Lysine-Cysteine-Serine-Tryptophan Inserted into the DNA-Binding Domain of Human Mineralocorticoid Receptor Increases Transcriptional Activation by Aldosterone

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Human MRs also contain mutations at two sites, codons 180 and 241, in the amino terminal domain (NTD). Together, there are five human MR genes in GenBank. Human MR-984, which was cloned in 1987, has been extensively studied. Human MR-988, cloned in 1995, contains KCSW in its DBD. Neither this human MR-988 nor the other human MR-988 genes have been studied for their response to aldosterone and other corticosteroids. Here, we report that transcriptional activation of human MR-988 by aldosterone is increased by about 50% compared to activation of human MR-984 in HEK293 cells transfected with the TAT3 promoter, while the half-maximal response (EC50) is similar for aldosterone activation of MR-984 and MR-988. The physiological responses in humans with MR genes containing KCSW and with differences in the NTD warrant investigation. mineralocorticoid receptor DNA-binding domain Duplicated mineralocorticoid receptors Aldosterone Cortisol Figures Figure 1 Figure 2 Figure 3 1. Introduction The mineralocorticoid receptor (MR) is a ligand-activated transcription factor that belongs to the nuclear receptor family, which arose in multicellular animals along with other vertebrate steroid receptors: the glucocorticoid receptor (GR), progesterone receptor (PR), androgen receptor (AR) and estrogen receptor (ER) [ 1 – 6 ]. The classical function of the MR in humans and other terrestrial vertebrates is to maintain electrolyte balance by regulating sodium and potassium transport in epithelial cells in the kidney and colon [ 7 – 12 ]. In addition, the MR also has important physiological functions in many other tissues, including brain, heart, skin and lungs [ 11 , 13 – 22 ]. The human MR sequence reported in 1987 by Arriza et al [ 1 ] contains 984 amino acids (MR-984). Inspection of the MR sequence revealed that, like other steroid receptors, the human MR is composed of four modular functional domains: a large amino-terminal domain (NTD) of about 600 amino acids, followed in the center by a DNA-binding domain (DBD) of about 65 amino acids, followed by a small hinge domain of about 60 amino acids that connected to the ligand-binding domain of about 250 amino acids at the C-terminus, where aldosterone, cortisol and other corticosteroids bind to activate transcription [ 1 , 2 , 23 – 25 ]. Analysis of the human MR sequence by Arriza et al. [ 1 ] revealed that the MR was closely related to the glucocorticoid receptor (GR), which was consistent with evidence that some corticosteroids, such as cortisol, corticosterone and 11-deoxycorticosterone were ligands for both the MR and GR [ 13 , 15 , 21 , 24 , 26 ] and that aldosterone, cortisol, corticosterone and 11-deoxycorticosterone have similar binding affinity for human MR [ 1 , 21 , 26 – 28 ]. Activation of the MR by cortisol and corticosterone, two steroids that are ligands for the GR [ 28 – 30 ], is consistent with the evolution of the GR and MR from a common ancestral corticoid receptor (CR) in a cyclostome (jawless fish) that evolved about 550 million years ago at the base of the vertebrate line [ 24 , 31 – 37 ]. Knowledge about the human MR family expanded in 1995, when Bloem et al. [ 38 ] cloned a human MR with 988 amino acids (MR-988). This human MR had four additional amino acids in the DBD due to alternative splicing between exons 3 and 4 of an in-frame insertion of 12 bp encoding Lys, Cys, Ser, Trp (KCSW) [ 39 , 40 ] (Fig. 1 ). As shown in Fig. 1 , the DBDs in terrestrial vertebrate MRs are highly conserved. Thus, humans were found to contain two almost identical MR transcripts, in which their DBDs differ by only four amino acids. The number of distinct human MR genes expanded after a BLAST analysis of GenBank with the human MR sequence identified differences at codons 180 and 241 in the NTD of MR-984 and MR-988 in humans [ 41 ] (Fig. 2 ). Human MR-984a, cloned by Arriza et al [ 1 ], has been studied for transcriptional activation by aldosterone and other corticosteroids [ 1 , 26 , 27 , 29 , 30 ]. However, it is not known if the KCSW insert in the DBD of human MR affects its transcriptional activation by corticosteroids, and if so, how? To answer these questions, we compared transcriptional activation by corticosteroids of human MR-984 to human MR-988 containing KCSW in the DBD transfected into in HEK293 cells in the presence of the TAT3 promoter. Here we report that compared to human MR-984, human MR-988, has about 50–90% increased transcriptional activation by aldosterone, cortisol, and other corticosteroids, when these MRs are transfected into HEK293 cells. The physiological responses to corticosteroids in humans with MR genes containing KCSW in the DBD and with differences at codons 180 and 241 in their NTD warrant investigation. 