(Hypertension. 1995;25:694-698.)
© 1995 American Heart Association, Inc.
Articles |
Mutational Inactivation of the Catalytic Domain of Guanylate Cyclase-A Receptor
From the Departments of Medicine and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio; and the Department of Medical Genetics, University of Toronto, Ontario, Canada (D.-L.S.).
Correspondence to Dr Chung-Ho Chang, Department of Medicine, Division of Hypertension, Case Western Reserve University School of Medicine, W 165, Cleveland, OH 44106.
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Abstract |
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Abstract Guanylate cyclase-A, the receptor for atrial natriuretic factor, contains a protein kinase–like domain and a catalytic domain in the intracellular region. To investigate the active site (the catalytic cavity) of guanylate cyclase-A, we amplified the catalytic domain plus three amino acids from the kinase-like domain of guanylate cyclase-A (GC-c) with polymerase chain reaction (PCR) and expressed it in Escherichia coli. During the screening of the PCR-cloned gene products with guanylate cyclase assay, a mutant that lacks enzyme activity was identified. Results of cDNA sequencing revealed that Leu 817 was replaced by an Arg residue in the mutated protein. The mutated GC-c bound to GTP-agarose as well as the wild-type protein, indicating that the binding capability of mutated GC-c to GTP is not significantly affected by the Arg substitution. Gel-filtration column chromatography showed that, like the wild-type GC-c, the mutated protein also formed a high-molecular-weight complex. Since mutation of Leu 817 to Arg abolishes the catalytic activity, Leu 817 is likely located near the active site of guanylate cyclase-A. These results demonstrate that the carboxyl fragment of guanylate cyclase-A is an ideal system for studying the active site of guanylate cyclase-A.
Key Words: guanylate cyclase • mutation • receptors, atrial natriuretic factor
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Introduction |
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Atrial natriuretic factor (ANF), synthesized primarily by atria, produces profound changes in renal and cardiovascular functions such as diuresis, natriuresis, and hypotension.1 2 3 One type of ANF receptors has been shown to be membrane-bound guanylate cyclase (GC-A).3 4 5 6 The binding of ANF to GC-A activates the enzyme leading to the elevation of intracellular cyclic GMP (cGMP) levels, which mediates most of the biological effects of ANF.1 2 3
GC-A contains an ANF-binding domain in the extracellular region and a kinase-like domain and catalytic domain in the intracellular region.3 4 However, although the catalytic domain of GC-A has been assigned to a 239–amino acid region,4 the exact location of the active site (or the catalytic cavity) is still unknown. The major obstacle is that the catalytic domain of GC-A does not contain G-x-G-x-x-G, the diagnostic consensus sequence of the nucleotide binding site that interacts with the phosphate chain of the nucleotide. The low levels of guanylate cyclase in tissues and cultured cells also make it difficult to locate the active site of GC-A with biochemical techniques such as photoaffinity labeling and protein modification. The catalytic domain plus a portion of the kinase-like domain of GC-A (293 amino acids) have been expressed in Escherichia coli.7 8 The recombinant proteins retain guanylate cyclase activity. Recently, we have further shown that the catalytic domain plus three amino acids from the kinase-like domain of GC-A (242 amino acids) are sufficient for performing the catalytic function (unpublished data, 1994). These studies indicate that the carboxyl 242–amino acid fragment of GC-A (GC-c), rather than the whole enzyme, may be a better system for studying the active site of guanylate cyclase. To locate the amino acid residues in the active site of GC-A, we screened the gene products of the polymerase chain reaction (PCR)–amplified GC-c and identified a mutant that lacked guanylate cyclase activity. Results of DNA sequencing revealed that Leu 817 was replaced by an Arg residue in the mutant, suggesting that Leu 817 may be located near the active site of GC-A.
