©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Analysis of Human Prorenin Prosegment Variants in Vitro and in Vivo(*)

Chantal Mercure , Gaetan Thibault , Suzanne Lussier-Cacan (1), Jean Davignon (1), Ernesto L. Schiffrin , Timothy L. Reudelhuber (§)

From the (1)Medical Research Council Multidisciplinary Research Group on Hypertension J. A. deSeve Laboratory of Hyperlipidemia and Atherosclerosis, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The aspartyl protease renin, an important modulator of blood pressure in humans, is present in the circulation not only in its active form, but also as an inactive precursor, prorenin, in which a 43-amino acid prosegment blocks access of the substrate to the active site of the enzyme. Site-directed mutagenesis of the prosegment has led to the following conclusions. 1) Maintenance of the enzymatically inactive state of prorenin requires a short peptide sequence between positions 10P and 20P (where P denotes prosegment and numbering is relative to amino terminus) of the prosegment; and 2) there is an inverse relationship between the ability of prosegment mutations to activate and their effect on the secretion of the various prorenins, suggesting that this same region of the prosegment plays a critical role in the biosynthesis of human prorenin. Since these results demonstrated that single amino acid mutations could activate human prorenin to varying degrees, mutations in this region of the renin gene could be clinically important in humans. To test this hypothesis, genomic screening was carried out on the corresponding region of the human renin gene (exon 2) in a cohort of patients selected for a likely familial component to their hypertension. While this study identified a novel polymorphism in exon 2 of the human renin gene, evidence was not obtained for either the presence of prosegment mutations or the association of the novel polymorphism with hypertension in the patient population studied. In conclusion, both structure-function studies and genetic screening suggest that mutation of the prorenin prosegment is an unlikely factor in activation of the renin-angiotensin system in humans.


INTRODUCTION

The renin-angiotensin system (RAS)()plays a critical role in the maintenance of blood pressure, and its activity has been implicated in the pathogenesis of a number of cardiovascular disorders including hypertension, cardiac hypertrophy, and vascular pathologies associated with diabetes and renal disease (1-4). The rate-limiting step in the RAS is the cleavage of angiotensinogen by the circulating aspartyl protease renin to release the decapeptide angiotensin I. Angiotensin-converting enzyme subsequently cleaves two amino acids from the carboxyl terminus of angiotensin I to release angiotensin II, a potent vasoconstrictor and regulator of aldosterone release.

Active renin in the circulation is derived almost exclusively from the juxtaglomerular cells of the kidney(5) . Within these cells, renin is first synthesized as a precursor, prorenin, in which a 43-amino acid amino-terminal prosegment maintains the enzyme in an inactive state, presumably by blocking access to the active site by steric hindrance (6). A portion of the prorenin made by juxtaglomerular cells is irreversibly activated by the proteolytic cleavage of the prosegment before secretion. Nevertheless, both the kidney and numerous other tissues secrete prorenin into the circulation, where it is present at 3-5 times the level of active renin(7) . Speculation abounds regarding the function of prorenin both in the circulation and in tissues for several reasons. First, the distribution of extrarenal tissues secreting prorenin in mammals is largely conserved, including the pituitary, ovaries, testes, adrenal gland, uterus, and placenta. Prorenin expression in the tissues is also modulated; during pregnancy, prorenin accumulates in the amniotic fluid at concentrations approaching 100 times that seen in the circulation(7) . Second, prorenin-secreting tissues also express other components of the RAS including angiotensinogen, angiotensin-converting enzyme, and angiotensin II receptors, leading to the suggestion that local or ``tissue'' RAS may exist and be physiologically important(8) . Third, prorenin can be transiently activated in vitro by either acidic conditions or extended storage in the cold(9, 10) , raising the possibility that such activation might occur under certain conditions in vivo.

