MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.

Kalyan C Tirupula,Dongmei Zhang,Appledene Osbourne,Sadashiva S Karnik,Russ Desnoyer,Arunachal Chatterjee,Belinda Willard,Laszlo Buday

doi:10.1371/journal.pone.0140872

Kalyan C Tirupula, Dongmei Zhang + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0140872

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Abstract

Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A ‘cardiac-specific finger print’ of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a ‘MAS-signalosome’ model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of ‘signalosome’ components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.

Highlights

MAS is a G protein-coupled receptor (GPCR) discovered as the product of Mas oncogene [1]
The biotinylated C-terminal tail (Ct) peptide was immobilized on NeutrAvidin agarose resin and Ct interacting proteins were isolated from HEK293, HL-1 and cardiac tissue lysates (Fig 1B; see methods)
To determine the success of the pull-down assay the samples were first analyzed on a western blot with a pan-MAGUK antibody that detects multiple proteins belonging to the membrane-associated guanylate kinases (MAGUK) sub-family of PDZ proteins

Summary

Introduction

MAS is a G protein-coupled receptor (GPCR) discovered as the product of Mas oncogene [1]. We showed that MAS activates G protein signaling pathways and undergoes functional desensitization in response to non-peptide ligands [23]. MAS signaling was atypical in response to endogenous peptide ligands. The physiological ligand, Neuropeptide FF (NPFF) produced functional selective MAS signaling without functional desensitization. The putative endogenous cardiovascular ligand angiotensin (1–7) potentiated an NPFF-like response of MAS only at non-physiological ligand concentration [23]. In both scenarios, a G protein independent component of signaling response of MAS was apparent. The molecular mechanism of atypical signaling, desensitization and G protein independent signaling observed in MAS is currently unknown

Methods

Results

Conclusion