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Methods |
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Expression of GC-c in E coli and Preparation of Bacterial Extract
The cDNA corresponding to the catalytic domain of guanylate cyclase-A (bp 2673 to 3401) was amplified with PCR from a rat brain library (Clonetech). The oligonucleotide primers used were (1) 5'-GCGCGCGGATCCAGCCGGCTGACCCTGAGT and (2) 5'-GCGCGCGGATCCTCAGCCTCGAGTGCTACATCC. For ease of subcloning, a BamHI cutting site was introduced into the 5' end of the primers. Reaction mixture for PCR contained 2 U Taq polymerase, 2 µmol/L primers, 5 mmol/L dNTP (dTTP, dGTP, dATP, dCTP), 10 mmol/L Tris (pH 8.4), 50 mmol/L KCl, 3 mmol/L MgCl2, 0.01% gelatin, 0.01% NP40, and 0.01% Triton X-100. PCR conditions were as follows: (1) denaturation: 94°C, 7 minutes; and (2) amplification: 94°C, 1 minute; 55°C, 1 minute; and 72°C, 3 minutes; 30 cycles. The sequence of amplified DNAs was determined by the dideoxy chain termination method. The amplified cDNAs were subcloned to the BamHI site of an expression vector, pQE-9, and transformed into an E coli strain M15 containing the lacI-producing plasmid pDM1.I. For induction of gene expression, isopropyl ß-D-thiogalactopyranoside (200 mg/mL) was added, and bacteria were grown in LB medium containing 100 µg/mL ampicillin and 25 µg/mL kanamycin at 30°C overnight. The bacteria were pelleted and resuspended in 25 mmol/L Tris-acetate buffer, pH 7.6, containing 250 mmol/L sucrose, 0.5 mmol/L EDTA, and lysozyme (1 mg/mL) at 4°C. Thirty minutes later, bacteria were sonicated in the presence of 5 mmol/L dithiothreitol, 0.1% phenylmethylsulfonyl fluoride, phosphoramidon (10 µg/mL), and leupeptin (10 µg/mL). The suspension was then centrifuged, and the supernatant was used for guanylate cyclase assay or Western blotting.
Induction of Rabbit Polyclonal Antiserum A2
The peptide GFELELRGDVEMKGK (3 mg, synthesized by Multiple
Peptide System) corresponding to 997-1011 of GC-A was conjugated to
keyhole limpet hemocyanin (10 mg) by glutaraldehyde (21 mmol/L) in the
presence of 0.1 mol/L phosphate buffer, pH 7.0. The conjugated peptide
(65 µg) was dialyzed against the phosphate buffer to remove
glutaraldehyde, mixed with an equal volume of complete Freund's
adjuvant, and subcutaneously injected into New Zealand White rabbit A2.
Every 4 weeks, rabbit A2 received a booster immunization with 32.5 µg
of the conjugated peptide in incomplete Freund's adjuvant. Antiserum
was collected 2 weeks after the third booster immunization.
Western Blotting
Bacterial extracts were subjected to sodium dodecyl
sulfate–polyacrylamide gel electrophoresis and electrotransferred to a
nitrocellulose membrane. The nitrocellulose membrane was incubated with
antiserum A2 (1:3000 dilution), which was raised against amino acids
997-1011 of GC-A, and then with alkaline phosphatase–conjugated goat
anti-rabbit immunoglobulin G (1:2500 dilution, Calbiochem). The
immunoreactive proteins were visualized after color development using
nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate
(Pierce).
Guanylate Cyclase Assay
Guanylate cyclase was assayed at 37°C in the presence of
50 mmol/L Tris, pH 7.6, 2 mmol/L isobutylmethylxanthine, 1 mmol/L GTP,
4 mmol/L MnCl2 or MgCl2, 0.1% (wt/vol)
bovine serum albumin, 25 mmol/L creatine phosphate, and 55 U/mL
creatine kinase (135 U/mg protein) in a final volume of 0.1 mL.
Reactions were initiated by the addition of the bacterial extract with
incubation for 3 minutes and terminated by the addition of 0.5 mL of 50
mmol/L chilled sodium acetate, pH 4.0. Generated cGMP was quantified by
radioimmunoassay.9 10 11
Gel-Filtration Chromatography
Bacterial extracts were concentrated by centrifugation with a
Centricon-10 and applied to a Sepharose 6B (Pharmacia) column (0.7x50
cm) that had been equilibrated with 50 mmol/L Tris buffer, pH 7.6, and
50 mmol/L sucrose. The column was eluted with the same buffer at 0.12
mL/min, and 0.4-mL fractions were collected. Each fraction was assayed
for guanylate cyclase activity. The column was calibrated with blue
dextran and the following protein standards (Sigma): ß-amylase (200
kD), alcohol dehydrogenase (150 kD), albumin (66 kD), carbonic
anhydrase (29 kD), and cytochrome c (12.4 kD).