Hypertension is a multifactorial disease whose etiology stems from both environmental and genetic factors. Studies have suggested that multiple genetic loci contribute to inheritable forms of hypertension in inbred strains of laboratory animals (11, 12, 13) and that as much as 20-40% of blood pressure variation in humans may be due to genetic factors(14) . Inappropriate activity of the RAS is both a major contributor to the pathophysiology of hypertension and an important target for pharmacologic intervention in its treatment(15) . Gene mutations could presumably lead to either increased expression or altered activity of RAS components. Indeed, prior studies have suggested a linkage between a polymorphism in the gene encoding angiotensinogen, circulating angiotensinogen levels, and hypertension in humans(16) . While a similar association has been reported for the renin gene locus in genetically hypertensive strains of laboratory rats(17) , no such linkage has been established to date for the human renin gene.

In this study, we have tested the hypothesis that some forms of inheritable hypertension might be due to mutations that cause prorenin to be partially active. Site-directed mutagenesis was used to identify the region of the human prorenin prosegment crucial for maintenance of the protease in its inactive state. This same region of the prosegment was found to play a critical role in the biosynthesis of prorenin. Genome sequencing of the portion of the human renin gene encoding the prosegment was subsequently carried out on a group of hypertensive patients with a likely familial inheritance of hypertension to test whether mutations in this region of the prosegment might lead to spurious activation of the renin-angiotensin system in humans.


EXPERIMENTAL PROCEDURES

Recombinant Plasmid Construction

The mammalian expression vector pRhR1100, which expresses the human prorenin cDNA under the control of the Rous sarcoma virus promoter/enhancer, has been described elsewhere(18) . Site-directed mutagenesis of amino acids in the prosegment of human prorenin was carried out by overlap extension polymerase chain reaction(19) . Amplified regions of the prorenin cDNA were verified by direct DNA sequencing(20) .

Cell Culture and Transfection

Chinese hamster ovary (CHO) cells were grown in Dulbecco's modified Eagle's medium supplemented with 200 µg/ml proline and 10% fetal calf serum. For expression studies, 5 10 cells were plated in a 35-mm dish, and transfection was carried out 24 h later by the calcium phosphate method (21) using 6 µg of the appropriate prorenin expression vector. Twenty-four hours after transfection, the medium was removed, and fresh medium was added. Sixteen hours later, the medium was collected and used immediately to determine renin and prorenin.

Measurement of Renin and Prorenin

Prorenin and renin content in tissue culture supernatants was determined by the angiotensin I generation assay as described previously(21) . Briefly, 16-h culture supernatants were incubated in the presence of excess semipurified sheep angiotensinogen either directly (active renin content) or after incubation with trypsin (total renin content (prorenin + renin)). Percent renin activity was calculated as (active renin content/total renin content) 100. Relative percent activity of the various mutant prorenins was compared with that of native human prorenin (see ). Statistical analysis was carried out by one-way analysis of variance with a posthoc Dunnet multiple comparison test. describes total renin content obtained by transfection of CHO cells with the various prosegment mutant prorenins as a percentage of the total renin content obtained with native prorenin (relative expression), representing the mean of three or more individual transfection experiments. To minimize for variation in transfection efficiency, total renin levels obtained with the various mutant prorenins were normalized to the levels exhibited by non-mutant (native) human prorenin obtained within the same experiment.

Labeling and Immunoprecipitation

Forty-eight hours after transfection of CHO cells as described above, cells were washed twice in prewarmed methionine-free Dulbecco's medium (Life Technologies, Inc.) and incubated in 500 µl of the same medium for 2 h at 37 °C. [S]Methionine (300 µC; >1000 µCi mmol; DuPont NEN) was added to each well, and the cells were incubated for 1 h, followed by the addition of 500 µl of complete medium. After a further 5 h of incubation, the culture supernatants were collected, and prorenin/renin was immunoprecipitated and displayed by gel fluorography as described previously(18) .

Patient Selection

Hypertensive patients were recruited from the Hypertension Clinic at the Clinical Research Institute of Montreal after provision of an informed consent to participate in the study and in adherence to a protocol approved by the institutional human ethics committee. In answer to a questionnaire, all participants reported having at least one hypertensive parent or sibling and either were receiving antihypertensive medication or had a diastolic blood pressure of >90 mm Hg or a systolic pressure of >140 mm Hg on at least two occasions. Blood was collected on EDTA and frozen at -20 °C until assayed.

