 flasks until confluence.
Competition Binding Experiments in Cell Monolayers-Equilibrium competition binding experiments were performed in intact cells at 4°C to obtain the apparent density (B max ) and apparent equilibrium dissociation constant (K i ) of ANP-specific binding sites as previously described (10,12). Cells plated in 24-well plates to near confluence were incubated for 3-4 h at 4°C with binding solution containing trace amounts of 125 I-ANP-  in the absence or presence of 0.01 nM to 0.5 M unlabeled ANP- . At the end of the incubation period, cells were washed twice with ice-cold washing solution, and membranebound 125 I-ANP-(1-28) was removed by incubation with a hypertonic acid solution (0.2 M acetic acid and 0.5 M NaCl) for 20 min at room temperature. The radioactivity released into the acid solution was counted in a ␥-counter. Specific binding was determined by the difference between total binding and binding of 125 I-ANP-  in the presence of excess ANP-(1-28) (0.5 M). Two additional wells in each 24-well plate were reserved for cell counting performed automatically in a Coulter cell counter or manually using a hemocytometer. At 4°C, the apparent dissociation constants (K i ) of ANP in transfected cells were below 1 nM, except in CHOGCAA506 and CHOGCAA505/A506, in which the values for the K i were ϳ3 and ϳ9 nM, respectively. The B max values were used to estimate the density of surface membrane receptors and are reported in the legends of Figs. 4 and 5.
Membrane Preparation-CHOGCAWT or CHOGCACYT Ϫ cells were grown to confluence in 850-cm 2 roller bottles. Cell monolayers were washed twice with ice-cold Hanks' balanced salt solution containing 5 mM HEPES and 3.7 g/liter NaHCO 3 (pH 7.4) and scraped with a rubber policeman into 40 ml of buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 250 mM sucrose, 10 g/ml leupeptin, 10 g/ml aprotinin, and 1 g/ml pepstatin A. The cells were pelleted at 250 ϫ g for 5 min, resuspended in buffer, and sonicated with a Polytron homogenizer. After centrifugation at 250 ϫ g for 3 min to remove unbroken cells and nuclei, the supernatant was recentrifuged at 100,000 ϫ g for 1 h. The resulting supernatant was discarded, and the final membrane pellet was resuspended in 2 ml of buffer and briefly homogenized with a Dounce homogenizer. The aliquots were frozen in liquid nitrogen and stored at Ϫ80°C until used.

Fate and Dissociation of Specifically Bound 125 I-ANP-(1-28) and Determination of Dissociation Constants in Intact Cells and Isolated
Membranes-The fate of specifically bound 125 I-ANP-(1-28) in intact cells was determined by chase experiments as previously described (10,12). Briefly, CHO cells stably transfected with GCAWT or mutant GCA receptors were grown to near confluence in six-well plates. Before the experiments, plates were washed with ice-cold washing solution and incubated for 2 h at 4°C with binding solution containing 0.1-0.5 Ci/ml 125 I-ANP-(1-28) . The wells were washed three times with icecold washing solution, and the cells of four wells were incubated with ice-cold binding solution containing 0.1 M unlabeled ANP-(1-28). The cells in the remaining two wells were incubated with 0.5 M ANP-(1-28) for nonspecific binding determination. The plates were then immediately transferred to a shaking water bath kept at 4, 22, or 37°C. Samples of supernatant were collected at several time intervals from 0.5 to 30 min. At the end of this period, cells were rapidly washed twice with ice-cold washing solution, and the radioactivity remaining bound to the surface membrane was determined by further incubation with the hypertonic acid solution as described above. Recovery experiments showed that, in all instances, the sum of 125 I-ANP-(1-28) radioactivity released to the medium and that remaining at the cell surface by the end of the experiment was Ͼ95% of the total radioactivity. More than 95% of the radioactivity released to the medium was precipitated by 10% trichloroacetic acid, and high performance liquid chromatography revealed that this radioactivity comigrated with 125 I-ANP-(1-28).