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Results |
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Identification of a Mutant of the Catalytic Domain of Guanylate Cyclase (GC-c-2-23) That Lacks Guanylate Cyclase Activity
To investigate the active site of GC-A, we amplified GC-c with PCR in the conditions that generate a higher mutation rate (high concentration of MgCl2 and dNTP, see "Methods"). The resulting cDNAs were expressed in E coli. Guanylate cyclase assay was used to screen 24 PCR-cloned gene products. Among them, the product of clone GC-c-2-23 had nondetectable guanylate cyclase activity, and the other gene products had enzyme activity comparable to that of the wild type (GC-c-2-12). Clone GC-c-2-23 was then sequenced. The result of cDNA sequencing revealed that a single mutation occurred at position 817 of clone GC-c-2-23, in which Leu 817 (CTT) was converted into an Arg residue (CGT). To examine whether the nondetectable enzyme activity in the bacterial extract of clone GC-c-2-23 is due to the resulting single point mutation or to the lack of protein expression in the cytosol, bacterial extracts from both GC-c-2-12 (wild type) and GC-c-2-23 were subjected to Western blot analysis using polyclonal antibody A2, which was raised against amino acids 997-1011 of GC-A. Fig 1 shows that both bacterial extracts contained recombinant GC-c, indicating that the mutated protein is expressed in the cytosol. Although the amount of GC-c-2-23 was about one third of that of GC-c-2-12, the activity of GC-c-2-23 was not detected by our enzyme assay, indicating that its activity is at least 100-fold less than that of the wild-type protein. Therefore, Leu 817 of GC-A may be essential for catalytic activity. These results also suggest that the minimal requirement of the carboxyl catalytically active fragment of GC-A should include Leu 817 (ie, at least 213 amino acid residues).
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Effects of Mutation on the High-Molecular-Weight Complex Formation
of GC-c and on the Binding of GC-c to the GTP-Agarose
GC-A and the carboxyl terminal 293–amino acid fragment of GC-A
have been reported to form oliogomers (dimer, tetramer, and possibly
higher order oligomers).8 12 To examine whether GC-c forms
oligomers, we applied GC-c-2-12 to a Sepharose 6B gel-filtration
column. Fig 2 (top panel) shows guanylate cyclase
activity in column fractions of this chromatographic procedure.
Guanylate cyclase activity was eluted as a single peak from the column,
and 80% to 90% of the applied activity was recovered. Compared with
the molecular masses of standard proteins, guanylate cyclase activity
eluted as a 200-kD protein. Fractions 31 through 34 that contained high
guanylate cyclase activity were further analyzed by Western blotting
with antiserum A2. Fig 2 (bottom) confirms the presence of GC-c-2-12 in
these fractions. In contrast, GC-c was not detected in fractions 50
through 55, which correspond to the molecular mass of monomeric GC-c
(data not shown). These results suggest that GC-c may form oligomers or
a complex with another protein. To examine whether mutation at position
817 affects GC-c oligomerization or association with another protein,
GC-c-2-23 was applied to the column. Since GC-c-2-23 did not possess
guanylate cyclase activity, we analyzed fractions 31 through 34 by
Western blotting. The result indicated that GC-c-2-23 also eluted as a
200-kD protein (Fig 2, bottom). Thus, although mutation at position 817
abolishes catalytic activity, it does not affect GC-c oligomerization
or association with another protein.
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The formation of cGMP by guanylate cyclase requires two processes: the binding of GTP to the active site and the catalytic conversion of GTP to cGMP. Alteration of one of them would lead to a loss of enzyme activity. On binding to the active site of guanylate cyclase, GTP is rapidly converted to cGMP. Therefore, it is not practical to perform the GTP binding assay or photoaffinity labeling to examine whether GTP binding to the active site of guanylate cyclase is affected by the substitution of Leu 817 with an Arg residue. However, since guanylate cyclase binds stably to GTP-agarose, we therefore can examine this possibility by comparing the binding of wild-type and the Arg 817 mutant to GTP-agarose. About 0.5 mg of the bacterial extracts of clone GC-c-2-12 and GC-c-2-23 were incubated with 0.5 mL GTP-agarose for 1 hour at 4°C. After incubation, the mixtures were centrifuged to separate the proteins that were bound (ie, associated with GTP-agarose) and unbound to GTP-agarose (ie, present in the supernatant). Bacterial extracts and the supernatants of both clone GC-c-2-12 and GC-c-2-23 were subjected to Western blotting with antiserum A2. Fig 3 shows that both GC-c-2-12 and GC-c-2-23 were not detected in the supernatant after incubation with GTP-agarose (Fig 3), indicating that, like the wild-type protein, the Arg 817 mutant binds to GTP-agarose.