A control patient population was obtained from healthy, predominantly French Canadian volunteers at Hydro-Quebec (Montreal, Quebec, Canada) who had participated in a previously reported study(22) . Criteria for participation were normal blood pressure at interview and age of >40 years.

Genome Sequencing

Genomic DNA was purified from 100 µl of whole blood using the DNA microextraction kit from Stratagene (La Jolla, CA). A 312-base pair DNA fragment corresponding to exon 2 of the human renin gene was selectively amplified by polymerase chain reaction with the following oligonucleotides: forward primer, CGGG-ATCCACGTTAAAGGTGGTTGTACTA; and reverse primer, CGGAAT-TCGCCTAGGTCCATGAGGGAAA. The amplified DNA fragment was subsequently digested with the restriction enzymes BamHI and EcoRI (underlined in the forward and reverse primer sequences, respectively) and cloned into the Bluescript II plasmid vector (Stratagene). Direct DNA sequencing was carried out on plasmids prepared by the alkaline lysis method (23) from random pools of bacterial colonies using the method of Sanger and Coulson (20) and the sequencing primer GGCTGAGCCAAGCACTC, which corresponds to a reverse primer in exon 2 of the human renin cDNA. All polymorphisms identified by this method were confirmed by a second round of genome amplification and sequencing.


RESULTS

Prosegment Sequences Involved in Renin Inactivation

As previously reported, expression of recombinant human prorenin in CHO cells leads to secretion of mostly active prorenin (, native). Recombinant human prorenin expressed in CHO cells migrates on SDS-polyacrylamide gel electrophoresis at the expected molecular weight for full-length prorenin (see Fig. 3)(24) . By contrast, expression of a human prorenin mutant in which the prosegment is deleted results in the secretion of constitutively active renin ()(18) . Transfection of CHO cells with expression vectors encoding human prorenin in which individual amino acids in the prosegment (Fig. 1) have been mutated results in the secretion of prorenins with varying amounts of renin activity (). The prosegment mutations tested can be subdivided into two categories. The first category of mutations involves charge reversals: Lys and Arg residues were converted to Asp, and Glu and Asp residues were converted to Arg. These results indicate that the effect of charge reversal on prorenin activation was not uniform over the prosegment, but rather clustered in the amino-terminal region from Arg-10P (where P denotes prosegment and numbering is relative to amino terminus) to Arg-20P. Within this region, the effect of charge reversal was most pronounced with Arg-10P (59% activation), Lys-14P (45% activation), and Arg-15P (53% activation) and least effective with reversal of Arg-20P (29% activation). The critical region for charge interaction is well defined since the reversal of Lys-9P and Glu-21P had no significant effect on prorenin activity. As well, charge reversals outside this region, at Lys-24P, Arg-26P, and Asp-29P, did not activate prorenin. Thus, it appears that charged amino acids in region 10P-20P in the prosegment contribute to the maintenance of the inactive state of human prorenin.


Figure 3: Immunoprecipitation of radiolabeled native and mutant prorenins from transfected CHO cell supernatants. Lane1, native prorenin (7.6% activity, 100% expression); lane2, Arg-10P Ala (15.9% activity, 34.4% expression); lane3, Leu-13P Ala (91.2% activity, 3.8% expression); lane4, control supernatant from nontransfected cells. Shown at left is the relative migration of size markers. Data for activity and expression are derived from Table II.




Figure 1: Schematic diagram representing the prosegment of human prorenin. The amino acid sequence is depicted using the one-letter code. Solidarrows denote those amino acids that, when mutated, led to a significant increase in prorenin activity (see Table I for details). Also denoted is the region of the protein encoded by exon 2 of the human renin gene (drawing not to scale).