The fate of specifically bound 125 I-ANP-(1-28) was also determined in membranes obtained from transfected CHOGCAWT and CHOGCA-CYT Ϫ cells. For this purpose, membranes were first diluted with 10 mM Tris (pH 7.4) to give a final protein concentration of 100 g/ml. 125 I-ANP-(1-28) (0.5-1 Ci/ml) was added to the incubation reaction, and equilibrium binding was attained after incubation at room temperature for 90 min. ANP-(1-28) (1 M) was then added to the incubation reaction, and the tubes were immediately transferred to a shaking water bath at 37°C. Aliquots (125 l) were taken at several time intervals from 0.5 to 30 min and immediately filtered through a Whatman GF/C filter precoated with 1% polyethyleneimine using a vacuum manifold. The filters were then washed three times with 10 mM Tris (pH 7.4). The radioactivity remaining in the filters was counted using a ␥-counter.
cGMP Assay in Cell Monolayers-Transfected CHO cells were plated in six-well plates and grown to near confluence. Washing and incubation solutions were the same as the binding solution described above without BSA. Cell monolayers were washed twice and preincubated for 15 min at 37°C with 1 ml of incubation solution to which 3-isobutyl-1methylxanthine was added to a final concentration of 0.25 mM. Cell monolayers were washed again, and incubation was initiated by adding 1 ml of incubation solution containing 0.25 mM 3-isobutyl-1-methylxanthine with or without 0.1 M ANP- . Incubation was carried out for 5 min at 37°C. At the end of this period, cGMP was extracted by adding 5% trichloroacetic acid to the incubation mixture. Water-saturated diethyl ether was used to remove trichloroacetic acid from the supernatant, and cGMP was determined by the [ 3 H]cGMP radioimmunoassay kit from Amersham Pharmacia Biotech using the procedure recommended by the vendor. Cells were lysed in 0.2% SDS, and the amount of protein was measured by the Bradford procedure (23). Using these assay conditions, Ͼ95% of cGMP generated in 5 min of incubation period was present inside the cells.
Guanylyl Cyclase Assay in Membranes-GC activity in crude membranes was assayed as described (24). Briefly, the reaction was initiated by addition of 30 g of membrane protein in a final volume of 0.3 ml of solution containing 50 mM Tris-HCl (pH 7.5), 2 mM 3-isobutyl-1-methylxanthine, 1 mM GTP, 4 mM MgCl 2 , 0.1% (w/v) BSA, 7.5 mM creatine phosphate, and 15 units/ml creatine phosphokinase (250 units/mg of protein). After incubation for 3 min at 37°C, the reaction was terminated by addition of 10 l of 1.5 M sodium acetate (pH 4.7), followed by boiling for 3 min in a water bath. Finally, the reaction was centrifuged at 12,000 rpm for 3 min, and cGMP generation was quantified by radioimmunoassay. To determine the effect of ANP-(1-28) and/or ATP, these substances were added directly to the reaction at concentrations 0.1 M and 0.5 mM, respectively. All experiments were done in triplicates and repeated at least twice.
Data Analysis Statistics-A nonlinear exponential curve fit was performed to determine the dissociation rate constant (K off ) of ANP from wild-type or mutant GCA receptors. The decay curves of specifically bound 125 I-ANP-(1-28) from the cell surface or from isolated membranes fitted a single-phase exponential decay with a high degree of reliability (r 2 Ͼ 0. 95). In several of the experiments, the decay curves fitted equally well a two-phase exponential decay. However, in this case, the major component accounted for Ͼ80% of the total dissociation. Thus, for simplicity, we chose to calculate an overall rate of dissociation using a single-phase exponential curve fit. Statistics were performed by analysis of variance with the Tukey-Kramer post-test. Differences were considered statistically significant when p Ͻ 0.05.  1B shows the corresponding appearance of intact 125 I-ANP-(1-28) in the medium. Practically all specifically bound 125 I-ANP-(1-28) that dissociated from surface membrane receptors was released in intact form to the medium, demonstrating that there was minimal, if any, receptor-mediated internalization or hydrolysis of ANP.

