Abstract
MAS is a G protein-coupled receptor (GPCR) implicated in multiple physiological processes. Several physiological peptide ligands such as angiotensin-(1–7), angiotensin fragments and neuropeptide FF (NPFF) are reported to act on MAS. Studies of conventional G protein signaling and receptor desensitization upon stimulation of MAS with the peptide ligands are limited so far. Therefore, we systematically analyzed G protein signals activated by the peptide ligands. MAS-selective non-peptide ligands that were previously shown to activate G proteins were used as controls for comparison on a common cell based assay platform. Activation of MAS by the non-peptide agonist (1) increased intracellular calcium and D-myo-inositol-1-phosphate (IP1) levels which are indicative of the activation of classical Gαq-phospholipase C signaling pathways, (2) decreased Gαi mediated cAMP levels and (3) stimulated Gα12-dependent expression of luciferase reporter. In all these assays, MAS exhibited strong constitutive activity that was inhibited by the non-peptide inverse agonist. Further, in the calcium response assay, MAS was resistant to stimulation by a second dose of the non-peptide agonist after the first activation has waned suggesting functional desensitization. In contrast, activation of MAS by the peptide ligand NPFF initiated a rapid rise in intracellular calcium with very weak IP1 accumulation which is unlike classical Gαq-phospholipase C signaling pathway. NPFF only weakly stimulated MAS-mediated activation of Gα12 and Gαi signaling pathways. Furthermore, unlike non-peptide agonist-activated MAS, NPFF-activated MAS could be readily re-stimulated the second time by the agonists. Functional assays with key ligand binding MAS mutants suggest that NPFF and non-peptide ligands bind to overlapping regions. Angiotensin-(1–7) and other angiotensin fragments weakly potentiated an NPFF-like calcium response at non-physiological concentrations (≥100 µM). Overall, our data suggest that peptide ligands induce atypical signaling and functional desensitization of MAS.
Highlights
IntroductionGene knockout studies have been instrumental in defining MAS function [2]
MAS is a G protein-coupled receptor (GPCR) encoded by the proto-oncogene MAS [1]
Generation of mutant MAS receptors for this study The choice of ligand binding residues to mutate was based on extensive review of structure-function relationship literature on rhodopsin and the Angiotensin II type 1 receptor (AT1R)
Summary
Gene knockout studies have been instrumental in defining MAS function [2]. MAS knockout mice are reported to have an overall impairment in cardiac function and vascular homeostasis as a result of pro-fibrotic changes and endothelial dysfunction, respectively [3,4,5,6,7,8]. MAS deficient mice exhibit renal and metabolic disorders, alterations in hemostasis and pathological changes in several other tissues and organs [9,10,11,12,13,14]. MAS deficiency is shown to offer protection from salt induced hypertension and inhibiting MAS function is shown to prevent ischemia/reperfusion injury in both kidney and heart [15,16,17,18]. MAS plays a key role in several physiological processes and is a potential target for development of novel therapeutics for multiple disorders
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