Production of polyclonal antibody to the bovine adrenal atrial natriuretic factor-R1 receptor.

Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/21/14 For personal use only.

JOURNAL OF RECEPTOR RESEARCH, 12(4), 485-505 (1992)

PRODUCTION OF POLYCLONAL ANTIBODY TO THE BOVINE ADRENAL ATRIAL NATRIURETIC FACTOR-R1 RECEPTOR

, No mand McNicolllI , Christine Jean-Ja ques Rondeau'' LordEf3, Louise Larose',', Sylvain Melochel Jean Gagnon4 , Huy Ong1r2 and Andrb De Lean* ",' 'Institut de Recherch s Cliniques de Montreal, 'Facultb de Pharmacie and 'Dbpartement de Pharmacologie, Universitg de Montreal , 4Biologie Structurale, CEA et CNRS URA 1333, Grenoble, France. ABSTRACT A polyclonal antibody monospecific for an intracellular epitope of the atrial natriuretic factor (ANF)-R1 receptor was produced. The receptor protein (200 pmoles) was purified to homogeneity from bovine adrenal zona glomerulosa (BAZG), reduced, alkylated and digested with trypsin. The tryptic fragments were purified by reverse-phase h.p.1.c. on a C18 column. Based on the sequence of one of these fragments, a peptide was chemically synthesized, coupled to thyroglobulin and injected into rabbits. The antibody obtained was shown to be specific for the R1-type as no receptor was detected in bovine red blood cells (RBC) (which are devoid of ANF receptors) and in NIH-3T3 cell membranes (where only the R2-type is expressed). Several other tissues were screened and comparison of the immunoreactive receptor density estimates with those obtained by ANF binding yielded a correlation coefficient (r2) of 0.965. The minimal detectable dose

*

Author to whom correspondence should be addressed: Dr Andre De Lean Dbpartement de Pharmacologie Universitb de Montreal C.P. 6128, Succursale A Montreal , (Quebec) CANADA H3C 3J7 485

Copyright 0 1992 by Marcel Dekker, Inc.


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was typically 3 fmoles/tube and the ED50 of the RIA was 30 fmolesltube. Cyanogen bromide digestion of the receptor was essential for antigenic detection, indicating that the epitope is probably hindered due to the tertiary structure of the native protein. Moreover, location of the epitope in the kinase homology domain of the receptor, combined with partial tryptic digestion, suggests that the proteolysis-sensitive region of the receptor is located between the transmembranespanning domain and the amino acid 586. This method of production of antibodies should be useful to precisely map the amino acids involved in various functions of the receptor. INTRODUCTION Atrial natriuretic factor is a 28 amino acid peptide showing vasodilatory, natriuretic and diuretic properties and inhibition of aldosterone biosynthesis and secretion (1-4). It has been shown that this hormone selectively activates a particulate guanylate cyclase (5) and increases cyclic GMP concentration in target tissues ( 5 - 9 ) . The biological effects of ANF are mediated through interaction with specific membrane receptors. Chemical cross-linking, photolabeling and pharmacological profiles have revealed two types of ANF receptors (10): the R1-type is a monomeric non-reducible Mr 130,000 protein, coupled to cGMP formation, which binds truncated forms of ANF with much less affinity and which mediates the biological effects of ANF. Amiloride enhances the binding of ANF to this receptor subtype (11). Copurification of particulate guanylate cyclase activity with ANF receptor from bovine adrenocortical cells (12), adrenal zona glomerulosa (13), rat lung (14), rat adrenocortical carcinoma cells (15) and elucidation of the primary structure of the receptor from various species and tissues by cDNA cloning (16-18) have shown that this subtype is a


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monomeric protein containing within its C-terminal domain a region showing high homology with the Mr 70,000 subunit of the bovine lung soluble guanylate cyclase (19). The R2-type is a Mr 130,000 homodimeric reducible protein composed of two Mr 65,000 subunits which bind truncated analogs of ANF with high affinity. This receptor has also been purified (20,21) and cloned from bovine aortic smooth muscle (22). This subtype is currently thought to act as a specific clearance binding site for the circulating hormone (23,24) or to transduce signal through interaction with a G-protein (25-27).

