MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.
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.
Department of Molecular Cardiology, Cleveland Clinic, Ohio, United States of America.
Kalyan C
KC
Tirupula
Proteomics Laboratory, Lerner Research Institute, Cleveland Clinic, Ohio, United States of America.
Dongmei
D
Zhang
Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland Clinic, Ohio, United States of America.
Appledene
A
Osbourne
Department of Molecular Cardiology, Cleveland Clinic, Ohio, United States of America.
Arunachal
A
Chatterjee
Department of Molecular Cardiology, Cleveland Clinic, Ohio, United States of America.
Russ
R
Desnoyer
Proteomics Laboratory, Lerner Research Institute, Cleveland Clinic, Ohio, United States of America.
Belinda
B
Willard
Department of Molecular Cardiology, Cleveland Clinic, Ohio, United States of America; Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland Clinic, Ohio, United States of America.
Sadashiva S
SS
Karnik
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism; D000074288:Discs Large Homolog 1 Protein; D051528:Guanylate Kinases; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009124:Muscle Proteins / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D054731:PDZ Domains / Q000502:physiology*; D040901:Proteomics; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; D008565:Membrane Proteins; D009124:Muscle Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; C091825:syntrophin alpha1; D051528:Guanylate Kinases; C113338:MAGI1 protein, human