Fig
The dissociation of 125 I-ANP-(1-28) from CHOGCAWT cells at physiological temperatures was very fast, with K off ϭ 0.49 Ϯ 0.02 min Ϫ1 , a value similar to that found for native GCA receptors in cultured glomerular and renomedullary interstitial cells (18). In CHOGCACYT Ϫ cells, the rate of dissociation decreased by Ͼ20-fold to 0.018 Ϯ 0.001 min Ϫ1 . Removal of the kinase-like and catalytic guanylyl cyclase domains also decreased receptor-ligand dissociation to the slow rates of 0.064 Ϯ 0.006 and 0.043 Ϯ 0.004 min Ϫ1 for CHOGCATK Ϫ and CHOG-CAGC Ϫ cells, respectively. These results show that the cytoplasmic domain of GCA is involved in the mediation of the fast receptor-ligand dissociation at physiological temperatures and that major deletions within the cytoplasmic domain markedly reduce the ability of GCA receptors to physiologically modulate receptor-ligand dissociation. Fig. 2 shows that in isolated membranes prepared from FIG. 1. Dissociation of specifically bound 125 I-ANP-(1-28) from CHO cells stably transfected with GCAWT, GCACYT ؊ , GCATK ؊ , and GCAGC ؊ . Transfected cells were grown to near confluence in six-well plates and incubated for 2 h at 4°C with binding solution containing 125 I-ANP-(1-28) (see "Experimental Procedures"). After washing to remove unbound radioligand, 2 ml of ice-cold binding solution containing 0.1 M unlabeled ANP-(1-28) was added, and the plates were immediately transferred to a shaking water bath kept at 37°C. Samples of supernatant (0.1 ml) were collected at several time intervals from 0.5 to 30 min. At the end of this period, the cells were rapidly washed, and the radioactivity remaining at the cell surface was removed by incubation of the cell monolayer with a hypertonic acid solution (see "Experimental Procedures"). A shows a time plot of radioactivity remaining at the cell surface as percent of specifically bound radioligand at time 0. The amount of radioactivity remaining at the cell surface at each time was determined by the difference between the trichloroacetic acid-precipitable radioactivity measured in the medium at that time and the radioactivity measured in the hypertonic acid solution at the end of the incubation period (30 min). The points were fitted to a single-phase monoexponential decay curve to calculate the off-rates of 125 I-ANP-(1-28) (see "Discussion" and Fig. 3). Note the rapidity of the dissociation of 125 I-ANP-(1-28) from CHOGCAWT compared with that from cytoplasmic domain-truncated receptors (see transfected cells, contrary to intact cells, the rate of dissociation of ANP from CHOGCACYT Ϫ (K off ϭ 0.038 Ϯ 0.002 min Ϫ1 ) was not different from that from CHOGCAWT (K off ϭ 0.035 Ϯ 0.006 min Ϫ1 ). This is due to the major decrease in the rate of dissociation of ANP from GCAWT in membrane preparations compared with intact cells at 37°C. In isolated membrane preparations, receptor-ligand dissociation was very slow and was not regulated by the cytoplasmic domain. This result suggests that factor(s) present in the intact cell and absent in the isolated membranes are responsible for interacting with the cytoplasmic domain of GCA to effectuate a fast dissociation rate of surface receptor-ligand complexes at 37°C. Fig. 3 summarizes the K off values of ANP from wild-type and mutant GCA receptors in intact cells at 37°C. In addition to the major decrease in ANP dissociation when the cytoplasmic domain of GCA is deleted or when the TK and catalytic GC domains are truncated (see Fig. 1), some point mutations in a putative ATP-binding site in Hanks' subdomain I of the TK domain resulted in significant (albeit relatively small) decreases in the receptor-ligand dissociation rate. Although the K off of ANP in CHOGCAA505 cells (0.50 Ϯ 0.06 min Ϫ1 ) was practically identical to that in CHOGCAWT cells (0.49 Ϯ 0.02 min Ϫ1 ), substitution of Ser 506 by Ala and particularly the double substitution of Gly 505 and Ser 506 by Ala resulted in significant decrease in K off to 0.36 Ϯ 0.03 and 0.24 Ϯ 0.02 min Ϫ1 , respectively (p Ͻ 0.01 versus CHOGCAWT). In no instance, however, did the K off value of these point mutants approach the low value observed in the cytoplasmic domain-truncated receptors.
