Unveiling the potential role of natriuretic peptide receptor a isoforms in fine-tuning the cGMP production and tissue-specific function

Figures

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  Figure 1. Schematic diagram of all three NPR-A receptor isoforms. (a) Alternative splicing of Isoform 1. (b) Exonisation of isoform 2. (c) Intronization of isoform 3. Top: Introns are represented by the horizonal line. Exons are represented by the colored bars. Bottom: mRNA containing the spliced product for WT NPR-A and the isoform.

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  Figure 2. Alignment of NPR-A nucleotide and amino acid sequences of human, rat, mouse and sheep. (a) NPR-A receptor isoform 1. The deleted exon 9 is underlined and the residues involved in phosphorylation are highlighted in grey. Differences in amino acid residues are indicated by red font. (b) NPR-A receptor isoform 2. Additional insertion of 9 nucleotides and three amino acid residues as exon 5a are underlined. Differences in the nucleotides are indicated by red bold font. The absence of critical GT sequence at 5’ end in human and sheep genes indicates that isoform 2 may not exist in them. (c) NPR-A receptor isoform 3. The deleted portion of exon 4 is highlighted in grey. Nucleotides AG in bold represent the sequence recognised by spliceosomes. Differences in the amino acid residues within the missing segments are indicated by red font.

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  Figure 3. NPR-A and receptor isoforms are expressed on the surface of the transfected cells in similar quantities and only cells containing WT NPR-A produce cGMP. (a) Western blot studies. Cells transfected with empty pCMV-4 plasmid were used as the negative control. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document}β-actin was used as the loading control. Full-size blot can be found in Supplementary Fig. S4a. (b) Immunofluorescence studies. The nuclei were stained blue with DAPI. Top panel: Cell surface glycoproteins were stained with WGA as the cell membrane marker. Middle panel: staining with antibodies targeting NPR-A/Isoforms. Bottom panel: Merge images show the co-localization of the receptors with the cell membrane marker. Untransfected cells and cells stained with only the secondary antibody were used as the two negative controls. Scale: 5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}…

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  Figure 4. Impact of co-transfection of receptor isoforms with WT NPR-A in 1%, 10% and 50% on cGMP production by WT NPR-A. The cGMP production induced by various concentrations of ANP was determined by ELISA. (a) Western blot illustrating the expression of both the receptors and the WT NPR-A in various percentage of co-transfection. Full-size blot can be found in Supplementary Fig. S4b. (b) Co-transfection of receptor isoforms 1 with WT NPR-A. (c) Co-transfection of receptor isoform 2 with WT NPR-A. (d) Co-transfection of receptor isoform 3 with WT NPR-A. Each point represents average ± S.D. of triplicates. These experiments were repeated on three independent days.

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  Figure 5. NPR-A receptor isoforms are non-functional guanylyl cyclases in the presence of (a) DGD-ANP and (b) DRD-ANP. The cGMP production induced by various concentrations of ANP was determined by ELISA. Each point represents average ± S.D. of triplicates.

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  Figure 6. Receptor isoforms reduce cGMP production of WT NPR-A in the presence of ANP analogues. The cGMP production induced by various concentrations of ANP was determined by ELISA. (a) & (b) Co-transfection of receptor isoforms 1 with WT NPR-A in the presence DGD-ANP and DRD-ANP respectively. (c) & (d) Co-transfection of receptor isoform 2 with WT NPR-A in the presence DGD-ANP and DRD-ANP respectively. (e) & (f) Co-transfection of receptor isoform 3 with WT NPR-A in the presence DGD-ANP and DRD-ANP respectively. Each point represents average ± S.D. of triplicates.