Differential phosphorylation of calreticulin affects AT1 receptor mRNA stability in VSMC

Abstract

The AT1 receptor plays a pivotal role for the pathogenesis of hypertension and atherosclerosis. AT1 receptor expression is regulated posttranscriptionally via destabilization of the AT1 receptor mRNA by mRNA binding proteins. Recently, we identified calreticulin as a novel binding protein within the 3′untranslated region of the AT1 receptor mRNA. Calreticulin phosphorylation is essential for binding of the AT1 receptor mRNA. In crosslink experiments, we identified src kinase as the key enzyme for calreticulin phosphorylation. Overexpression of src sense DNA resulted in vascular smooth muscle cells (VSMC) in destabilization, overexpression of src antisense resulted in stabilisation of the AT1 receptor mRNA. Furthermore, phosphorylation/dephosphorylation sites of calreticulin and their impact on the AT1 receptor mRNA stability were investigated. VSMC were stimulated with AngII before tyrosine phosphorylation as well as serine phosphorylation of calreticulin were analysed via immunoprecipitation. Stimulation of VSMC with AngII resulted in enhanced tyrosine and reduced serine phosphorylation. Both effects are essential for AT1 mRNA stability as assessed by use of pharmacological inhibitors of serine dephosphorylation (cantharidin/ocadaic acid) or tyrosine phosphorylation (tyrphostin/orthovanadat). These findings imply an important role of serine dephosphorylation and tyrosine phosphorylation on calreticulin mediated AT1 receptor mRNA stability.