title: A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.
abstract: The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection.
authors.affiliation: School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom.
authors.forename: Falguni S
authors.initials: FS
authors.lastname: Vyas
authors.affiliation: School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Hargreaves
authors.affiliation: School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom.
authors.forename: Philip L R
authors.initials: PL
authors.lastname: Bonner
authors.affiliation: John van Geest Cancer Research Centre, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Boocock
authors.affiliation: John van Geest Cancer Research Centre, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom.
authors.forename: Clare
authors.initials: C
authors.lastname: Coveney
authors.affiliation: School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom. Electronic address: john.dickenson@ntu.ac.uk.
authors.forename: John M
authors.initials: JM
authors.lastname: Dickenson
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists / Q000494:pharmacology*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D032386:Myoblasts, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D051381:Rats; D043682:Receptor, Adenosine A1 / Q000097:blood / Q000378:metabolism*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D058907:Adenosine A1 Receptor Agonists; D004791:Enzyme Inhibitors; D006657:Histones; D047428:Protein Kinase Inhibitors; D043682:Receptor, Adenosine A1; C000717609:Tgm2 protein, rat; C048599:N(6)-cyclopentyladenosine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D006593:Hexokinase; D019204:GTP-Binding Proteins; D000241:Adenosine
keywords: A(1) adenosine receptor; Cytoprotection; G protein-coupled receptor; SWATH mass spectrometry; Transglutaminase 2