Study of the primary structure of the ANF-R1 receptor has allowed to grossly subdivide it into three functional domains: an extracellular domain ( containing the binding site of ANF), a protein kinase domain ( where several consensus sequences for nucleotide binding site are found) and a guanylate cyclase domain at the C-terminal end of the receptor. Previous work from our group have shown that limited proteolysis converted the Mr 130,000 protein to a Mr 70,000 membrane-associated fragment when exposed to low trypsin concentrations. This fragment showed a slight increase in affinity for ANF, a loss of guanylate cyclase activity (28) and of the negative regulation of ATP on the binding of ANF to its receptor (29). These results indicate that proteolytic cleavage occurs in the protein kinase domain.

In this paper, we report the production of a polyclonal antibody against a 17 amino acid peptide located in the protein kinase homology domain of the ANF-R1 receptor. The sequence of this peptide was derived from that of a proteolytic fragment obtained by tryptic digestion of purified bovine adrenal zona

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glomerulosa ANF-R1 receptor. This antibody was used to develop a specific RIA for assaying the R1 receptor independently of the occupancy by ANF of its binding domain and which is devoid of cross-reactivity for soluble guanylate cyclase and ANF-R2 receptor. It also allowed us to localize the trypsin-sensitive region relatively to the epitope. It constitutes the first reported antibody specific to the R1-type and for which the epitope is known. Production of monoclonal antibodies to the receptor was reported but they were raised against a partially purified preparation (30). MATERIALS N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)treated trypsin (type XIII) , bovine serum albumin and thyroglobulin, phenylmethylsulfonyl fluoride (PMSF), succinyl cyclic GMP tyrosine methylester (ScGMP-TME), l-ethyl-3-[3-dimethylaminopropyl] carbodiimide-HC1 and alumina (type WN-3) were obtained from Sigma. Rat ANF (99-126) was from Institut Armand-Frappier (Laval, Canada). Antiserum to cyclic GMP was kindly provided by Dr. Alain BBlanger (Centre hospitalier de Carrier-free l'Universit6 Laval, QuBbec, Canada) Na1251 was from Amersham Corp. Iodo-beads were purchased from Pierce Chemical Co.. Electrophoresis reagents and molecular weight standards were from BioRad

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METHODS Iodination of PeDtides and ScGMP-TME The beads. 100 p 1 started

synthetic fragment was iodinated with IodoTypically, 10 pg of peptide was dissolved in of 0.5 M KH2P04, pH 7.0. The reaction was by the addition of 1 mCi Na1251 and two Iodo-

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beads and the mixture was incubated at 4OC for 15 min. The radiolabeled peptide was then purified by h.p. 1.c. on a 15 x 0.46 cm reverse-phase Exsil C18 column (CSC co.) using a 60 min linear 15-45% CH3CN gradient in 20 mM triethylamine-phosphate buffer, pH 6.0. ScGMP-TME was (31)

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iodinated as described previously

Purification of Bovine Adrenal ANF-R1 Receptor The ANF-R1 receptor was solubilized in Triton X-100 from bovine adrenal zona glomerulosa (BAZG) and purified by affinity chromatography on ANF-agarose as described previously (13). Trylstic Diqestion of the Purified Recelstor The pure receptor (200 pmoles) eluted from the ANFagarose affinity column was concentrated by dialysis against 20% polyethylene glycol (PEG) 35,000 in 20 mM NH4HC03 to 3.5 ml and evaporated to 0.15 ml. The concentrate was diluted with 0.15 ml of Laemmli sample buffer 2X and runned on a SDS-polyacrylamide gel. The Mr 130,000 protein was then eluted from the gel in H20 and reduced in 0.1 M NH4HC03, 5 mM dithiothreitol under N2 atmosphere for 2 hours at 25OC. The receptor was then alkylated under N2 atmosphere for 1 hour at 25OC in the dark with 10 mM iodoacetic acid. The preparation was dialyzed against 10 mM NHqHC03, 0.01% SDS, evaporated to 0.25 ml and incubated for 17 hours at 25OC in 0.1 M NH4HC03, 2 mM CaC12 with TPCK-treated trypsin (1 pg/50 pg of protein). Trifluoroacetic acid (TFA) was added to a final concentration of 1% and the sample was injected on a microbore C18 column, washed with 0.06% TFA at 0.2 ml/min, eluted in 100 % CH3CN,


