Role of specific protein kinase C isoforms in modulation of β 1- and β 2-adrenergic receptors

Abstract

The function of β-adrenergic receptor (βAR) is modulated by the activity status of α 1-adrenergic receptors (α 1ARs) via molecular crosstalk, and this becomes evident when measuring cardiac contractile responses to adrenergic stimulation. The molecular mechanism underlying this crosstalk is unknown. We have previously demonstrated that overexpression of α 1B-adrenergic receptor (α 1BAR) in transgenic mice leads to a marked desensitization of βAR-mediated adenylyl cyclase stimulation which is correlated with increased levels of activated protein kinase C (PKC) β, δ and ε [J. Mol. Cell. Cardiol. 30 (1998) 1827]. Therefore, we wished to determine which PKC isoforms play a role in heterologous βAR desensitization and also which isoforms of the βAR were the molecular target(s) for PKC. In experiments using constitutively activated PKC expression constructs transfected into HEK 293 cells also expressing the β 2AR, constitutively active (CA)-PKC overexpression was first confirmed by immunoblots using specific anti-PKC antibodies. We then demonstrated that the different PKC subtypes lead to a decreased maximal cAMP accumulation following isoproterenol stimulation with a rank order of PKCα≥PKCζ>PKCε>PKCβII. However, a much more dramatic desensitization of adenylyl cyclase stimulation was observed in cells co-transfected with different PKC isoforms and β 1AR. Further, the modulation of β 1AR by PKC isoforms had a different rank order than for the β 2AR: PKCβII>PKCα>PKCε>PKCζ. PKC-mediated desensitization was reduced by mutating consensus cAMP-dependent protein kinase (PKA)/PKC sites in the third intracellular loop and/or the carboxy-terminal tail of either receptor. Our results demonstrate therefore that the β 1AR is the most likely molecular target for PKC-mediated heterologous desensitization in the mammalian heart and that modulation of adrenergic receptor activity in any given cell type will depend on the complement of PKC isoforms present.