{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T01:01:48Z","timestamp":1792026108025,"version":"4.4.0"},"reference-count":25,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5476","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2000,7,7]]},"abstract":"<jats:p>Hypertension and pregnancy-related hypertension are major public health problems of largely unknown causes. We describe a mutation in the mineralocorticoid receptor (MR), S810L, that causes early-onset hypertension that is markedly exacerbated in pregnancy. This mutation results in constitutive MR activity and alters receptor specificity, with progesterone and other steroids lacking 21-hydroxyl groups, normally MR antagonists, becoming potent agonists. Structural and biochemical studies indicate that the mutation results in the gain of a van der Waals interaction between helix 5 and helix 3 that substitutes for interaction of the steroid 21-hydroxyl group with helix 3 in the wild-type receptor. This helix 5\u2013helix 3 interaction is highly conserved among diverse nuclear hormone receptors, suggesting its general role in receptor activation.<\/jats:p>","DOI":"10.1126\/science.289.5476.119","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:48:21Z","timestamp":1027763301000},"page":"119-123","source":"Crossref","is-referenced-by-count":582,"title":["Activating Mineralocorticoid Receptor Mutation in Hypertension Exacerbated by Pregnancy"],"prefix":"10.1126","volume":"289","author":[{"given":"David S.","family":"Geller","sequence":"first","affiliation":[{"name":"Howard Hughes Medical Institute,"},{"name":"Departments of Genetics, Internal Medicine (Nephrology) and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anita","family":"Farhi","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute,"},{"name":"Departments of Genetics, Internal Medicine (Nephrology) and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikki","family":"Pinkerton","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute,"},{"name":"Departments of Genetics, Internal Medicine (Nephrology) and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Fradley","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute,"},{"name":"Departments of Genetics, Internal Medicine (Nephrology) and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Moritz","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Albert Einstein College of Medicine, New York, NY 10461, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adrian","family":"Spitzer","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Albert Einstein College of Medicine, New York, NY 10461, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gretchen","family":"Meinke","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute,"},{"name":"Molecular Biophysics and Biochemistry, Yale University School of Medicine, Boyer Center for Molecular Medicine, Room 154, 295 Congress Avenue, New Haven, CT 06510, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francis T. F.","family":"Tsai","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute,"},{"name":"Molecular Biophysics and Biochemistry, Yale University School of Medicine, Boyer Center for Molecular Medicine, Room 154, 295 Congress Avenue, New Haven, CT 06510, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul B.","family":"Sigler","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute,"},{"name":"Molecular Biophysics and Biochemistry, Yale University School of Medicine, Boyer Center for Molecular Medicine, Room 154, 295 Congress Avenue, New Haven, CT 06510, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard P.","family":"Lifton","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute,"},{"name":"Departments of Genetics, Internal Medicine (Nephrology) and"},{"name":"Molecular Biophysics and Biochemistry, Yale University School of Medicine, Boyer Center for Molecular Medicine, Room 154, 295 Congress Avenue, New Haven, CT 06510, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","unstructured":"P. August and M. D. Lindheimer in Hypertension: Pathophysiology Diagnosis and Management J. H. Laragh and B. M. Brenner Eds. (Raven New York 1995) pp. 2407\u20132426."},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.272.5262.676"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1038\/966"},{"key":"e_1_3_1_5_2","unstructured":"Seventy-five index cases with early onset of severe hypertension many with suppressed renin and\/or aldosterone levels were screened for mutation by single-stranded conformational polymorphism (SSCP) analysis of all coding regions of MR (3). Identified variants were subjected to DNA sequence analysis (3). All clinical studies were approved by the Yale Human Investigation Committee."},{"key":"e_1_3_1_6_2","unstructured":"Sequence alignment was performed with the Megalign Clustal method software (DNAStar)."},{"key":"e_1_3_1_7_2","unstructured":"A. Sinaiko in (1) pp. 209\u2013225."},{"key":"e_1_3_1_8_2","unstructured":"Lod scores were calculated with the LINKAGE program (20) specifying early onset of severe hypertension as an autosomal dominant trait with complete penetrance a phenocopy rate of 0.001 and a mutant allele frequency of 0.0001."