2. Results Corticosteroid-dependent activation of human MR-984 and human MR-988 containing KCSW. We have used human MR-984a (accession AAA59571) [ 1 ] for our studies of the human MR [ 27 ]. For this project, as described in the Methods Section, we constructed MR-984b (accession XP_054206038), MR-988b (accession XP_054206037), MR-984c (accession NP_000892), MR-988c (accession XP_01530277). We also constructed MR-988a by insertion of KCSW into the DBD of MR-984a. In Fig. 3 , we show the concentration dependence of transcriptional activation by corticosteroids of these six human MRs transfected into HEK293 cells with a TAT3 luciferase promoter. Luciferase levels were used to calculate an EC50 and fold-activation for each steroid (Table 1). These results reveal that the three human MR-988 genes with KCSW in the DBD have a substantial increase in transcriptional activation compared to their MR-984 counterparts, which lack KCSW, while there is only a modest change in EC50s of corticosteroids for MR-988 compared to MR-984. As shown in Fig. 3 and Table 1, there also were differences in the level of transcriptional activation of MR-984a, MR-984b and MR-984c (Fig. 3 A-D), as well as for MR-988a, MR-988b and MR-9848c (Fig. 3 E-H), which have different amino acids at codons 180 and 241 in their NTD. Compared to activation by aldosterone of the three human MRs with 984 amino acids, aldosterone increased fold activation by about 50% for MR-988a, MR-988b and MR-9848c, all of which contain KCSW in the DBD. As shown in Table 1, cortisol, which activates the MR in organs that lack 11b-hydroxysteroid dehydrogenase [ 7 , 42 – 44 ], increases fold activation by 29% for MR-988a compared to MR984a, by 73% for MR-988b compared to 984b and by 69% for MR-988c compared to MR-984c. Corticosterone increases fold activation by 94% for MR-988a compared to MR984a, by 78% for MR-988b compared to 984b and by 111% for MR-988c compared to MR-984c. 11-Deoxycorticosterone increases fold activation by 94% for MR-988a compared to MR984a, by 78% for MR-988b compared to 984b and by 90% for MR-988c compared to MR-984c. The differences in human MR at codons 180 and 241 in the NTD influences corticosteroid activation of transcription. MR-984b and MR-988b, which have Ile-180 and Val-241 in the NTD, have the highest activation, and MR-984c and MR-988c, which have Val-180 and Val-241 in the NTD have the lowest fold activation. Transcriptional activation by aldosterone, cortisol and 11-deoxycorticosterone of MR-984a and MR-988a, which have Ile-180, Ala-241 in the NTD, is between that of MR-984b and MR-984c. An exception is corticosterone, which has similar activation of MR-988a and MR-988c. These results indicate that the NTD has an allosteric influence on transcriptional activation by corticosteroids of human MR. 3. Discussion The human MR cloned by Arriza et al [ 1 ] has 984 amino acids (MR984a), and this is the MR that has been widely studied [ 7 , 9 – 11 , 13 , 15 , 24 ]. However, our BLAST search of GenBank [ 41 ] revealed that MR984a is not the only human MR in GenBank (Fig. 2 ). GenBank also contains two human MRs (MR-988b, MR-988c) with KCSW beginning at 634 in the DBD and with different amino acids at codons 180 and 241 in the NTD, than are found in MR-988a, providing total of five distinct human MR sequences. To begin to understand the effects, if any, of the sequence differences among these human MRs, we studied corticosteroid activation of MR-984b, MR-988b, MR-984c and MR-988c for comparison with each other and with MR-984a. We also constructed MR988a for comparison with human MR984a and the other four human MR genes. We find a clear increase in transcriptional activation by aldosterone and other corticosteroids of human MRs with 988 amino acids and KCSW in the DBD. Thus, insertion into an ancestral MR of KCSW by alternative splicing in the DBD resulted in increased transcriptional activation of human MR by aldosterone and other corticosteroids [Figure 3 , Table 1]. This increase of MR transcription by aldosterone and other corticosteroids may also occur in other terrestrial vertebrate MRs with KCSW in the DBD. We find that mutations at codons 180 and 241 in the NTD of human MR also affect transcriptional activation by aldosterone and other corticosteroids. This allosteric effect of two mutations in the human NTD was surprising. Compared to chimpanzee, gorilla and orangutan MRs, human MRs are unique in containing an MR with either (Ile-180, Ala-241) or (Val-180, Val-241) in their NTD [ 41 ]. That is, chimpanzees, gorillas and orangutans lack an MR with either (Ile-180, Ala-241) or (Val-180, Val-241) in their NTD. We propose that MRs with (Ile-180, Ala-241) or (Val-180, Val-241) in their NTD evolved in humans after divergence from chimpanzees and the evolution of these MRs. In the light of the diverse physiological functions of the MR in humans in regulating transcription in kidney, brain, heart, skin, lungs and other tissues [ 11 , 13 – 22 ], the evolution of human MRs with (Ile-180, Ala-241) or (Val-180, Val-241) in their NTD may have been important in the evolution of humans from chimpanzees [ 41 ]. The interaction of the MR with the GR to form heterodimers is important [ 47 – 51 ]. The response to aldosterone and other corticosteroids to heterodimers of human MRs containing KCSW in the DBD, as well as either (Ile-180, Ala-241), (Ile-180, Val-241) or (Val-180, Val-241) with human GR needs to be explored. 4. Materials & Methods Construction of plasmid vectors Full-length mineralocorticoid receptor (MR) of human as registered in Genbank (accession number: XP_054206038) was used as MR-984 to construct MR-984b, MR-984c, MR-988a, MR-988b, MR-988c. The insertion of 4-amino acids (Lys-Cys-Ser-Trp) into the DBD after codon 633 of human MR was performed using KOD-Plus-mutagenesis kit (TOYOBO). The nucleic acid sequences of all constructs were verified by sequencing. Chemical reagents Cortisol, corticosterone, 11-deoxycorticosterone and aldosterone were purchased from Sigma-Aldrich. For reporter gene assays, all hormones were dissolved in dimethyl-sulfoxide (DMSO); the final DMSO concentration in the culture medium did not exceed 0.1%. Transactivation assays and statistical analyses Transfection and reporter assays were carried out in HEK293 cells, as described previously [ 27 ]. The cells were transfected with 100 ng of receptor gene, reporter gene containing the Photinus pyralis luciferase gene and pRL-tk, as an internal control to normalize for variation in transfection efficiency; pRL-tk contains the Renilla reniformis luciferase gene with the herpes simplex virus thymidine kinase promoter. Each assay had a similar number of cells, and assays were done with the same batch of cells in each experiment. All experiments were performed in triplicate. Promoter activity was calculated as firefly ( P. pyralis )-lucifease activity/sea pansy ( R. reniformis )-lucifease activity. The values shown are mean ± SEM from three separate experiments, and dose-response data, which were used to calculate the half maximal response (EC50) for each steroid, were analyzed using GraphPad Prism. Declarations Funding: This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (23K05839) to Y.K., and the Takeda Science Foundation to Y.K. Competing interests: The authors have declared that no competing interests exist. Contributions . Yoshinao Katsu: Investigation, Conceptualization, Supervision, Formal Analysis, Writing – original draft, Writing – review & editing. Jiawen Zhan: Data curation, Investigation, Methodology. Michael E. Baker: Conceptualization; Formal analysis, Supervision, Writing – original draft, Writing – review & editing. References Arriza JL, Weinberger C, Cerelli G, et al. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science. 1987;237(4812):268-275. doi:10.1126/science.3037703, (n.d.). Evans RM. The steroid and thyroid hormone receptor superfamily. 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PMID: 27655894; PMCID: PMC5056104., (n.d.). Pooley JR, Rivers CA, Kilcooley MT, Paul SN, Cavga AD, Kershaw YM, Muratcioglu S, Gursoy A, Keskin O, Lightman SL. Beyond the heterodimer model for mineralocorticoid and glucocorticoid receptor interactions in nuclei and at DNA. PLoS One. 2020 Jan 10;15(1):e0227520. doi: 10.1371/journal.pone.0227520. PMID: 31923266; PMCID: PMC6953809., (n.d.). Kiilerich P, Triqueneaux G, Christensen NM, et al. Interaction between the trout mineralocorticoid and glucocorticoid receptors in vitro. J Mol Endocrinol. 2015;55(1):55-68. doi:10.1530/JME-15-0002, (n.d.). Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4095590","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":279324214,"identity":"22c99646-3b78-41be-9fd3-8ab14bf06007","order_by":0,"name":"Yoshinao Katsu1","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yoshinao","middleName":"","lastName":"Katsu1","suffix":""},{"id":279324485,"identity":"4bee2fff-abf5-4728-b441-bd80418dc340","order_by":1,"name":"Jiawen Zhang","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiawen","middleName":"","lastName":"Zhang","suffix":""},{"id":279324486,"identity":"a6f884b4-6e89-4459-945e-98dca4f2fac1","order_by":2,"name":"Michael Baker","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYDACCQZmIGkDYhowMLARryWNdC2HSdCiO7v5scHHPecT+/sPb2D4UHaYsBazO8eME2c8u50440ZaAeOMc8RouZFgfJjnwO3EDRI8Bsy8bURpSf98+M+Bc4kb+M8YMP8lTkuOcTLDgQOJGxhyDJgZidJy50yxYc+BZGOQXw72nEsnQsvt9s0SPw7YyQJDbOODH2XWhLWggAMkqh8Fo2AUjIJRgAsAAN5VQM5s8cz4AAAAAElFTkSuQmCC","orcid":"","institution":"University of California, San Diego","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Baker","suffix":""}],"badges":[],"createdAt":"2024-03-13 19:36:42","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false,"coiExplicitlySet":false},"doi":"10.21203/rs.3.rs-4095590/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4095590/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52695497,"identity":"a2d18bac-50ae-47a9-a842-c0f1bf7693b5","added_by":"auto","created_at":"2024-03-14 15:54:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the DNA-binding domains on human MR-984, human MR-988, platypus MR-990, platypus MR-994-, turtle MR-993, turtle MR-997, Xenopus MR-979, Xenopus MR-983, lungfish MR-985, zebrafish MR-970, elephant shark MR-956. \u003c/strong\u003eThe DBD of human MR-988 has an insertion of KCSW that is absent in human MR-984. Otherwise, the rest of the sequences of human MR-984 and human MR-988 are identical. A four amino acid insertion also is present at a corresponding position in the DBD of platypus MR turtle MR, and \u003cem\u003eXenopus\u003c/em\u003eMR. Moreover, except for the four amino acid insert in the DBD, the rest of the DBD sequences of human MR, platypus MR and turtle MR are identical. Differences between the DBD sequence in human MR and selected vertebrate MRs are shown in red. Protein accession numbers are XP_054206038 for human MR-984, XP_054206037 for human MR-988, XP_007669969 for platypus MR-990, XP_016083764 for platypus MR-994, XP_044874191 for turtle MR-993, XP_044874189 for turtle MR-997, NP_001084074 for Xenopus MR-979, XP_018098693 for \u003cem\u003eXenopus\u003c/em\u003e MR-983, BCV19931 for lungfish MR-985, NP_001093873 for zebrafish MR-970 and XP_007902220 for elephant shark MR-956.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4095590/v1/2c17950f2268ec61ffe9b4ee.png"},{"id":52695117,"identity":"3de240eb-0cd7-4ffb-81f5-15e2bc32c0cb","added_by":"auto","created_at":"2024-03-14 15:46:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuman MR Genes in GenBank.\u003c/strong\u003e There are three human MR genes with 984 amino acids and two human MR genes with 988 amino acids in GenBank. These MRs contain either isoleucine-180, alanine-241 MR-984a (accession AAA59571), isoleucine-180, valine-241 MR-984b (accession XP_054206038), or valine-180, valine-241 MR-984c (accession NP_000892), in the NTD. \u0026nbsp;GenBank also contains two human MRs with 988 amino acids containing KCSW in the DBD and either isoleucine-180, valine-241 MR-988b (accession XP_054206037), or valine-180, valine-241 MR-988c (accession XP_01530277) in the NTD. \u0026nbsp;A human MR with isoleucine-180 and alanine-241 and a KCSW insert in the NTD was not found in GenBank. MR-988a is our construction of this human MR, which we studied for activation by corticosteroids along with the other five human MR genes that are found in GenBank.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4095590/v1/0e7f465a94bbc6e35d2415b2.png"},{"id":52695115,"identity":"583e149f-8efb-4828-bf7a-b863eb47cc56","added_by":"auto","created_at":"2024-03-14 15:46:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConcentration-dependent transcriptional activation by corticosteroids of human MR-984a, b and c and human MR-988a, b and c.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids for human MR-984a, b, c and human MR-988a, b, c were expressed in HEK293 cells with a TAT3-luciferase promoter [2,45,46]. Cells were treated with increasing concentrations of either aldosterone, cortisol, 11-deoxycorticosterone, corticosterone, or vehicle alone (DMSO). Results are expressed as means ± SEM, n=3. Y-axis indicates fold-activation compared to the activity of vector with vehicle (DMSO) alone as 1. A. Human MR-984 with aldosterone. B. Human MR-984 with cortisol. C. Human MR-984 with corticosterone. D. Human MR-984 with 11-deoxycorticosterone. E. Human MR-988 with aldosterone. F. Human MR-988 with cortisol. G. Human MR-988 with corticosterone. H. Human MR-988 with 11-deoxycorticosterone.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4095590/v1/a50f429784976b23d4e73057.png"},{"id":52696124,"identity":"79281a63-966e-45e6-9f07-00b41d19eff2","added_by":"auto","created_at":"2024-03-14 16:02:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":461108,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4095590/v1/1bc66824-cdcc-4584-ab92-cc73abb32c58.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eLysine-Cysteine-Serine-Tryptophan Inserted into the DNA-Binding Domain of Human Mineralocorticoid Receptor Increases Transcriptional Activation by Aldosterone\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe mineralocorticoid receptor (MR) is a ligand-activated transcription factor that belongs to the nuclear receptor family, which arose in multicellular animals along with other vertebrate steroid receptors: the glucocorticoid receptor (GR), progesterone receptor (PR), androgen receptor (AR) and estrogen receptor (ER) [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The classical function of the MR in humans and other terrestrial vertebrates is to maintain electrolyte balance by regulating sodium and potassium transport in epithelial cells in the kidney and colon [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, the MR also has important physiological functions in many other tissues, including brain, heart, skin and lungs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe human MR sequence reported in 1987 by Arriza et al [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] contains 984 amino acids (MR-984). Inspection of the MR sequence revealed that, like other steroid receptors, the human MR is composed of four modular functional domains: a large amino-terminal domain (NTD) of about 600 amino acids, followed in the center by a DNA-binding domain (DBD) of about 65 amino acids, followed by a small hinge domain of about 60 amino acids that connected to the ligand-binding domain of about 250 amino acids at the C-terminus, where aldosterone, cortisol and other corticosteroids bind to activate transcription [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis of the human MR sequence by Arriza et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] revealed that the MR was closely related to the glucocorticoid receptor (GR), which was consistent with evidence that some corticosteroids, such as cortisol, corticosterone and 11-deoxycorticosterone were ligands for both the MR and GR [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and that aldosterone, cortisol, corticosterone and 11-deoxycorticosterone have similar binding affinity for human MR [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Activation of the MR by cortisol and corticosterone, two steroids that are ligands for the GR [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], is consistent with the evolution of the GR and MR from a common ancestral corticoid receptor (CR) in a cyclostome (jawless fish) that evolved about 550\u0026nbsp;million years ago at the base of the vertebrate line [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35 CR36\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKnowledge about the human MR family expanded in 1995, when Bloem et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] cloned a human MR with 988 amino acids (MR-988). This human MR had four additional amino acids in the DBD due to alternative splicing between exons 3 and 4 of an in-frame insertion of 12 bp encoding Lys, Cys, Ser, Trp (KCSW) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the DBDs in terrestrial vertebrate MRs are highly conserved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThus, humans were found to contain two almost identical MR transcripts, in which their DBDs differ by only four amino acids. The number of distinct human MR genes expanded after a BLAST analysis of GenBank with the human MR sequence identified differences at codons 180 and 241 in the NTD of MR-984 and MR-988 in humans [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHuman MR-984a, cloned by Arriza et al [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], has been studied for transcriptional activation by aldosterone and other corticosteroids [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, it is not known if the KCSW insert in the DBD of human MR affects its transcriptional activation by corticosteroids, and if so, how? To answer these questions, we compared transcriptional activation by corticosteroids of human MR-984 to human MR-988 containing KCSW in the DBD transfected into in HEK293 cells in the presence of the TAT3 promoter. Here we report that compared to human MR-984, human MR-988, has about 50\u0026ndash;90% increased transcriptional activation by aldosterone, cortisol, and other corticosteroids, when these MRs are transfected into HEK293 cells. The physiological responses to corticosteroids in humans with MR genes containing KCSW in the DBD and with differences at codons 180 and 241 in their NTD warrant investigation.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003eCorticosteroid-dependent activation of human MR-984 and human MR-988 containing KCSW.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have used human MR-984a (accession AAA59571) [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e] for our studies of the human MR [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. For this project, as described in the Methods Section, we constructed MR-984b (accession XP_054206038), MR-988b (accession XP_054206037), MR-984c (accession NP_000892), MR-988c (accession XP_01530277). We also constructed MR-988a by insertion of KCSW into the DBD of MR-984a.\u003c/p\u003e\n\u003cp\u003eIn Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, we show the concentration dependence of transcriptional activation by corticosteroids of these six human MRs transfected into HEK293 cells with a TAT3 luciferase promoter. Luciferase levels were used to calculate an EC50 and fold-activation for each steroid (Table 1). These results reveal that the three human MR-988 genes with KCSW in the DBD have a substantial increase in transcriptional activation compared to their MR-984 counterparts, which lack KCSW, while there is only a modest change in EC50s of corticosteroids for MR-988 compared to MR-984. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Table 1, there also were differences in the level of transcriptional activation of MR-984a, MR-984b and MR-984c (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D), as well as for MR-988a, MR-988b and MR-9848c (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE-H), which have different amino acids at codons 180 and 241 in their NTD.\u003c/p\u003e\n\u003cp\u003eCompared to activation by aldosterone of the three human MRs with 984 amino acids, aldosterone increased fold activation by about 50% for MR-988a, MR-988b and MR-9848c, all of which contain KCSW in the DBD. As shown in Table\u0026nbsp;1, cortisol, which activates the MR in organs that lack 11b-hydroxysteroid dehydrogenase [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e], increases fold activation by 29% for MR-988a compared to MR984a, by 73% for MR-988b compared to 984b and by 69% for MR-988c compared to MR-984c. Corticosterone increases fold activation by 94% for MR-988a compared to MR984a, by 78% for MR-988b compared to 984b and by 111% for MR-988c compared to MR-984c. 11-Deoxycorticosterone increases fold activation by 94% for MR-988a compared to MR984a, by 78% for MR-988b compared to 984b and by 90% for MR-988c compared to MR-984c.\u003c/p\u003e\n\u003cp\u003eThe differences in human MR at codons 180 and 241 in the NTD influences corticosteroid activation of transcription. MR-984b and MR-988b, which have Ile-180 and Val-241 in the NTD, have the highest activation, and MR-984c and MR-988c, which have Val-180 and Val-241 in the NTD have the lowest fold activation. Transcriptional activation by aldosterone, cortisol and 11-deoxycorticosterone of MR-984a and MR-988a, which have Ile-180, Ala-241 in the NTD, is between that of MR-984b and MR-984c. An exception is corticosterone, which has similar activation of MR-988a and MR-988c. These results indicate that the NTD has an allosteric influence on transcriptional activation by corticosteroids of human MR.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe human MR cloned by Arriza et al [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] has 984 amino acids (MR984a), and this is the MR that has been widely studied [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, our BLAST search of GenBank [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] revealed that MR984a is not the only human MR in GenBank (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). GenBank also contains two human MRs (MR-988b, MR-988c) with KCSW beginning at 634 in the DBD and with different amino acids at codons 180 and 241 in the NTD, than are found in MR-988a, providing total of five distinct human MR sequences. To begin to understand the effects, if any, of the sequence differences among these human MRs, we studied corticosteroid activation of MR-984b, MR-988b, MR-984c and MR-988c for comparison with each other and with MR-984a. We also constructed MR988a for comparison with human MR984a and the other four human MR genes.\u003c/p\u003e\u003cp\u003eWe find a clear increase in transcriptional activation by aldosterone and other corticosteroids of human MRs with 988 amino acids and KCSW in the DBD. Thus, insertion into an ancestral MR of KCSW by alternative splicing in the DBD resulted in increased transcriptional activation of human MR by aldosterone and other corticosteroids [Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;1]. This increase of MR transcription by aldosterone and other corticosteroids may also occur in other terrestrial vertebrate MRs with KCSW in the DBD.\u003c/p\u003e\u003cp\u003eWe find that mutations at codons 180 and 241 in the NTD of human MR also affect transcriptional activation by aldosterone and other corticosteroids. This allosteric effect of two mutations in the human NTD was surprising. Compared to chimpanzee, gorilla and orangutan MRs, human MRs are unique in containing an MR with either (Ile-180, Ala-241) or (Val-180, Val-241) in their NTD [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. That is, chimpanzees, gorillas and orangutans lack an MR with either (Ile-180, Ala-241) or (Val-180, Val-241) in their NTD. We propose that MRs with (Ile-180, Ala-241) or (Val-180, Val-241) in their NTD evolved in humans after divergence from chimpanzees and the evolution of these MRs. In the light of the diverse physiological functions of the MR in humans in regulating transcription in kidney, brain, heart, skin, lungs and other tissues [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the evolution of human MRs with (Ile-180, Ala-241) or (Val-180, Val-241) in their NTD may have been important in the evolution of humans from chimpanzees [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe interaction of the MR with the GR to form heterodimers is important [\u003cspan additionalcitationids=\"CR48 CR49 CR50\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e–\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The response to aldosterone and other corticosteroids to heterodimers of human MRs containing KCSW in the DBD, as well as either (Ile-180, Ala-241), (Ile-180, Val-241) or (Val-180, Val-241) with human GR needs to be explored.\u003c/p\u003e"},{"header":"4. Materials \u0026 Methods","content":"\u003cp\u003e \u003cb\u003eConstruction of plasmid vectors\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFull-length mineralocorticoid receptor (MR) of human as registered in Genbank (accession number: XP_054206038) was used as MR-984 to construct MR-984b, MR-984c, MR-988a, MR-988b, MR-988c. The insertion of 4-amino acids (Lys-Cys-Ser-Trp) into the DBD after codon 633 of human MR was performed using KOD-Plus-mutagenesis kit (TOYOBO). The nucleic acid sequences of all constructs were verified by sequencing.\u003c/p\u003e\u003cp\u003e \u003cb\u003eChemical reagents\u003c/b\u003e \u003c/p\u003e\u003cp\u003eCortisol, corticosterone, 11-deoxycorticosterone and aldosterone were purchased from Sigma-Aldrich. For reporter gene assays, all hormones were dissolved in dimethyl-sulfoxide (DMSO); the final DMSO concentration in the culture medium did not exceed 0.1%.\u003c/p\u003e\u003cp\u003e \u003cb\u003eTransactivation assays and statistical analyses\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTransfection and reporter assays were carried out in HEK293 cells, as described previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The cells were transfected with 100 ng of receptor gene, reporter gene containing the \u003cem\u003ePhotinus pyralis\u003c/em\u003e luciferase gene and pRL-tk, as an internal control to normalize for variation in transfection efficiency; pRL-tk contains the \u003cem\u003eRenilla reniformis\u003c/em\u003e luciferase gene with the herpes simplex virus thymidine kinase promoter. Each assay had a similar number of cells, and assays were done with the same batch of cells in each experiment. All experiments were performed in triplicate. Promoter activity was calculated as firefly (\u003cem\u003eP. pyralis\u003c/em\u003e)-lucifease activity/sea pansy (\u003cem\u003eR. reniformis\u003c/em\u003e)-lucifease activity. The values shown are mean ± SEM from three separate experiments, and dose-response data, which were used to calculate the half maximal response (EC50) for each steroid, were analyzed using GraphPad Prism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (23K05839) to Y.K., and the Takeda Science Foundation to Y.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eYoshinao Katsu: Investigation, Conceptualization, Supervision, Formal Analysis, Writing – original draft, Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eJiawen Zhan: Data curation, Investigation, Methodology.\u003c/p\u003e\n\u003cp\u003eMichael E. Baker: Conceptualization; Formal analysis, Supervision, Writing – original draft, Writing – review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArriza JL, Weinberger C, Cerelli G, et al. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science. 1987;237(4812):268-275. doi:10.1126/science.3037703, (n.d.).\u003c/li\u003e\n \u003cli\u003eEvans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988;240(4854):889-895. doi:10.1126/science.3283939, (n.d.).\u003c/li\u003e\n \u003cli\u003eBridgham JT, Eick GN, Larroux C, et al. Protein evolution by molecular tinkering: diversification of the nuclear receptor superfamily from a ligand-dependent ancestor. PLoS Biol. 2010;8(10):e1000497. Published 2010 Oct 5. doi:10.1371/journal.pbio.1000497, (n.d.).\u003c/li\u003e\n \u003cli\u003eBaker ME, Nelson DR, Studer RA. 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J Mol Endocrinol. 2015;55(1):55-68. doi:10.1530/JME-15-0002, (n.d.).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mineralocorticoid receptor, DNA-binding domain, Duplicated mineralocorticoid receptors, Aldosterone, Cortisol","lastPublishedDoi":"10.21203/rs.3.rs-4095590/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4095590/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDue to alternative splicing in the DNA-binding domain (DBD) of the mineralocorticoid receptor (MR), humans contain two almost identical MR transcripts with either 984 amino acids (MR-984) or 988 amino acids (MR-988), in which their DBDs differ by only four amino acids, Lys,Cys,Ser,Trp (KCSW). Human MRs also contain mutations at two sites, codons 180 and 241, in the amino terminal domain (NTD). Together, there are five human MR genes in GenBank. Human MR-984, which was cloned in 1987, has been extensively studied. Human MR-988, cloned in 1995, contains KCSW in its DBD. Neither this human MR-988 nor the other human MR-988 genes have been studied for their response to aldosterone and other corticosteroids. Here, we report that transcriptional activation of human MR-988 by aldosterone is increased by about 50% compared to activation of human MR-984 in HEK293 cells transfected with the TAT3 promoter, while the half-maximal response (EC50) is similar for aldosterone activation of MR-984 and MR-988. The physiological responses in humans with MR genes containing KCSW and with differences in the NTD warrant investigation.\u003c/p\u003e","manuscriptTitle":"Lysine-Cysteine-Serine-Tryptophan Inserted into the DNA-Binding Domain of Human Mineralocorticoid Receptor Increases Transcriptional Activation by Aldosterone","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-14 15:46:37","doi":"10.21203/rs.3.rs-4095590/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a109eae3-80a7-48f9-8a46-e10fc531b0ba","owner":[],"postedDate":"March 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-14T15:46:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-14 15:46:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4095590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4095590","identity":"rs-4095590","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}