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reinjected on a Vydac C18 column and eluted in a CH3CN linear gradient of 15-25% for 20 minutes followed by 25-60% in 140 min. in 0.06% TFA at 0.5 ml/min. Absorbance was monitored at 214 nm. Microsequencinq of Proteolvtic Fraqments Automated Edman degradation was performed ising an Applied Biosystems Model 477A liquid-pulse protein sequencer and amino acid phenylthiohydantoin derivatives were identified according to the protocol recommended by the manufacturer on a Model 120A HPLC system. Preparation of Membranes Bovine adrenal zona glomerulosa membranes were prepared as described previously (13). The LLC-PK1 and NIH-3T3 cells membranes were prepared as described by FethiPre and coworkers (32). Bovine red blood cells (RBC) were prepared from fresh blood collected in flasks coated with EDTA. The RBC were washed three times by centrifugation at 4OC in 0.9% NaC1. Hemolysis was performed by resuspension of the pellet in 2 mM EDTA in H20. The ghost cells were centrifuged at 30,000 x g for 15 min. at 4OC and pellets were homogenized with a Dounce homogenizer in 20 volumes of buffer A (20 mM NaHC03, 2 mM EDTA, 1 pM leupeptin, 1 pM aprotinin, 100 nM pepstatin A and 100 pM PMSF. Membranes were centrifuged again at 30,000 x g for 15 min. at 4OC. The homogenization and wash steps were repeated 4 times. The final pellet was resuspended in buffer B (50 mM Tris-HC1, pH 7.4, 4OC, 0.1 mM EDTA, 250 mM sucrose and 1 mM MgC12) and frozen at -7OOC until used.


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Partially purified liver membranes were prepared from Sprague-Dawley rats according to the method of Neville ( 3 3 ) . Bovine lungs were perfused with 0.9% NaCl before homogenization with a Brinkman Polytron and a Dounce homogenizer in buffer A . All steps were done at 4OC. Membranes were filtered on a cheese cloth and the filtrate was centrifuged at 720 x g for 10 min. The supernatant was centrifuged at 30,000 x g for 15 min. The resulting pellet was washed twice with homogenization buffer and finally resuspended in 2 ml per g of tissue of buffer B. ANF Bindins and Photolabelinq Adrenal zona glomerulosa membranes (20 pg/ml) were incubated with 10 pM 1251-ANF(99-126) for 90 min. at 25OC in 50 mM Tris-HC1, pH 7 . 4 , 5 mM MnC12, 0.1 mM EDTA and 0.5% bovine serum albumin (BSA). Bound 1251-ANF was separated from free ligand by centrifugation at 30,000 x g for 20 min. Photoaffinity cross-linking was then performed as previously described (34). Production of Antibodies Based on the sequence of the tryptic fragment located in the protein kinase domain of the ANF-R1 receptor (RP), a peptide was chemically synthesized (with a tyrosine at the N-terminus to make iodination possible) and coupled to thyroglobulin using the crosslinking agent l-ethyl-3-[3-dimethylaminopropyl] carbodiimide-HC1. 100 pg of conjugate were emulsified in complete Freund adjuvant and injected subcutaneously to 1.5 kg male rabbits. Antibodies were collected after 2-3 boosts.

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RIA Procedures Digestion of the receptor. Membranes were solubilized in 70% HCOOH, CNBr was added in equivalent amount of proteins and incubated at room temperature for 24 hours under N2 atmosphere. The solution was then diluted 15 times with 0.1% TFA and passed on a CN-Sep-Pak cartridge preequilibrated in 0.1% TFA. The column was then washed with 20 volumes 0.1% TFA and the fragments eluted with 5 volumes 50% isopropylic alcohol in 0.1% TFA The eluate was evaporated to dryness and Samples were diluted resuspended in 10% Triton X-100. with RIA buffer before RIA in order to reduce detergent concentration to 2% in the sample (final concentration of 0 . 8 % ) .

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RIA The RIA was performed in 50 mM NaH2P04, 50 mM NaC1, 0.1% BSA, 0.02% NaN3, 0.1% Triton X-100, pH 7.4. The samples (200 pl) were incubated with 16 pM 1251-RP and antibody in a final volume of 0.5 ml overnight at 4OC. Bound fraction was estimated by precipitation by adding 75 pl 1.5% bovine gamma-globulins, 750 p1 20% PEG in H20 and pelleting at 1500 x g for 12 min at 4OC. The supernatant was discarded and pellets counted in a gamma-counter.

