Fiona Freeman

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

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 &bgr;2‐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 &bgr;2‐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 &bgr;2‐adrenoceptor‐induced modulation of TG2 represents a novel paradigm in &bgr;2‐adrenoceptor cell signalling, expanding the repertoire of cellular functions responsive to catecholamine stimulation.

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