},{"key":"e_1_3_1_9_2","unstructured":"The plasmid pRShMR NX (21) encodes MR expressed from the Rous sarcoma virus long terminal repeat promoter. Site-specific mutations were generated in pRShMR NX by using the Quikchange procedure (Stratagene) with the desired mutagenic primers. Each resulting mutant MR gene was sequenced in its entirety to ensure that no undesired mutations were introduced. The plasmid pMTV-Luc (21) encodes luciferase under control of the MR-sensitive MMTV promoter. pSV2 (Promega) encodes \u03b2-galactosidase (\u03b2-Gal) from the SV40 early promoter. Cos-7 cells were cultured in Dulbecco's minimal essential medium (DMEM; Gibco-BRL) supplemented with 10% heat-inactivated fetal calf serum (FCS). On the night before transfection cells were plated at 2 \u00d7 10 5 cells per well. Cells in each well were transfected with 2 \u03bcg of pMTV-Luc 2 \u03bcg of either MR WT or MR L810 and 0.05 \u03bcg of pSV2. Transfection was performed with cationic liposomes (Lipofectamine-Plus; Life Technologies) after which cells were incubated in DMEM supplemented with 10% FCS for 24 hours. Cells were washed and then serum-free DMEM containing the test steroid was added. The cells were incubated for an additional 16 hours before assay. Luciferase and \u03b2-Gal activities were measured as described (22). Luciferase activity was normalized to \u03b2-Gal activity to correct for transfection efficiency and is expressed as a percentage of the MR WT activity at 1 nM aldosterone. All results are the mean of at least nine independent transfections. For heterodimer experiments the absolute levels of activity induced by aldosterone with MR WT and MR L810 were indistinguishable and multiple independent preparations of both plasmids yielded indistinguishable results in heterodimer experiments."},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/0922-4106(93)90072-H"},{"key":"e_1_3_1_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0955-0674(98)80015-X"},{"key":"e_1_3_1_12_2","doi-asserted-by":"publisher","DOI":"10.1210\/jcem-53-1-58"},{"key":"e_1_3_1_13_2","unstructured":"Additional data are available at www.sciencemag.org\/feature\/data\/1051091.shl."},{"key":"e_1_3_1_14_2","doi-asserted-by":"publisher","DOI":"10.1038\/30775"},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/17.12.3317"},{"key":"e_1_3_1_16_2","unstructured":"The MR WT and MR L810 models were generated from the PR crystal structure (13) with the Turbo Frodo molecular graphics program (). Residues abutting the ligand were altered to match the MR sequence (G773A M810S Y941F and M960L). In addition the E774G substitution was incorporated to reflect the rotational freedom G774 confers on helix 3 in MR. The geometry was regularized with Turbo Frodo. Coordinates for aldosterone (23) were superimposed upon progesterone. Parameters for aldosterone were generated with HIC-Up (24). The 21-hydroxyl group was rotated to form a favorable interaction with the N770 side chain (14)."},{"key":"e_1_3_1_17_2","unstructured":"COS-7 cells were transfected in 100-mm plates with 10 \u03bcg of receptor plasmid by using Lipofectamine 2000 (Life Technologies). On the day after transfection serum-free media was substituted and the cells were grown for an additional 24 hours. Cells were harvested in 40 mM tris-HCl (pH 7.5) 150 mM NaCl 1 mM EDTA and lysed by freeze-thaw treatment in hypotonic buffer containing 10 mM tris-HCl (pH 7.8) 10 mM NaCl 1 mM EDTA 10 mM Na 2 MoO 4 5 mM dithiothreitol antipain (5 \u03bcg\/ml) leupeptin (5 \u03bcg\/ml) chymostatin (5 \u03bcg\/ml) pepstatin A (5 \u03bcg\/ml) and 500 \u03bcM phenylmethylsulfonyl fluoride. After centrifugation at 15 000 g for 15 min extracts were adjusted to 100 mM NaCl and 5% glycerol (binding buffer). Extracts were incubated overnight with [ 3 H]aldosterone and competitor steroid at 0\u00b0C in a total volume of 200 \u03bcl and then incubated with 100 \u03bcl of a 50% slurry of hydroxyapatite in binding buffer. Samples were centrifuged washed twice in binding buffer and then resuspended in ethanol and prepared for scintillation counting. The value for 100% binding was determined by subtracting the number of counts per minute bound in the presence of 500-fold excess of unlabeled aldosterone from the counts bound in the absence of competitor. Nonspecific binding was determined with a 500-fold excess of unlabeled aldosterone. No specific binding was seen in mock-transfected cells."},{"key":"e_1_3_1_18_2","doi-asserted-by":"publisher","DOI":"10.1038\/39645"},{"key":"e_1_3_1_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1097-2765(00)80424-4"},{"key":"e_1_3_1_20_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.3037703"},{"key":"e_1_3_1_21_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.81.11.3443"},{"key":"e_1_3_1_22_2","first-page":"597","volume":"7","author":"Rupprecht R.","year":"1993","unstructured":"Rupprecht R., et al., Mol. Endocrinol. 7, 597 (1993).","journal-title":"Mol. Endocrinol."},{"key":"e_1_3_1_23_2","first-page":"271","volume":"19","author":"Igarashi P.","year":"1996","unstructured":"Igarashi P., Whyte D. A., Li K., Nagami G. T., J. Biol. Chem. 19, 271 (1996).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_24_2","doi-asserted-by":"publisher","DOI":"10.1021\/ja00770a050"},{"key":"e_1_3_1_25_2","first-page":"1119","volume":"54","author":"Kleywegt G. J.","year":"1998","unstructured":"Kleywegt G. J., Jones T. A., Acta Crystallogr. D54, 1119 (1998).","journal-title":"Acta Crystallogr."},{"key":"e_1_3_1_26_2","unstructured":"We thank members of the family studied for their invaluable contribution to this project; C. Nelson-Williams K. Choate P. Igarashi R. Denton and P. Dannies for technical advice; R. Evans for plasmids pRShMR NX and pMTV-Luc; and P. Igarashi for providing pSV2. Supported in part by an NIH Specialized Center of Research grant in hypertension. R.P.L. is an investigator of the Howard Hughes Medical Institute. Dedicated to the memory of Paul Sigler whose passion for science remains a source of inspiration."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.289.5476.119","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T10:40:28Z","timestamp":1705142428000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.289.5476.119"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2000,7,7]]},"references-count":25,"journal-issue":{"issue":"5476","published-print":{"date-parts":[[2000,7,7]]}},"alternative-id":["10.1126\/science.289.5476.119"],"URL":"https:\/\/doi.org\/10.1126\/science.289.5476.119","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2000,7,7]]}}}