Epitope-tagged and phosphomimetic mouse models for investigating natriuretic peptide-stimulated receptor guanylyl cyclases

Figures

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  FIGURE 1. Modifications of the NPR1 and NPR2 proteins in HA-NPR1, HA-NPR2, NPR1-8E, and NPR2-7E mice. Diagrams show the domains of each protein, including the HA epitope tag at the N-terminus (A,B) and the glutamate substitutions for regulatory phosphosites within the kinase homology domain (C,D).

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  FIGURE 2. Generation of the HA-NPR1 mouse strain using CRISPR. The Npr1 signal peptide sequence is shown in blue with the PAM site for CRISPR underlined. The HA tag sequence is shown in purple, and the sequence of the 3 amino acid spacer is shown in pink. The Npr1 exon 1 coding sequence is shown in black.

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  FIGURE 3. Western blots of NPR1 and NPR2 proteins in native tissues from mice expressing HA-NPR1 and HA-NPR2. Tissue samples from homozygous HA-NPR1 (A) and HA-NPR2 (B) mice were prepared similarly (see section “Methods”). Except as indicated, 20 μg of protein was loaded per lane (determined by a BCA assay). Three exposures of each blot are shown. Bands corresponding to NPR1 and NPR2 are seen only in the samples from the mice with the HA tags, not in those from wild-type mice. Doublet bands at ∼120 and 130 kDa represent differentially glycosylated and phosphorylated forms (Koller et al., 1993; Dickey et al., 2016; Shuhaibar et al., 2016). A non-specific band is present at ∼110 kDa. The figure shows only the top half of the blots, where NPR1 and NPR2 are located; the complete range of molecular weights for the blots in (A) is shown in Supplementary Figure 1A. Supplementary Figures 1B,C shows corresponding Ponceau stained blots and densitometry measurements, confirming that equal amounts of protein were loaded per lane.

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  FIGURE 4. Unlike 12-week-old NPR1-8E male mice, 2-year-old NPR1-8E male mice do not have increased ejection fractions or decreased end systolic volumes. 12-week-old (A,B,E,F) and 2-year old (C,D,G,H) male and female mice were assessed by echocardiography for ejection fractions and end systolic volumes as described by Wagner et al. (2022a). Data for NPR1-WT and NPR1-8E mice are shown by black and red symbols, respectively. Long horizontal bars indicate the mean and vertical bars indicate SEM. Statistical differences were determined using a two-tailed student’s t-test with associated p-values shown in each panel; asterisks indicate significant differences *p < 0.05 and **p < 0.01. Heart rates at the time of imaging were the same for all groups (Supplementary Table 2). (A,B,E,F) Reproduced, as permitted by the publisher, from Wagner et al. (2022a).