Limited Proteolvsis Limited proteolysis was performed as described previously (28). Briefly, zona glomerulosa membranes were washed in 50 mM Tris-HC1, pH 7 . 4 and resuspended in the same buffer at a protein concentration of 2 mg/ml. Proteolysis was initiated at 25OC by addition of an equal volume of buffer containing TPCK-treated After 30 min, the trypsin ( 5 pg/mg of protein).


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digestion was stopped by addition of PMSF (1 mM final concentration) and membranes were washed twice by centrifugation at 4OC at 30,000 x g for 20 min. The RIA was performed on each fraction after digestion with CNBr

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Guanvlate Cvclase Assav Guanylate cyclase activity was assayed as described by Garbers and Murad (35). Membranes were incubated at 37OC for 10 min in 100 pl of buffer containing 5 0 mM Tris-HC1, pH 7.6, 10 mM theophylline, 2 mM 3-isobutyl1-methylxanthine, 10 mM creatine phosphate, 10 units of creatine phosphokinase, 1 mM GTP and 4 mM MnC12. The reaction was initiated with the addition of sample and terminated by the addition of 100 pl 120 mM EDTA followed by immersion in boiling water for 3 min. The mixture was centrifugated and cyclic GMP was quantified in the supernatant by radioimmunoassay (36,37) after separation on alumina column (38). SDS-Polvacrvlamide Gel Electrophoresis Samples were solubilized in Laemmli sample buffer (62 mM Tris-HC1, p H 6.8, 2% SDS, 10% glycerol, 5% mercaptoethanol and 0.001% bromophenol blue) and heated at 100°C for 3 min. Electrophoresis was performed according to Laemmli (39) on a 7.5%-acrylamide running gel. Data Analvsis The density of sites estimated from ANF binding was calculated from the competition binding curves of 1251-ANF by ANF which were analyzed by least-squares regression analysis (40).

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Fig.1 Purification of tryptic fragments of the ANF-R1 receptor. The purified receptor (200 pmoles) was reduced, alkylated and digested with TPCK-treated trypsin (1 p g / 5 0 pg of protein) for 17 hours at 2 5 O C as described in the Methods section. The sample was then injected on a Vydac C18 column eluted at 0.5 ml/min in a CH3CN gradient in 0.06% TFA. Absorbance was monitored at 214 nm. The arrow indicates the position of the 17 amino acid fragment from which a peptide (RP) was synthesized.

bR1 (RP) hR1 rR1 mR1

Fig.2 Homology of the tryptic fragment from bovine adrenal zona glomerulosa. The amino acid sequence of the fragment is aligned with the ANF-R1 from rat brain (rR1) (16), human kidney (hR1) (17) and murine Leydig tumor cells (mR1) (18). Identical amino acids are boxed.

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RESULTS AND DISCUSSION


Microsequence of ANF-R1 Trvptic Fracrments Figure 1 shows the chromatogram of the fragments obtained from the ANF-R1 receptor purified from bovine zona glomerulosa and digested with trypsin. Several peaks were microsequenced. One of the longest peptides sequenced was: GSLQDILENEEITLDNM. The comparison of this sequence (referred to as RP) with those deduced from the cDNA clones of ANF receptor indicates that RP is located in the kinase domain (amino acid 586-602, fig.2) Monospecific antibodies were produced against RP 1) because this peptide is relatively hydrophilic, having a reasonable probability of being located at the surface of the native protein and thereby immunoreactive; 2) because its length was appropriate for antibody production. Moreover, this peptide was specific to the ANF-R1 receptor, since it is absent in soluble guanylate cyclase domain as well as in the ANFR2 receptor. Other tryptic fragments from this and subsequent experiments have been microsequenced and have enabled us to establish homologies between the bovine adrenal receptor and other cloned ANF-R1 receptors (manuscript in preparation). Specificity of Anti-ANF-R1 Antibodv Specificity of the polyclonal antibody produced is illustrated in fig.3 where the binding of the radioactive peptide on the antibody was inhibited by a solubilized fraction of bovine adrenal zona glomerulosa or bovine red blood cell membranes after CNBr digestion. The competition curve obtained with dilutions of solubilized zona glomerulosa membranes is parallel to that observed with the synthetic peptide, suggesting

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Fig.3 Specificity of the antibody. Membranes (10 mg/ml) were digested with CNBr, washed on a CN-Sep-Pak cartridge, evaporated to dryness and resuspended in Triton X-100 and RIA buffer as escribed in the Methods section. Displacement of 12'I-RP (16 pM) from the antibody by increasing amounts of proteins or cold RP was estimated by precipitation. Shown here are representative curves observed with the peptide ( 0 ) , bovine adrenal zona glomerulosa membranes ( ) or bovine red blood cell membranes ( A ) Curves were fitted by non-linear least square regression using a four parameters logistic equation.