To determine whether changes in receptor-ligand interactions were related to receptor activity, we measured basal and ANP-stimulated guanylyl cyclase activity or cGMP levels in CHO cells stably transfected with wild-type or mutant GCA receptors. Fig. 4A shows basal, ATP-, ANP-, and (ANP ϩ ATP)stimulated guanylyl cyclase activity in isolated membranes obtained from CHOGCAWT and CHOGCATK Ϫ cells. Basal guanylyl cyclase in CHOGCAWT cells was very low, and ANP ϩ ATP produced a major (Ͼ30-fold) activation of guanylyl cyclase. ATP alone was without effect, and ANP alone had only a modest effect. In membranes obtained from CHOGCATK Ϫ cells, basal levels of guanylyl cyclase activity were markedly elevated, reaching levels similar to those in isolated membranes from CHOGCAWT cells maximally stimulated with ANP ϩ ATP. In this truncated mutant, ATP slightly but consistently decreased guanylyl cyclase activity (p Ͻ 0.01 versus CHOGCAWT), whereas ANP or ANP ϩ ATP did not further increase guanylyl cyclase activity from its high basal level. As expected, CHOGCACYT Ϫ and CHOGCAGC Ϫ cells showed no significant ANP-(1-28)-stimulated guanylyl cyclase activity or increases in cellular cGMP levels (data not shown). Fig. 4B shows the results of the studies on cGMP levels in intact CHOGCAWT and CHOGCATK Ϫ cells. Deletion of the kinase-like domain also led to marked increases in basal cGMP levels and unresponsiveness to ANP. However, in intact cells, contrary to the observation in isolated membranes, the basal levels of cGMP in CHOGCATK Ϫ cells were significantly lower than the maximally ANP-stimulated levels of cGMP in CHOG-CAWT cells, even when the results were normalized by the density of surface receptors. We interpret this finding to suggest that under sustained constitutive activation of GCA in intact cells, there are adaptive mechanisms that increase the hydrolysis or extrusion of cGMP from the cells. It is of interest that despite the chronically elevated basal levels of cGMP, we could not detect a change in growth rate of cultured CHOG-CATK Ϫ cells compared with CHOGCAWT cells (data not shown).   was loaded onto isolated cell membranes (100 g of protein/ml of incubation mixture) obtained from transfected CHOGCAWT and CHOGCACYT Ϫ cells (see "Experimental Procedures"). Equilibrium binding was obtained after 90 min of incubation at room temperature. Excess unlabeled ANP-(1-28) was then added to the incubation mixture to a final concentration of 1 M to initiate receptorligand dissociation, and the tubes were rapidly placed in a shaking water bath at 37°C. Samples were taken at several time intervals and rapidly filtered through Whatman GF/C membranes using a vacuum manifold. The GF/C membranes were washed three times with 3 ml of ice-cold 10 mM Tris solution (pH 7.4). The radioactivity remaining in the GF/C membranes was counted in a ␥-counter. Results (mean of triplicates) show the time course of specific 125 I-ANP-(1-28) radioactivity remaining in the membranes as percent of specific 125 I-ANP-(1-28) radioactivity bound at time 0. The points were fitted to a single-phase monoexponential decay curve to calculate K off . The rates of dissociation of 125 I-ANP-(1-28) from membranes prepared from CHOGCAWT and CHOGCACYT Ϫ were 0.089 Ϯ 0.019 and 0.063 Ϯ 0.013 min Ϫ1 , respectively (p Ͼ 0.05). A repeat experiment gave similar results. main I on basal and ANP-stimulated cGMP levels. Basal levels of cGMP were similar in all transfected cells, except in CHOG-CAA505/A506 cells, in which there was an ϳ5-fold increase compared with CHOGCAWT cells (p Ͻ 0.01). However, this higher basal level of cGMP in CHOGCAA505/A506 was still far lower than that observed in CHOGCATK Ϫ (see also Fig. 4B). Maximal ANP-stimulated cGMP was significantly decreased in CHOGCAA506 and, in a more pronounced manner, in the double mutant CHOGCAA505/A506.