The second category of mutations tested involved alanine substitution. Only substitution of alanine for either Leu-13P or Arg-15P resulted in a significant activation of prorenin (). These residues lie in the same region as identified using charge reversal (see above). In addition, with the exception of the substitution of Arg-15P, which leads to a minor activation of prorenin (21%), alanine substitution of charged amino acids within region 10P-20P resulted in only a slight, but nonsignificant activation of prorenin. By contrast, the rather conservative alanine substitution of Leu-13P led to a major activation of prorenin (91%), whereas alanine substitution on either side of Leu-13P (for Phe-12P and Lys-14P) had only a slight, but nonsignificant activating potential on prorenin. Substitution of a charged amino acid (Asp) for Leu-31P did not lead to further activation of prorenin. Taken together, these results suggest that prorenin inactivation involves charged amino acids between positions 10P and 20P on the prosegment and is critically dependent on a leucine residue at position 13P.

Role of the Prosegment in the Biosynthesis of Human Prorenin

Several groups have reported the expression of recombinant active renin in tissue culture cells transfected with a prorenin-encoding expression vector in which the prosegment is selectively deleted(18, 25, 26) , suggesting that the prosegment is not absolutely required for biosynthesis of the active enzyme. Nevertheless, in AtT-20 cells transfected with a similar construction, we have previously reported that expression of the resulting active renin was severely suppressed(18) . Comparison of the overall production of total renin (prorenin + renin) from native and prosegment-deleted prorenins in transfected CHO cells confirms this observation: removal of the prosegment results in an apparent 20-fold decrease in biosynthetic capacity (). One possible explanation of this finding is that the prosegment, while not essential, nevertheless plays a role in the biosynthesis or stability of the renin molecule as has been suggested with other proteases(27) . Plotting total renin production achieved by transfection of the various mutant prorenin expression vectors () against their corresponding percent activities () reveals an inverse relationship (Fig. 2), i.e. the more a prorenin molecule is activated by a given mutation, the lower are its levels of secretion in the culture supernatant of transfected cells. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis analysis of selected mutant prorenins demonstrated a protein with the expected molecular weight for intact prorenin (M 49,000)(24) . In addition, the quantity of immunoprecipitable prorenin secreted into the supernatant of transfected cells correlated well with the total renin production as determined by the enzymatic assay (Fig. 3). Thus, while these data should only be considered semiquantitative due to the inherent variation in efficiencies of transfection assays, they identify an inverse relationship between prosegment inactivation and prorenin secreted.


Figure 2: Relationship between mutation-induced activation and secretion of the various recombinant prorenins studied. Arrows denote the positions of native prorenin and prorenin in which the prosegment has been removed by site-directed mutagenesis (ProDel.). Data are from Table II.



Sequence Analysis of Renin Gene Exon 2 in Human Hypertensive Patients

The short region of the prorenin prosegment responsible for the maintenance of the inactive state of human renin is located within exon 2 of the human renin gene (Fig. 1)(28) . To test whether mutations of this region might account for some forms of hypertension due to partial activation of prorenin, a method was developed to allow the direct sequencing of this portion of the genome. Specific oligonucleotides designed to flank exon 2 were used to amplify and clone this region of the genome into bacterial plasmids. Subsequent DNA sequencing of the cloned insert DNA allowed the determination of the complete sequence of exon 2 in the patients tested. Sixty-five patients were selected according to the following criteria: hypertension as defined by either a diastolic pressure of >90 mm Hg or a systolic pressure of >140 mm Hg or a history of hypertension requiring at least two medications for control and at least one parent or sibling with hypertension. In the 65 patients analyzed, no mutations were detected that would lead to a change in the peptide sequence encoded by exon 2. However, a novel polymorphism (CAA to CAC) resulting in the creation of a StyI restriction site (data not shown) at the position in the exon corresponding to Thr-2 of the active renin protein was detected in 14 of the 65 subjects analyzed (I). Preliminary tests to determine whether this polymorphism might serve as a marker for hypertension were negative in a French Canadian normotensive population (I). In summary, there is no evidence for mutation of prosegment sequences responsible for maintaining the inactive state of human renin in the hypertensive patient population analyzed in this study.


