title: Atypical signaling and functional desensitization response of MAS receptor to peptide ligands.
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.
authors: Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.
authors: Kalyan C
authors: KC
authors: Tirupula
authors: Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.
authors: Russell
authors: R
authors: Desnoyer
authors: Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, Florida, United States of America.
authors: Robert C
authors: RC
authors: Speth
authors: Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.
authors: Sadashiva S
authors: SS
authors: Karnik
mesh_terms: D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009479:Neuropeptides / Q000378:metabolism / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D007295:Inositol Phosphates; D008024:Ligands; D009479:Neuropeptides; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; D000803:Angiotensin I; C053651:phenylalanyl-leucyl-phenylalanyl-glutaminyl-prolyl-glutaminyl-arginyl-phenylalaninamide; C118790:angiotensin I (1-7); D002118:Calcium