We also tested receptor-ligand dissociation and receptor activity (cGMP generation) in cells stably transfected with GCA receptors that had point mutations in other conserved Hanks' subdomains within the TK domain. GCAA535, GCA551, and GCAA646 are mutants in which Lys 535 (subdomain II), Glu 551 (subdomain III), and Asp 646 (subdomain VII) were mutated to Ala, respectively. In this series, maximal ANP-induced generation of cGMP in CHOGCAWT was 19.8 Ϯ 4.3 pmol/5 min/10 9 surface GCA receptors. The values for the mutants were as follows: CHOGCAA535, 1.8 Ϯ 0.6 pmol/5 min/10 9 receptors (p Ͻ 0.001 versus CHOGCAWT); CHOGCAA646, 6.1 Ϯ 0.4 (p Ͻ 0.05 versus CHOGCAWT); and CHOGCAA551, 10.1 Ϯ 2.0 (not significantly different from CHOGCAWT, p Ͼ 0.05). The dissociation of 125 I-ANP-(1-28) from these mutants was determined in intact cells at 37°C in the same manner as described above (see Fig. 1A). The K off values of ANP from CHOGCAA535 (0.64 Ϯ 0.05 min Ϫ1 ), CHOGCAA551 (0.53 Ϯ 0.14 min Ϫ1 ), and CHOGCAA646 (0.57 Ϯ 0.05 min Ϫ1 ) were not significantly different (p Ͼ 0.05) from the K off of ANP from CHOGCAWT (0.48 Ϯ 0.04 min Ϫ1 ). Finally, we tested the effects of the dele- Results are means Ϯ S.E. of four to five wells obtained in two separate experiments. ATP alone did not significantly increase guanylyl cyclase activity; ANP alone had a significant (albeit small) effect; and ANP ϩ ATP dramatically increased guanylyl cyclase activity to Ͼ30-fold basal activity (bars labeled B). Deletion of the kinase-like domain led to full constitutive activation of guanylyl cyclase with basal levels similar to those obtained in wild-type receptors exposed to a maximal concentration of ANP. Under these conditions, ATP had a small but consistent inhibitory effect, and the TK domain-deleted receptor became unresponsive to ANP. tion of an amino acid sequence between Hanks' subdomains I and II (from Val 520 to Lys 528 ). This deletion mutant was completely unable to generate cGMP upon maximal stimulation with ANP, whereas the dissociation of 125 I-ANP-(1-28) was decreased by only ϳ40% to 0.30 Ϯ 0.02 min Ϫ1 (p Ͻ 0.01 versus CHOGCAWT). Fig. 6 shows the temperature dependence of the rate of dissociation of 125 I-ANP-(1-28) from wild-type and mutant GCA receptors stably transfected into CHO cells. Between 22 and 37°C, the dissociation rate increased from 0.063 Ϯ 0.005 to 0.49 Ϯ 0.04 min Ϫ1 (Q 10 ϭ 5.2), whereas between 4 and 22°C, the increase was only from 0.04 Ϯ 0.001 to 0.063 Ϯ 0.005 min Ϫ1 (Q 10 ϭ 1.0). Fig. 6A shows that this remarkable increase in dissociation of ANP from CHOGCAWT at near physiological temperatures was abolished in CHOGCACYT Ϫ , CHOGCATK Ϫ , and CHOGCAGC Ϫ cells. Fig. 6B shows the temperature dependence of the dissociation of ANP from transfected CHO cells expressing GCA receptors with point mutations in Hanks' subdomain I. Although the K off values of ANP from CHOGCAA506 and CHOGCAA505/A506 at 37°C were significantly lower than those from CHOGCAWT and CHOGCAA505, all point mutant receptors were still able to show the disproportionate increase in receptor-ligand dissociation rates at near physiological temperatures.
It is noteworthy that the major differences in the rate of dissociation of ANP from wild-type and cytoplasmic domaintruncated receptors at 37°C virtually disappeared at 4°C, a temperature at which dissociation from all receptors was similar and very slow (Fig. 6). Accordingly, the measured apparent equilibrium dissociation constants (K i ) in transfected cells at 4°C were similarly low in wild-type and cytoplasmic domaintruncated receptors, amounting to 0.54 Ϯ 0.06, 0.26 Ϯ 0.12, 0.69 Ϯ 0.44, and 0.68 Ϯ 0.58 nM for CHOGCAWT, CHOGCA-CYT Ϫ , CHOGCATK Ϫ , and CHOGCAGC Ϫ , respectively.