DISCUSSION

The results of this study suggest that the region of the human prorenin prosegment from positions 10P to 20P plays an important role in the maintenance of the inactive state of the zymogen. As CHO cells do not cleave the prosegment of prorenin(24) , the most likely explanation for this activation would be that certain of these mutations result in a disruption of both electrostatic and hydrophobic interactions, which are important for the binding of the prosegment to the body of renin. This would presumably have the effect of causing an unfolding of the prosegment, thereby exposing the active site of the enzyme to create enzymatically active prorenin. Analysis of the acid activation kinetics of recombinant prorenin whose prosegment had been subjected to limited proteolysis has likewise led to the suggestion that amino acids 10P-14P contribute to the binding of the prosegment to the body of renin(29) . Since the activation of prorenin by prosegment mutation is not an ``all or none'' phenomenon, but rather follows a continuum (), it is likely that the induced mutations shift the equilibrium of prosegment binding in the active site. That prorenin exists in an equilibrium between a closed and open conformation in vitro has previously been suggested on the basis of its activa-tion kinetics (9) and active-site ``trapping'' experiments in which purified prorenin was incubated with active site-directed inhibitors(29, 30) .

The degree of mutation-induced prorenin activation also exhibits an inverse relationship with the amount of total renin (prorenin + renin) detected in culture supernatants (Fig. 2). Thus, as has been described in other protease families(27) , the prosegment of prorenin (and perhaps other aspartyl proteases) may play a dual role as both an inhibitor and a folding ``catalyst'' during biosynthesis of the active enzyme. These results also suggest that even though prorenin can be activated to varying degrees by single amino acid mutations in the prosegment, the effect of these mutations on the overall physiological activity of the RAS might be offset by the decreased secretion of such mutant proteins. Indeed, the combined effect of these two factors in vitro is a normalization of active (pro)renin levels (, corrected activity). In addition to these structure-function studies, our inability to detect coding mutations in a limited survey of patients selected for a likely familial component to their hypertension suggests that prosegment mutations resulting in activated prorenin are an unlikely explanation for inappropriately high renin activity such as has been reported in some hypertensive patients(31) .

To date, there has been only one published report of a protein-altering mutation in human prorenin(32) . In the family identified, affected individuals were heterozygous for a mutation inducing a premature stop codon within the prorenin coding sequence. Interestingly, these individuals had compensated for their prorenin hemizygosity by increasing the expression level from their unaffected allele. In addition, no homozygotes for this or any other protein-altering mutation in prorenin have been reported, and although several variants of angiotensinogen (the renin substrate) have been described(16) , none affect the region of the protein that codes for the angiotensin I peptide released by renin. Taken together, these findings may suggest an additional role for the RAS in development such that mutations that would inactivate renin (or the RAS) in humans would be lethal.

This study also defines a previously undescribed polymorphism in exon 2 of the human renin gene. While this sequence alteration leads to the appearance of a novel StyI restriction site in exon 2 (data not shown), there are numerous additional StyI sites in the human renin gene, making the detection of this polymorphism difficult in the context of the whole gene. However, by using direct DNA sequencing, a selective polymerase chain reaction amplification strategy, or StyI digestion of the polymerase chain reaction-amplified exon 2 region (data not shown), it has been possible to catalogue the frequency of this polymorphism in populations of hypertensive and normotensive patients (I). While we have not found any obvious correlation with hypertension within the limitations of this study, two points merit further comment. First, we detected the StyI polymorphism in only 1 of 35 (2.9%) ethnically heterogeneous normotensive volunteers drawn from laboratory workers of our institution (data not shown). It is possible, therefore, that the StyI polymorphism will display some degree of ethnic segregation, being more prevalent in the French Canadian population. Ethnic heterogeneity has been reported for two other restriction fragment length polymorphisms in the human renin 5`-flanking DNA in Afro-Caribbeans(33) , reinforcing the importance of selecting an ethnically comparable control population in these studies. Second, the multifactorial nature of hypertension and the implied difficulty in assigning a sufficiently precise clinical subclassification, the contribution of environmental factors, and the potential variation in penetrance of contributing genes will likely require efforts to identify genetic causes in individual families with approaches such as combinations of polymorphisms to increase the power of the statistical analysis. In this effort, the StyI polymorphism described herein may be of greater use in combination with other previously described restriction fragment length polymorphisms in the human renin gene(33, 34, 35) .