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Conservation of Leu 817 in Guanylate Cyclases and Adenylate
Cyclases
To examine whether Leu 817 is conserved in other guanylate
cyclases, we compared amino acids 809-826 of GC-c-2-23 and GC-A with
the corresponding region of particulate guanylate cyclases from rat
brain (GC-B),13 14 rat small intestine
(GC-C),15 Strongylocentrotus purpuratus
(SP-GC),16 Arbacia punctulata
(AP-GC),17 and of soluble guanylate cyclase 70 kD
(SGC-70)18 19 and 80 kD (SGC-80)
subunits20 21 from bovine and rat lung. Fig 4A shows that Leu 817 of GC-A is conserved in the
corresponding position of various guanylate cyclases except GC-C. In
GC-C, the residue corresponding to Leu 817 of GC-A is replaced by a Gln
residue. The catalytic domain of GC-A is also homologous to that of
various adenylate cyclases. At least eight mammalian adenylate cyclases
have been cloned, including type I,22 type
II,23 type III,24 type IV,25
type V-1,26 27 type V-2,28 type
VI-1,26 and type VI-2.29 Two, type V and type
VI, have been cloned by different groups and are temporarily assigned
types V-1, V-2, VI-1, and VI-2 here. Comparison of amino acids 809-826
of GC-c-2-23 and GC-A with the corresponding region of various
adenylate cyclases is shown in Fig 4B. The residue corresponding to Leu
817 of GC-A is conserved in various adenylate cyclases except type III.
Thus, this Leu residue is conserved among many guanylate cyclases and
adenylate cyclases.
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Discussion |
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Little is known about the active site of guanylate cyclase, although it is assigned to a 239–amino acid region (catalytic domain).4 Since the catalytic domain of guanylate cyclase (GC-c) can be functionally expressed as a soluble protein in E coli, it, rather than the whole enzyme, may be an ideal system for studying the active site of guanylate cyclase. Using guanylate cyclase assay, we screened several PCR-cloned gene products of GC-c and identified a mutant that lacks enzyme activity. Results of cDNA sequencing revealed that Leu 817 is converted into an Arg residue in the mutant. Sequence comparison reveals that Leu 817 is conserved in the corresponding position of most known guanylate cyclases and adenylate cyclases. These results suggest that Leu 817 of guanylate cyclase may play an important functional or structural role.
GC-A has been reported to form oliogomers (dimer, tetramer, and possibly higher order oligomers) in the absence of ANF, and the extracellular receptor sequence has been suggested to be necessary and sufficient for oligomer formation.12 On the other hand, the carboxyl terminal 293–amino acid fragments of GC-A forms dimers,8 and GC-c forms a high-molecular-weight complex when expressed in E coli, indicating that the intracellular region of GC-A may also contribute to protein-protein interaction such as receptor oligomerization or association with another protein. Compared with GC-c, the carboxyl 293–amino acid fragments contain an additional 51 amino acids (amino acids 737-787) located in the kinase-like domain. Since GC-c forms a high-molecular-weight complex, it seems that removal of these 51 amino acids further facilitates oligomerization of the catalytic domain of GC-A or its interaction with another protein. However, it should be noted that bacteria-expressed GC-c may lack appropriate posttranslational modification such as protein phosphorylation. Therefore, whether the intracellular region of GC-A plays a physiological role in protein oligomerization requires further investigation.
The active site of an enzyme consists of residues involved in binding the substrate and residues involved in the catalytic process. Since the mutated enzyme is still capable of binding to GTP-agarose, Leu 817 may be directly or indirectly involved in catalyzing the conversion of GTP to cGMP rather than in binding GTP. We cannot rule out the possibility that the loss of the enzyme activity of GC-c-2-23 may be due to the global structural effect induced by the mutation. However, since the introduced mutation does not affect protein oligomerization and GTP binding, the results suggest that the mutation may not significantly alter the conformation of GC-c. Therefore, Leu 817 may be near the active site of guanylate cyclase.
In summary, we have shown that replacement of Leu 817 with an Arg residue abolishes catalytic activity but not GTP-binding capability or protein-protein interaction. Therefore, Leu 817 may be directly or indirectly involved in the catalytic process. This study demonstrates that the carboxyl fragment (GC-c) is an ideal system to locate the active site on GC-A and to identify the critical amino acid residues involved in GTP binding, enzyme catalysis, or protein oligomerization.
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Acknowledgments |
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This work was supported by the American Heart Association, the American Lung Association, the Leonard C. Rosenberg Renal Research Foundation, the Diabetes Association of Greater Cleveland, and the National Heart, Lung, and Blood Institute (HL-41618). We are grateful to Norma Brown for her careful reading of the manuscript.
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