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that it is free from interferences and specific for the epitope. No displacement of 1251-RP from the antibody is observed with red blood cell membranes, indicating that the curve observed with zona glomerulosa is specific since red blood cells are known to be devoid of ANF receptors. Several tissues were studied following the same protocol and the measured receptor densities are shown in table 1. The standard curve for this antibody gives typically an ED50 of 30 fmoles/tube and a minimum detectable dose of 3 fmoles/tube.

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TABLE 1 Comparison of the ANF-R1 Receptor Densities Obtained by RIA and by ANF Binding. ESTIMATED ANF-R1 RECEPTOR DENSITY (FMOL/MG) RIA BAZG 470 f 22 BAZG (TREATED)d 40 f 1 RBC N D ~ LLC-PK1 41 & 5 NIH-3T3 < 3 RAT LIVER 26 f 3 BOVINE LUNG 94 ? 7

ANF BINDING (n=4)a (n=2)

517 f 4 4 (n=3) 580 ? 92 (n=2) ND

(n=3) (n=2) (n=2) (n=4)

98 (n=1) N D ~ ND 60' (n=1)

a Results expressed as the mean ? S.E.M. Not detectable ANF binding estimated in resence of the specific ~ N F - R Zreceptor ligand [ C y s ' l ] -ANF(102-116) -NH2. Membranes treated with trypsin (10 pg/mg of proteins) as described in the methods section. ND Not detectable.

The antibody obtained could not detect the native receptor, either in its membrane or Triton X-100 solubilized forms. This indicates that the epitope is not readily accessible to the antibody probably due to the tertiary structure of this region. Digestion with CNBr was essential for exposing the epitope. Denaturation in 70 % HCOOH by itself was insufficient (data not shown). Since we have shown that binding of ANF to the receptor induced a conformational change in the proteolysis-sensitive region leading to enhanced sensitivity to trypsin (28) we tested the effect of ANF binding to the intact receptor on its detectability by the antibody. A similar phenomenon was not observed for the binding of the antibody to the native receptor,

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either in its membrane or solubilized forms (data not shown). In order to validate this method, the values estimated by RIA were compared with those obtained with ANF binding. We estimated ANF binding in the presence of 1 pM R2-type-specific ligand [ CYS”~] -ANF (102-116)NH2 in NIH-3T3 and bovine lung membranes in order to block the ANF-Rz receptors. The estimates calculated from each method are compared in table 1. The correlation coefficient (r2) was 0.965. The lack of detection of the R2-type is in agreement with the exclusive positioning of the epitope in the R1-type since the former has only a small intracellular tail ending before the protein kinase homology domain (22). Effect of Limited Proteolysis on ANF-R1 Immunoreacti-

& The localization of the proteolysis-sensitive region relative to the epitope was further confirmed in the bovine receptor by studying the effect of limited proteolysis on the detection of immunoreactive RP and of guanylate cyclase activity. Treatment of membranes with increasing amounts of trypsin led to a gradual loss of the membrane-associated ANF-R1 receptor detected by the antibody and this loss of immunoreactivity is superimposable with that for the gradual loss of membrane guanylate cyclase activity (fig.4). A corresponding increase in immunodetectable epitope in the supernatant was observed. Since there is no trypsin cleavage site in RP, the loss of membrane-bound RP indicates that the proteolysissensitive region is located in the cytoplasmic domain between the transmembrane domain and the RP epitope. The simultaneous loss of membrane guanylate cyclase

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Fig.4 Effect of partial trypsin digestion on the detection of membrane receptor by RIA. Membranes of BAZG ( 2 mg/ml) were incubated in 50 mM Tris-HC1, pH 7 . 4 with increasing amounts of TPCK-treated trypsin for 30 min at 25OC. The digestion was stopped by addition of 1 mM PMSF and the membranes were centrifuged at 30,000 x g for 15 tnin at 4OC. Aliquots of these membranes (160 p g ) were digested with CNBr, evaporated to dryness and resuspended in Triton X-100 and RIA buffer as described in the methods section. Supernatants were evaporated to dryness before digestion with CNBr. Fragment of receptor containing the RP peptide ( o ) and guanylate cyclase activity ( 0 ) still attached to the membranes after trypsin treatment were evaluated. The presence of RP peptide was also estimated in the supernatants ( A ) . Curves were analyzed simultaneously as in fig.3 and ED50 were estimated to 4 . 0 pg of trypsin/mg of protein.

