Figure 1.
Secondary structure model of MAS highlighting the mutation sites.
The myc-tag on the receptor and the locations of mutations used in the study are shown on the secondary structure model of MAS receptor. The transmembrane helices (TM) I - VII are predicted by bioinformatics analysis. Also shown in the model are the predicted ligand binding residues that were mutated in this study.
Figure 2.
Expression of WT and mutant MAS in stable cell lines.
Total expression of myc-tagged MAS was evaluated by (A and B) confocal microscopy. Images are labeled in green, red and blue for MAS, membranes and nuclei, respectively. (A) MAS expression is seen only in induced cells (bottom panel) compared to un-induced cells (top panel). (B) Expression of mutant MAS in ligand binding mutants. The anti-c-myc (9E10) antibody (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) was used for imaging the receptor.
Figure 3.
Calcium, IP1 and luciferase assay responses in WT MAS stable cell line.
Dose dependent changes in (A) calcium flux and (B) IP1 levels in cells upon stimulation with AR-agonist, AR-inverse agonist (AR-inverse) and NPFF in induced WT stable cell line. (C) Complete inhibition of AR-agonist and NPFF dose-response curves upon pre-treatment with 25 µM of AR-inverse agonist (AR-inverse) in calcium assays. (D) Dose dependent changes in luciferase expression upon stimulation with AR-agonist, AR-inverse agonist (AR-inverse) and NPFF in induced WT stable cell line. Representative curves from a single experiment wherein measurements are made in triplicate are shown as mean±SEM. The number of independent experiments is: N> = 3 in panels A, B and D; N = 2 in panel C.
Table 1.
Summary of IC50 and EC50 values for different ligands in multiple functional assays.
Table 2.
NPFF and its analogs along with corresponding EC50 values in calcium assays.
Figure 4.
Differential MAS signaling upon treatment with AR-agonist and NPFF in calcium assays.
The distinct signaling profiles of AR-agonist and NPFF are evident in (A and B) calcium flux kinetics, (C and D) antagonism and (E and F) re-stimulation assays. Calcium flux kinetics upon treatment with AR-agonist (in green) and NPFF (in blue) in the (A) absence and (B) presence of BIM-46187. The horizontal dashed line indicates the approximate fluorescence value which is half of the maximum observed upon ligand treatment. The kinetic parameter (t1/2) which is time taken to reach half of the maximal calcium response is also indicated for both ligands. This AR-agonist calcium response was subtracted from that of NPFF to highlight (in black and dashed line) the faster component in NPFF treated cells. In the antagonism assays the cells were initially treated with AR-inverse agonist (AR-inverse) at 25 µM (in dark olive green), 5 µM (in light olive green) and no ligand control (in magenta) and then challenged with (C) AR-agonist or (D) NPFF. In the re-stimulation assays the cells were initially treated with AR-agonist (in green), NPFF (in blue) and no ligand control (in magenta) and then challenged with (E) AR-agonist or (F) NPFF. In all the panels, vertical dashed lines indicate addition of ligands that are added at t = 0s and also at t = 600s in antagonism and re-stimulation assays. Data in B to F is normalized to maximum calcium response in the presence of agonists, AR-agonist or NPFF. Representative curves from a single experiment wherein measurements are made in triplicate (duplicate in case of antagonism and re-stimulation assays) are shown as mean±SEM. The number of independent experiments is N> = 3 in all the panels.
Figure 5.
Calcium and IP1 signaling in ligand binding MAS mutants.
Dose-response curves for (A) AR-inverse agonist (AR-inverse) treatment in ligand binding domain mutant MAS stable cell lines. Dose response curves for (B, C) AR-agonist and (D, E) NPFF treatment measured as function of intracellular (B, D) calcium and (C, E) IP1 levels. Representative curves from a single experiment wherein measurements are made in triplicate are shown as mean±SEM. The number of independent experiments is N> = 3 in all the panels.
Table 3.
EC50 and t1/2 values for different ligands in WT and mutant MAS stable cell lines.
Figure 6.
Evaluating response of Ang(1–7), Ang(1–7) analogs and angiotensin metabolites in MAS expressing stable cell lines.
Ang(1–7) dose-response curves for MAS stable cell line in (A) calcium (B) IP1 and (C) luciferase assays. (D) Dose-response curves for Ang(1–7) analogs (Sar1-Ang(1–7)-NH2 and Ang(1–7)-NH2) and Angiotensin metabolites (AngIII, AngIV, AngIV-amide and Ang(3–7)) in calcium assays. (E) Inhibition of Ang(1–7), Sar1-Ang(1–7)-NH2 and AngIV-amide dose-response curves upon pre-treatment with 25 µM of AR-inverse agonist (AR-inverse) in calcium assays. Representative curves from a single experiment wherein measurements are made in triplicate are shown as mean±SEM. The number of independent experiments is: N> = 3 in panels A, B and C; N> = 2 in panel D; N> = 1 in panel E.
Table 4.
EC50 and t1/2 values for Ang(1–7), Ang(1–7) analogs and angiotensin metabolites.
Figure 7.
Schematic model for distinct signaling mechanisms of MAS.
(A) Major G protein signaling pathways are constitutively activated by MAS. (B) Non-peptide agonist elevates MAS-mediated G protein activation beyond the constitutive activity and potentially promotes subsequent functional desensitization of MAS. (C) Physiological peptide agonists poorly activate MAS-mediated G protein activation beyond the constitutive activity and weakly promote functional desensitization of MAS. Strong calcium potentiation with very weak increase in intracellular IP1 levels along with unique calcium kinetics suggests that MAS possibly engages an unknown effector protein upon stimulation with peptide ligands.