DISCUSSION
The present results demonstrate that GCA receptors stably transfected into CHO cells are not endocytosed at appreciable rates and do not mediate lysosomal hydrolysis of ANP. Extremely rapid receptor-ligand dissociation in intact cells at 37°C, but not at subphysiological temperatures, terminates the interaction of ANP with GCA. These results are in full agreement with our previous observation with native GCA receptors in primary cultures of glomerular mesangial and renomedullary interstitial cells (18). In contrast, they are at variance with reported studies showing receptor internalization for GCA in Leydig tumor cells and, more recently, for transiently transfected GCA in COS-7 cells, an SV40-transformed cell line (25,26). Although we do not have a definitive explanation for this apparent discrepancy, it is not surprising that tumor cells or SV40-transformed (COS-7) cells would have enhanced endocytosis, resulting in internalization of constitutive membrane proteins nonspecifically entrapped in coated pits or other endocytic regions of the cell. In this regard, it is noteworthy that GCA lacks know internalization signals in its cytoplasmic tail, a feature consistent with our finding that these receptors do not undergo specific endocytosis (18). Our results cannot be attributed to a putative defect of CHO cells to effectuate receptor endocytosis or to an intrinsic decrease in receptor affinity in these cells. Indeed, we have previously shown that clearance receptors of natriuretic peptides stably transfected into CHO cells undergo robust endocytosis and have very low receptor-ligand dissociation at 37°C (12). Thus, the lack of appreciable endocytosis and the rapid receptorligand dissociation of native GCA in primary culture cells or of GCA stably transfected into CHO cells are likely to reflect the dynamics of this receptor under physiological conditions.
The dissociation of ANP from wild-type GCA receptors at 37°C is markedly slower in isolated membranes than in intact cells. On one hand, this novel finding demonstrates that temperature per se cannot account for the dramatic increase in ANP dissociation from GCA receptors in intact cells at near physiological temperatures. On the other hand, it suggests that temperature-dependent interaction of cytosolic factors with the intracellular domain of GCA results in conformational changes that favor rapid receptor-ligand dissociation under physiological conditions. Under nonphysiological conditions, such as in isolated membrane preparations or in intact cells at subphysiological temperatures, the extracellular domain of GCA loses its conformational control by the cytoplasmic domain, resulting in exceedingly slow rates of ligand dissociation.
As previously shown by several investigators (14,(27)(28)(29)(30)(31)(32)(33) and confirmed in this study (Fig. 4), signaling of GCA is dependent on ATP. Studies by several laboratories, including our own, indicated that ATP is also involved in the modulation of receptor-ligand dissociation and in receptor affinity (18, 27, 28, 34 -36). In isolated membranes from bovine zona glomerulosa cells, addition of ATP or ATP␥S significantly increases the half-time of a fast component of the dissociation of ANP from GCA, whereas amiloride competitively counteracts this effect (35,36). On the basis of these results, it was postulated that ATP interacts with the cytoplasmic domain of GCA and by an allosteric effect switches these receptors from a high affinity to a low affinity state. In our previous study (18), we also found that amiloride markedly decreased the dissociation of ANP from GCA in intact glomerular mesangial and renomedullary interstitial cells, a finding consistent with the above interpretation.
As a whole, the studies referred to above strongly indicate that ATP participates in the modulation of receptor-ligand dissociation. However, this study shows for the first time that ATP alone cannot fully account for rapid GCA receptor-ligand dissociation. The dissociation of ANP from wild-type GCA receptors in intact cells at 37°C was ϳ10-fold faster than in isolated membranes at 37°C. Addition of 1 mM ATP to the isolated membranes approximately doubled the overall dissociation rate (data not shown) to values similar to those reported for the effect of ATP on the fast component of ANP dissociation reported in the earlier work by Larose et al. (35). Thus, even in the presence of ATP, the dissociation rate of ANP in isolated membranes at 37°C is 4-5 times slower than in intact cells at physiological temperatures. Moreover, it is unlikely that differences in cellular concentration of ATP between 22 and 37°C are of such magnitude as to explain the major increase in receptor-ligand dissociation between these temperatures (Fig.  6). The nature of cytoplasmic factors other than ATP that contribute to the rapid receptor-ligand dissociation remains to be elucidated. It is possible, even likely, that temperaturedependent interactions between GCA and cytosolic components such as the cytoskeleton and chaperone proteins contribute to the physiological regulation of receptor-ligand dissociation. In this regard, the recent findings demonstrating that hsp90 interacts with the cytoplasmic domain of GCA and that geldanamycin, an inhibitor of hsp90, significantly decreases ANPinduced activation of GCA (37) suggest intriguing new possibilities to explain not only the modulation of GCA activity, but also the unique properties of GCA receptor-ligand dissociation described in the present study. Further studies are needed to test this hypothesis.