activity suggests that the cleavage(s) in the trypsinsensitive region released a fragment bearing both the epitope and the catalytic domain of the guanylate cyclase. No guanylate cyclase activity could be detected in the supernatant. This suggests that even if limited proteolysis left intact the Mr 7 0 , 0 0 0 fragment containing the ANF binding domain, further breakdown of the cytoplasmic domain released from the membrane receptor into fragments still occured and

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resulted in a more cytoplasmic domain.

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It can be seen in table 1 that partial proteolysis of BAZG membranes with trypsin (10 pg/mg of proteins) decreases dramatically the membrane-bound immunoreactivity without affecting the receptor density estimated by ANF binding. This observation further confirms that the epitope is a specific fragment of the ANF-R1 receptor located in the cytoplasmic domain and that the antibody only detect the intact receptor. The protease-sensitive region was previously located between the membrane-spanning sequence and the our cytoplasmic guanylate cyclase domain ( 2 8 ) . immunological approach has further documented the position of this region, between the membrane anchor sequence (amino acids 442-462) and amino acid 5 8 6 . This specific antibody also allowed us to develop a specific and sensitive RIA for the ANF-R1 receptor. This approach should prove to be useful for future functional mapping of the receptor, e.g. locating the binding site of ANF and the sites involved in the effects of amiloride or ATP. ACKNOWLEDGMENTS This work was supported by a grant from the Medical Research Council of Canada. The expert help in manuscript preparation by Isabelle Blain is gratefully acknowledged. J.-J. R. is a recipient of a studentship from the Medical Research Council of Canada. A.D.L. is a scientist of the Medical Research Council of Canada REFERENCES 1. Needleman, P.; Adams, S . P . ; Cole, B.R.; Currie, M.G.; Geller, D.M.; Michener, M.L.; Saper, C.B.; Schwartz, D.; and Standaert, D.G. Atriopeptins as cardiac hormones, Hypertension 7, 4 6 9 - 4 8 2 , 1985.

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2. Cantin, M.; and Genest, J . The heart and the atrial natriuretic factor, Endocr. Rev 6, 107-126, 1985. 3. Atlas, S.A. Atrial natriuretic factor: a new hormone of cardiac origin, Recent Prog Horm. Res. 42, 207-249, 1986. 4. Lang, R.E.; Unger, T.; and Ganten, D. Atrial natriuretic peptide: a new factor in blood pressure control, J. Hypertens. 5, 255-271, 1987. 5. Waldman, S.A.; Rapoport, R.M.; and Murad, F. Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues, J. Biol. Chem. 259, 14332-14334, 1984. 6. Hamet, P.; Tremblay, J . ; Pang, S.C.; Garcia, R.; Thibault, G.; Gutkowska, J.; Cantin, M.; and Genest, J . Effect of native and synthetic atrial natriuretic factor on cyclic GMP, Biochem. Biophys. Res. Commun. 123, 515-527, 1984.

7. Waldman, S . A . ; Rapoport, R.M.; Fiscus, R.R.; and Murad, F. Effect of atriopeptin on particulate guanylate cyclase from rat adrenal, Biochim Biophys. Acta 845, 298-303, 1985. 8. Winquist, R . J . ; Faison, E.P.; Waldman, S.A.; Schwartz, K.; Murad, F.; and Rapoport, R.M. Atrial natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle, Proc. Natl Acad. Sci. USA

81, 7661-7664 , 1984.

9. Tremblay, J.; Gerzer, R.; Vinay, P.; Pang, S.C.; BBliveau, R.; and Hamet, P. The increase of cGMP by atrial natriuretic factor correlates with the distribution of particulate guanylate cyclase, FEBS Lett. 181, 17-22, 1985. 10. Leitman, D.C.; Andresen, J . W . ; Kuno, T.; Kamisaki, Y.; Chang, J.-K.; and Murad, F. Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells, J . Biol. Chem. 261, 11650-11655, 1986. 11. De Ldan, A. Amiloride potentiates atrial natriuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa, Life Sci 39, 1109-1116, 1986.


