β2-adrenoceptor-induced modulation of transglutaminase 2 transamidase activity in cardiomyoblasts

Eur J Pharmacol. 2017 Oct 15:813:105-121. doi: 10.1016/j.ejphar.2017.07.043. Epub 2017 Jul 25.

Abstract

Tissue transglutaminase 2 (TG2) is modulated by protein kinase A (PKA) mediated phosphorylation: however, the precise mechanism(s) of its modulation by G-protein coupled receptors coupled to PKA activation are not fully understood. In the current study we investigated the potential regulation of TG2 activity by the β2-adrenoceptor in rat H9c2 cardiomyoblasts. Transglutaminase transamidation activity was assessed using amine-incorporating and protein cross-linking assays. TG2 phosphorylation was determined via immunoprecipitation and Western blotting. The long acting β2-adrenoceptor agonist formoterol induced time- and concentration-dependent increases in TG2 transamidation. Increases in TG2 activity were reduced by the TG2 inhibitors Z-DON (Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-L-valinyl-L-prolinyl-L-leucinmethylester) and R283 ((1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride). Responses to formoterol were blocked by pharmacological inhibition of PKA, extracellular signal-regulated kinase 1 and 2 (ERK1/2), or phosphatidylinositol 3-kinase (PI-3K) signalling. Furthermore, the removal of extracellular Ca2+ also attenuated formoterol-induced TG2 activation. Fluorescence microscopy demonstrated TG2-induced biotin-X-cadaverine incorporation into proteins. Formoterol increased the levels of TG2-associated phosphoserine and phosphothreonine, which were blocked by inhibition of PKA, ERK1/2 or PI-3K signalling. Subsequent proteomic analysis identified known (e.g. lactate dehydrogenase A chain) and novel (e.g. Protein S100-A6) protein substrates for TG2. Taken together, the data obtained suggest that β2-adrenoceptor-induced modulation of TG2 represents a novel paradigm in β2-adrenoceptor cell signalling, expanding the repertoire of cellular functions responsive to catecholamine stimulation.

Keywords: ERK1/2; H9c2 cardiomyocytes; Phosphorylation; Protein kinase A; Transglutaminase 2; β(2)-adrenoceptor.

MeSH terms

  • Animals
  • Calcium / metabolism
  • Catecholamines / pharmacology
  • Cell Line
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • GTP-Binding Proteins / antagonists & inhibitors
  • GTP-Binding Proteins / metabolism*
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / enzymology
  • Myocytes, Cardiac / metabolism*
  • Phosphorylation / drug effects
  • Protein Glutamine gamma Glutamyltransferase 2
  • Rats
  • Receptors, Adrenergic, beta-2 / metabolism*
  • Signal Transduction / drug effects
  • Transglutaminases / antagonists & inhibitors
  • Transglutaminases / metabolism*

Substances

  • Catecholamines
  • Enzyme Inhibitors
  • Receptors, Adrenergic, beta-2
  • Tgm2 protein, rat
  • Protein Glutamine gamma Glutamyltransferase 2
  • Transglutaminases
  • Cyclic AMP-Dependent Protein Kinases
  • GTP-Binding Proteins
  • Calcium