Ca2+-Sensor Neurocalcin δ and Hormone ANF Modulate ANF-RGC Activity by Diverse Pathways: Role of the Signaling Helix Domain

Figures

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  FIGURE 1. (A) ANF-RGC region amino acid residues N783-P822 exists as a α-helix. Three-dimensional structural model for ANF-RGC region comprising amino acid residues N783-P822 was built using I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) protein modeling software. The top-ranked model is shown. The protein is displayed in a solid ribbon style with a continuous gradient coloring scheme that starts with dark blue at the N-terminus (NT) progressing to white and ends as dark red at the C-terminus (CT). (B) ANF-RGC region amino acid residues N783-P822 forms an anti-parallel dimer. The dimer model for the ANF-RGC region amino acid residues N783-P822 was built using a protein-protein docking program, M-ZDOCK (Symmetric Multimer Docking software; http://zdock.umassmed.edu/m-zdock). Top-ranked M-ZDOCK predicted complex is shown. The E801R802T803 – triad that is important for the dimer interface is shown in stick form and marked with green, blue and red colors. The protein chains coloring style is same as the figure. (C) ANF-RGC region amino acid residues N783-P822 forms an anti-parallel coiled coil. Fourth ranked M-ZDOCK complex was identified by SOCKET…

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  FIGURE 2. Deletion of individual heptads but not of the entire SHD of the ANF-RGC affects the cyclase basal catalytic activity. ANF-RGC deletion mutants were prepared as described in Section “Materials and Methods.” Wt-ANF-RGC and the mutants were individually expressed in COS cells which membranes were assayed for guanylate cyclase activity. The experiment was done in triplicate and repeated three times. The results presented are average ± SD of these experiments. The expression of the proteins was monitored by Western blot. The intensities of the ANF-RGC immunoreactive bands of the expressed proteins are shown at the top of the figure.

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  FIGURE 3. The SHD is obligatory for ANF/ATP signaling of the ANF-RGC catalytic activity. COS cells were induced to express the ΔSHD mutant and their membranes were assayed for guanylate cyclase activity in the presence of 10-7 M ANF and indicated concentrations of ATP. Membranes of COS cells expressing wt-ANF-RGC were processed identically as control. The experiment was done in triplicate and repeated three times. The results presented are average ± SD of these experiments. The error bars are indicated.

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  FIGURE 4. ANF/ATP effect on the catalytic activity of wt-ANF-RGC and its ΔH1 or ΔH2 mutants. COS cells were individually transfected with ANF-RGC, ΔH1 or ΔH2 mutants’ cDNAs and their membrane fractions were assessed for guanylate cyclase activity in the presence of 10-7 M ANF and increasing concentrations of ATP. (A,C,E) Show the specific guanylate cyclase activities of ANF-RGC, ΔH1 and ΔH2 mutants respectively, while figures (B,D,F), their x-fold stimulations. The experiment was done in triplicate and repeated four times. The results shown are mean ± SD of these experiments. The error bars for (A,C,E) are shown whereas for (B,D,F) they are within the size of the symbols.

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  FIGURE 5. ANF/ATP effect on the catalytic activity of ANF-RGC ΔH3 and R802C mutants. Membranes of COS cells individually expressing ΔH3 or R802C mutants were assayed for guanylate cyclase activity in the presence of 10-7 M ANF and increasing concentrations of ATP. (A,C) Show respectively, the specific guanylate cyclase activities of the ΔH3 and R802C mutants, while figures (B,D), their x-fold stimulations. The experiment was done in triplicate and repeated three times. The results shown are mean ± SD of these experiments. The error bars for (A,C) are shown whereas for (B,D) they are within the size of the symbols.

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  FIGURE 6. ANF/ATP effect on the catalytic activity of ΔH4 and ΔH5 mutants. COS cells were individually transfected with ΔH4 or ΔH5 mutants and their membrane fractions were assayed for guanylate cyclase activity in the presence of 10-7 M ANF and increasing concentrations of ATP. (A,C) Show the specific guanylate cyclase activities of the ΔH4 and ΔH5 mutants, respectively while figures (B,D), their x-fold stimulations. The experiment was done in triplicate and repeated three times. The results shown are mean ± SD of these experiments. The error bars for (A,C) are shown whereas for (B,D) they are within the size of the symbols.

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  FIGURE 7. The SHD has no role in Ca2+/myr-NCδ signaling of the ANF-RGC catalytic activity. COS cells were individually induced to express wt-ANF-RGC or its ΔSHD mutant. Membranes of transfected cells were exposed to increasing concentrations of myr-NCδ in the presence of 1 μM Ca2+ and assayed for the guanylate cyclase activity. (A,C) Show the specific guanylate cyclase activities of ANF-RGC and its ΔSHD mutant, respectively while figures (B,D), their x-fold stimulations. The experiment was done in triplicate and repeated three times. The results shown are mean ± SD of these experiments. The error bars for (A,C) are shown whereas for (B,D) they are within the size of the symbols.

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  FIGURE 8. The effect of myr-NCδ on ANF-RGC heptad deletion (ΔH1 – ΔH5) and R802C mutants. COS cells were induced to individually express each of the ANF-RGC heptad deletion mutants: ΔH1, ΔH2, ΔH3, ΔH4, ΔH5, or the substitution R802C mutant. Membranes of transfected cells were exposed to increasing concentrations of myr-NCδ in the presence of 1 μM Ca2+ and assayed for the guanylate cyclase activity. (A) Shows the specific guanylate cyclase activities of the mutants whereas figure (B), their x-fold stimulations. The experiment was done in triplicate and repeated three times. The results shown are mean ± SD of these experiments. The error bars are within the size of the symbols.

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  FIGURE 9. Two independent signaling pathways of ANF-RGC activity. (A) Ca2+/myr-NCδ. ANF-RGC is a single-transmembrane spanning homodimer protein. The dashed lines at the right show the defined boundaries of its segments: LS, leader sequence; ExtD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain. The functional domains housed in ICD are: JMD, juxtamembrane domain; ARM, ATP regulated module; SHD, signaling helix domain; CCD, core catalytic domain. The site targeted by neurocalcin δ (NCΔ) (encircled) is located within the CCD. Thus, the Ca2+/myr-NCδ signal originates and is transduced at the CCD. (B) ANF/ATP. The trajectory of ANF signaling pathway is shown with a red dashed arrow. From its origin at the ExtD, it passes through the structural domains of TM, ARM (ATP binds to ARM in response to ANF signal), and SHD on its way to CCD. The figure was modified from (Duda et al., 2012b).