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12. Takayanagi, R.; Inagami, T.; Snajdar, R.M.; Imada, T.; Tamura, M.; and Misono, K.S. Two distinct forms of receptors for atrial natriuretic factor in bovine adrenocortical cells. Purification, ligand binding, and peptide mapping, J. Biol. Chem. 262, 12104-12113, 1987. 13. Meloche, S.; McNicoll, N.; Liu, B.; Ong, H.; and De Lban, A. Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride, Biochemistry 27, 8151-8158, 1988. 14. Kuno, T.; Andresen, J.W.; Kamisaki, Y.; Waldman, S.A.; Chang, L.Y.; Saheki, S.; Leitman, D.C.; Nakane, M.; and Murad, F. Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung, J. Biol. Chem. 261, 5817-5823, 1986. 15. Paul, A.K.; Marala, R.B.; Jaiswal, R.K.; and Sharma, R.K. Coexistence of guanylate cyclase and atrial natriuretic factor receptor in a 1 8 0 - k D protein, Science 235, 1224-1226, 1987.

16. Chinkers, M.; Garbers, D.L.; Chang, M.-S.; Lowe, D.G.; Chin, H.; Goeddel, D.V.; and Schulz, S. A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor, Nature 338, 78-83, 1989. 17. Lowe, D.G. ; Chang, M.-S. ; Hellniss, R. ; Chen, E. ; Singh, S.; Garbers, D.L.; and Goeddel, D.V. Human atrial natriuretic peptide receptor defines a new paradigm for second rnessenger signal transduction, EMBO J. 8, 1377-1384, 1989.

18. Pandey, K.N.; and Singh, S. Molecular cloning and expression of murine guanylate cyclase/atrial natriuretic factor receptor cDNA, J. Biol. Chem. 265, 12342-12348, 1990. 19. Koesling, D.; Herz, J.; Gausepohl, H.; Niroomand, F.; Hinsch, K.D.; Mulsch, A.; Bohme, E.; Schultz, G.; and Frank, R . The primary structure of the 70 kDa subunit of bovine soluble guanylate cyclase, FEBS Lett. 239, 29-34, 1988. 20. Schenk, D.B.; Phelps, M.N.; Porter, J.G.; Fuller, F.; Cordell, B.; and Lewicki, J.A. Purification and subunit composition of atrial natriuretic peptide receptor, Proc. Natl Acad. Sci. USA 84, 1521-1525, 1987.


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Regulation of adrenal atrial natriuretic hormone receptor subtypes.

Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope.

Atrial natriuretic factor recognizes two receptor subtypes in endothelial cells cultured from bovine pulmonary artery.

Characterisation of atrial natriuretic peptide receptors in bovine ventricular sarcolemma.

Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization.

cDNA sequence of the human atrial natriuretic peptide clearance receptor.

Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase. Effects on guanylate cyclase activity and ligand-binding specificity.

Muscarinic receptor subtypes in bovine adrenal medulla.

Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production.

Characterization of a polyclonal anti-peptide antibody to the angiotensin II type-1 (AT1) receptor.

Novel MicroRNA Regulators of Atrial Natriuretic Peptide Production.

Comparison of aldosterone synthesis in adrenal cells, effect of various AT1 receptor blockers with or without atrial natriuretic peptide.

Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide.

Atrial natriuretic factor mRNA and binding sites in the adrenal gland.

Dietary fatty acid modulates glomerular atrial natriuretic peptide receptor.

Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells.

Response to atrial natriuretic peptide, endopeptidase 24.11 inhibitor and C-ANP receptor ligand in the rat.

Pro-Atrial Natriuretic Peptide: A Novel Guanylyl Cyclase-A Receptor Activator That Goes Beyond Atrial and B-Type Natriuretic Peptides.

Differential regulation of natriuretic peptide biosynthesis in bovine adrenal chromaffin cells.

Plasma concentrations of adrenomedullin and atrial and brain natriuretic peptides in patients with adrenal pheochromocytoma.

Atrial natriuretic peptide inhibition of calcium ionophore A23187-stimulated aldosterone secretion in rat adrenal glomerulosa cells.

Cloning of neuromedin B and its receptor in the rabbit and generating a polyclonal antibody to the neuromedin B protein.

A polyclonal antibody to the rat oestrogen receptor expressed in Escherichia coli: characterization and application to immunohistochemistry.

Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli.