Research ArticleSteroid Receptors

Evolution of human, chicken, alligator, frog, and zebrafish mineralocorticoid receptors: Allosteric influence on steroid specificity

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Science Signaling  03 Jul 2018:
Vol. 11, Issue 537, eaao1520
DOI: 10.1126/scisignal.aao1520

Figures

  • Fig. 1 Steroids and MRs.

    (A) The structures of the steroid ligands. These steroids are called 3-ketosteroids because they contain a ketone at C3. With the exception of Spiron, all of the other steroids are natural ligands. (B) Phylogeny of vertebrates investigated for MR activation by Prog. The phylogeny was constructed with the neighbor-joining method (71) after sequences were aligned by ClustalW (72). Values for 1000 bootstrap runs are shown as percentages at each node. MRs activated by Prog are indicated with (+), whereas those inhibited by Prog are indicated with (−). Those tested in this study are indicated by an asterisk. GenBank accession numbers are as follows: human MR (NP_000892), chicken MR (ACO37437), alligator MR (NP_001274242), Xenopus MR (NP_001084074), zebrafish MR (NP_001093873), trout MRa (NP_001117955), sturgeon MR (BAV17690), and gar MR (BAV17691). (C) Comparison of the domains in vertebrate MRs. Domains A/B (NTD), C (DBD), D (hinge), and E (LBD) on MRs from the indicated species are compared. Shown are the number of amino acids in each domain and the percentage of identical amino acids compared to the human MR. GenBank accession numbers are as follows: human MR (NP_000892), chicken MR (ACO37437), alligator MR (NP_001274242), Xenopus MR (NP_001084074), and zebrafish MR (NP_001093873).

  • Fig. 2 Corticosteroid activation of human, chicken, alligator, Xenopus, and zebrafish full-length MRs and LBD MRs.

    (A to E) Full-length (left) and truncated (right) human (A), chicken (B), alligator (C), Xenopus (D), and zebrafish (E) MRs. Full-length MR constructs were coexpressed in HEK293 cells with an MMTV-Luc reporter. Plasmids corresponding to the truncated MRs containing the D and E domains fused to a GAL4-DBD were coexpressed in HEK293 cells with a luciferase reporter containing a GAL4-binding site. The cells were treated with the indicated concentrations of ligand or vehicle alone (DMSO) for 44 hours before reporter gene activity was assessed. Data are means ± SEM of three independent experiments and are expressed as the fold activation compared to the activity of cells expressing the control vector and treated with vehicle (DMSO) alone, which was set as 1.

  • Fig. 3 Prog, 19norProg, or Spiron activation of human, chicken, alligator, Xenopus, and zebrafish full-length and truncated MRs.

    (A to E) Full-length (left) and truncated (right) human (A), chicken (B), alligator (C), Xenopus (D), and zebrafish (E) MRs. HEK293 cells were transfected with the appropriate constructs and reporters as described for Fig. 2. The cells were treated with the indicated concentrations of Prog, 19norProg, or Spiron or vehicle alone (DMSO) for 44 hours before gene reporter activity was measured. Data are means ± SEM of three independent experiments and are expressed as the fold activation compared to the activity of cells expressing the control vector and treated with vehicle (DMSO) alone, which was set as 1.

  • Fig. 4 Concentration-dependent transcriptional activation by corticosteroids of full-length human, chicken, alligator, Xenopus, and zebrafish MRs.

    HEK293 cells were cotransfected with plasmids encoding the indicated full-length MRs together with the reporter plasmid. The cells were then treated with increasing concentrations of the indicated steroids or with vehicle (DMSO) alone before gene reporter activity was measured. Data are means ± SEM of three independent experiments and are expressed as fold activation compared to the activity of cells expressing the control vector and treated with vehicle (DMSO) alone, which was set as 1.

  • Fig. 5 Concentration-dependent transcriptional activation of truncated human, chicken, alligator, Xenopus, and zebrafish MRs.

    HEK293 cells were cotransfected with plasmids encoding the GAL4-DBD fused to the D and E domains of the indicated MRs together with the reporter plasmid. The cells were then treated with increasing concentrations of the indicated steroids or with vehicle (DMSO) alone before gene reporter activity was measured. Data are means ± SEM of three independent experiments and are expressed as fold activation compared to the activity of cells expressing the control vector and treated with vehicle (DMSO) alone, which was set as 1.

  • Fig. 6 Competition by Prog, 19norProg, Spiron, or 11-deoxycortisol for transcriptional activation by ALDO of truncated human, alligator, and Xenopus MRs.

    HEK293 cells were cotransfected with plasmids encoding the GAL4-DBD fused to the D and E domains of the indicated MRs together with reporter plasmid. Cells expressing human or alligator MR were treated with 0.5 nM ALDO and increasing concentrations of the competing ligands Prog, 19norProg, Spiron, or 11-deoxycortisol, as indicated. Cells expressing Xenopus MR were treated with 5 nM ALDO and with increasing concentrations of the competing ligands Prog, 19norProg, Spiron, 11-deoxycortisol, or 11-deoxycorticosterone. E2 was used a control in all experiments. Data are means ± SEM of three independent experiments and are expressed as fold activation compared to the activity of cells expressing the control vector and treated with vehicle (DMSO) alone, which was set as 1.

Tables

  • Table 1 EC50 values for the 3-ketosteroid transcriptional activation of full-length vertebrate MRs.

    The percentage relative induction is compared to the maximal response to ALDO.

    MRALDOCorticosteroneCortisol11-Deoxycorticosterone11-Deoxycortisol
    EC50 (M)EC50 (M)EC50 (M)EC50 (M)EC50 (M)
    Human2.7 × 10−101.2 × 10−95.5 × 10−94.2 × 10−103.6 × 10−9
    100%119%133%74%42%
    Chicken6.2 × 10−115.1 × 10−112.8 × 10−103.4 × 10−116.7 × 10−10
    100%109%128%110%112%
    Alligator2.8 × 10−103.6 × 10−106.9 × 10−92.3 × 10−102.7 × 10−9
    100%138%176%85%45%
    Xenopus4.6 × 10−106.2 × 10−101.1 × 10−87.6 × 10−109.1 × 10−9
    100%105%126%59%31%
    Zebrafish8.2 × 10−113.0 × 10−104.4 × 10−106.3 × 10−114.0 × 10−10
    100%112%123%103%94%
  • Table 2 EC50 values for the 3-ketosteroid transcriptional activation of GAL4-DBD-MR-LBD of vertebrate MRs.

    The percentage relative induction is compared to the maximal response to ALDO.

    MRALDOCorticosteroneCortisol11-Deoxycorticosterone11-Deoxycortisol
    EC50 (M)EC50 (M)EC50 (M)EC50 (M)EC50 (M)
    Human2.8 × 10−105.9 × 10−103.2 × 10−91.8 × 10−9
    100%95%74%44%8%
    Chicken1.3 × 10−101.6 × 10−106.9 × 10−101.7 × 10−104.7 × 10−9
    100%92%75%92%36%
    Alligator3.5 × 10−103.8 × 10−102.3 × 10−95.2 × 10−10
    100%88%68%51%8%
    Xenopus1.5 × 10−91.9 × 10−91.2 × 10−8
    100%74%37%10%6%
    Zebrafish2.7 × 10−111.5 × 10−103.1 × 10−101.0 × 10−109.1 × 10−10
    100%96%77%99%67%

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