  
Table: 0p4in p < 0.05.

  
Table: Relationship between activity and expression in prorenin prosegment mutations

Percent active renin values are from Table I. Percent expression is calculated by dividing the average level of total renin (prorenin + renin) obtained for each mutant prorenin by the average level of expression of native prorenin in the same experiment times 100. Corrected activity is calculated by multiplying percent activity by percent expression.


  
Table: Characteristics of hypertensive/normotensive patient populations used in this study

French Canadian origin was determined by interview.



FOOTNOTES

*
This work was supported in part by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of the Merck-Frosst Chair in Molecular and Clinical Pharmacology. To whom correspondence should be addressed: Clinical Research Inst. of Montreal, 110 Pine Ave. West, Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5716; Fax: 514-987-5717.

The abbreviations used are: RAS, renin-angiotensin system; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We thank Micheline Vachon for expert technical assistance; Michelle LaPointe for patient interviews and blood sampling; Richard Larivière and Anne Minnick for assistance in sample procurement; W. N. Chu for assistance with some of the plasmid constructions; Ken Morgan, Michael N. G. James, and John D. Baxter for helpful advice; and Christian F. Deschepper for critical reading of the manuscript.


REFERENCES
  1. Lindpaintner, K., and Ganten, D. (1991) Cardiology79, Suppl. 1, 32-44
  2. Griendling, K. K., Murphy, T. J., and Alexander, R. W. (1993) Circulation87, 1816-1828 [Medline] [Order article via Infotrieve]
  3. Danser, A. H., van den Dorpel, M. A., Deinum, J., Derkx, F. H., Franken, A. A., Peperkamp, E., de Jong, P. T., and Schalekamp, M. A. (1989) J. Clin. Endocrinol. Metab.68, 160-167 [Abstract]
  4. Neuringer, J. R., and Brenner, B. M. (1993) Am. J. Kidney Dis.22, 98-104 [Medline] [Order article via Infotrieve]
  5. Sealey, J. E., and Rubattu, S. (1989) Am. J. Hypertens.2, 358-366 [Medline] [Order article via Infotrieve]
  6. Baxter, J. D., Duncan, K., Chu, W., James, M. N., Russell, R. B., Haidar, M. A., DeNoto, F. M., Hsueh, W., and Reudelhuber, T. L. (1991) Recent Prog. Horm. Res.47, 211-257 [Medline] [Order article via Infotrieve]
  7. Hsueh, W. A., and Baxter, J. D. (1991) Hypertension (Dallas) 17, 469-479 [Abstract]
  8. Frohlich, E. D., Iwata, T., and Sasaki, O. (1990) Am. J. Med.87, Suppl. 6B, 6B-19S-6B-23S
  9. Derkx, F. H., Schalekamp, M. P., and Schalekamp, M. A. (1987) J. Biol. Chem.262, 2472-2477 [Abstract/Free Full Text]
  10. Pitarresi, T. M., Rubattu, S., Heinrikson, R., and Sealey, J. E. (1992) J. Biol. Chem.267, 11753-11759 [Abstract/Free Full Text]
  11. Creager, M. A., Roddy, M. A., Holland, K. M., Hirsch, A. T., and Dzau, V. J. (1991) Hypertension (Dallas) 17, 989-996 [Abstract]
  12. Dahl, L. K., Heine, M., and Tassinari, L. (1962) Nature194, 480-482 [Medline] [Order article via Infotrieve]
  13. Williams, R. R. (1989) Hypertension (Dallas) 14, 610-613 [Medline] [Order article via Infotrieve]