Deletion of the kinase-like domain leads to a major constitutive activation of GCA, which then becomes insensitive to further stimulation with ANP (14,17). We confirmed and extended this observation by showing that in the kinasetruncated receptor, when the results were normalized by the number of surface receptors, guanylyl cyclase activity was enhanced to levels similar to those obtained with a maximal stimulation of wild-type GCA receptors with ANP (Fig. 4). Deletion of the kinase-like like domain also markedly slowed receptor-ligand dissociation in intact cells at 37°C to the same level as observed in cells at 4°C or in isolated membranes at 37°C and abolished the temperature dependence of this process. Surprisingly, deletion of the guanylyl cyclase sequence decreased receptor-ligand dissociation to the same extent as deletion of the kinase-like domain. None of the point mutations performed in this study had such a dramatic effect on receptorligand dissociation as the deletions of the kinase-like domain and the catalytic guanylyl cyclase sequence (see below). This suggests that structural integrity and/or receptor oligomerization rather than specific sites within the intracellular domain of GCA determines the interaction of cytosolic factors that modulate receptor-ligand dissociation. It is also possible that the common feature of the two major truncations in the cytoplasmic domain of GCA is the disruption of the hinge region between the kinase-like sequence and the guanylyl cyclase domain, a region that has been shown to participate in GCA oligomerization (15). In performing the deletion of the guanylyl cyclase domain, we also deleted the major portion of the hinge region. In the deletion of the kinase-like domain, this region was preserved, but we cannot rule out that the mutation led to a conformational change that impeded a putative participation of the hinge region in the modulation of receptor-ligand dissociation. Whatever the case, loss of rapid receptor-ligand dissociation is not directly related to the state of activation of the receptor since deletion of the kinase-like domain leads to full constitutive activation, whereas deletion of the guanylyl cyclase domain precludes activation of GCA.
In an attempt to further test the relationship between receptor activation and receptor-ligand dissociation, we also performed discrete mutations within the kinase-like domain that were previously reported to markedly reduce ANP-induced generation of cGMP (30, 38 -40). We mutated Gly 505 and Ser 506 , which belong to a putative ATP-binding site in Hanks' subdomain I of the kinase-like domain, the so-called ATPregulated module (28). Ser 506 is a constitutively phosphorylated residue that, once dephosphorylated, is involved in the desensitization of GCA receptors (39,40). Substitution of Ser 506 and particularly the double substitution of Gly 505 and Ser 506 by Ala markedly decreased ANP-induced cGMP generation (Fig. 5), whereas the effect of these mutations on receptor-ligand dissociation was relatively modest, albeit significant (Fig. 3). Moreover, the remarkable temperature dependence of receptor-ligand dissociation was retained in point mutant GCA receptors (Fig. 6). The deletion of an eight-amino acid sequence between Hanks' subdomains I and II had previously been shown to be a splice variant of GCA in Anguilla japonica and led to complete unresponsiveness to ANP (41). This study confirms this finding and shows for the first time that receptorligand dissociation rate is significantly decreased in this mutant. However, in this mutant, the off-rate of ANP from GCA is not nearly as slow as in the mutant with deletion of the entire kinase-like domain. Several point mutations within the kinaselike domain also inhibited ANP-induced increases in cGMP, but were without effect on receptor-ligand dissociation (see "Results"). As a whole, these results further indicate that receptor activation and receptor-ligand dissociation are at least to some extent independently modulated.
These results point out the importance of using near physiological conditions to study the dynamics of interaction of ANP with GCA receptors. Cell integrity, physiological temperatures, and integrity of the cytoplasmic domain of GCA receptors are all essential for an appropriate stimulus-response homeostasis of atrial natriuretic peptides in the regulation of cardiovascular and renal functions. The results support the notion that GCA receptors function in a "staccato" mode (2). The rapid dissociation of ANP from GCA receptors, combined with the removal of dissociated ligand by the abundant clearance receptors, assures the availability of unoccupied receptors for prompt responses as plasma levels of the hormone rise and rapid termination of responses as plasma levels of ANP fall and impedes sustained desensitization of GCA receptors under physiological conditions.