  14. Ward, R. (1990) in Hypertension: Pathophysiology, Diagnosis, and Treatment (Laragh, J. H., and Brenner, B. M., eds) pp. 81-100, Raven Press, Ltd., New York
  15. Schalekamp, M. A., Derkx, F. H., and van den Meiracker, A. H. (1992) J. Hypertens.10, S157-S164
  16. Jeunemaitre, X., Soubrier, F., Kotelevtsev, Y. V., Lifton, R. P., Williams, C. S., Charru, A., Hunt, S. C., Hopkins, P. N., Williams, R. R., Lalouel, J. M., and Corval, P. (1992) Cell71, 169-180 [Medline] [Order article via Infotrieve]
  17. Rapp, J. P., Wang, S. M., and Dene, H. (1989) Science243, 542-544 [Medline] [Order article via Infotrieve]
  18. Chu, W. N., Baxter, J. D., and Reudelhuber, T. L. (1990) Mol. Endocrinol.4, 1905-1913 [Abstract]
  19. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 61-68 [CrossRef][Medline] [Order article via Infotrieve]
  20. Sanger, F., and Coulson, A. R. (1975) J. Mol. Biol.94, 441-447 [Medline] [Order article via Infotrieve]
  21. Chu, W. N., Mercure, C., Baxter, J. D., and Reudelhuber, T. L. (1992) Hypertension (Dallas) 20, 782-787 [Abstract]
  22. Xhignesse, M., Lussier-Cacan, S., Sing, C. F., Kessling, A. M., and Davignon, J. (1991) Arterioscler. Thromb.11, 1100-1110 [Abstract]
  23. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. Fritz, L. C., Haidar, M. A., Arfsten, A. E., Schilling, J. W., Carilli, C., Shine, J., Baxter, J. D., and Reudelhuber, T. L. (1987) J. Biol. Chem.262, 12409-12412 [Abstract/Free Full Text]
  25. Harrison, T. M., Chidgey, M. A., Brammar, W. J., and Adams, G. J. (1989) Proteins Struct. Funct. Genet.5, 259-265 [Medline] [Order article via Infotrieve]
  26. Nagahama, M., Nakayama, K., Hori, H., and Murakami, K. (1989) FEBS Lett.259, 202-204 [CrossRef][Medline] [Order article via Infotrieve]
  27. Baker, D., Shiau, A. K., and Agard, D. A. (1993) Curr. Opin. Cell Biol.5, 966-970 [Medline] [Order article via Infotrieve]
  28. Hardman, J. A., Hort, Y. J., Catanzaro, D. F., Tellam, J. T., Baxter, J. D., Morris, B. J., and Shine, J. (1984) DNA (N. Y.) 3, 457-468 [Medline] [Order article via Infotrieve]
  29. Heinrikson, R. L., Hui, J., Zürcher-Neely, H., and Poorman, R. A. (1989) Am. J. Hypertens.2, 367-380 [Medline] [Order article via Infotrieve]
  30. Derkx, F. H., Deinum, J., Lipovski, M., Verhaar, M., Fischli, W., and Schalekamp, M. A. (1992) J. Biol. Chem.267, 22837-22842 [Abstract/Free Full Text]
  31. Alderman, M. H., Madhavan, S., Ooi, W. L., Cohen, H., Sealey, J. E., and Laragh, J. H. (1991) N. Engl. J. Med.324, 1098-1104 [Abstract]
  32. Villard, E., Lalau, J. D., van Hooft, I. S., Derkx, F. H., Houot, A. M., Pinet, F., Corvol, P., and Soubrier, F. (1994) J. Biol. Chem.269, 30307-30312 [Abstract/Free Full Text]
  33. Barley, J., Carter, N. D., Cruickshank, J. K., Jeffery, S., Smith, A., Charlett, A., and Webb, D. J. (1991) J. Hypertens.9, 993-996 [Medline] [Order article via Infotrieve]
  34. Okura, T., Kitami, Y., and Hiwada, K. (1993) J. Hum. Hypertens.7, 457-461 [Medline] [Order article via Infotrieve]
  35. Jeunemaitre, X., Rigat, B., Charru, A., Houot, A. M., Soubrier, F., and Corvol, P. (1992) Hum. Gen.88, 301-306 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.