P440Targeting subcellular GRK2 localization. (15th July 2014)

• Record Type: Journal Article
• Authors: Anastasio, A; Carillo, AL; Ciccareli, M; Trimarco, B; Iaccarino, G; Sorriento, D
• Is Part Of: Cardiovascular research. Volume 103(2014)Supplement 1
• Page Start: S81
• Page End: S81
• Publication Date: 2014-07-15
• Subjects: Cardiovascular system -- Diseases -- Periodicals; Cardiovascular system -- Periodicals; 616.1
• DOI: 10.1093/cvr/cvu091.119
• Languages: English
• ISSNs: 0008-6363

Abstract

Abstract: GRK2 is a kinase involved in the regulation of G protein coupled receptor signaling and in particular phosphorylates beta adrenergic receptors at plasma membrane leading to desensitization. In animal and human models of heart failure, its increased level and activity are associated with a worse prognosis. Recent findings show that GRK2 localizes into mitochondria where it plays a protective role on cell function by promoting biogenesis and increasing ATP production. Aim of this study is to identify the minimum effective sequence of the pleckstrin homology domain of GRK2 (PH), able to displace the kinase from plasma membrane and favour its localization into mitochondria. To this aim we carried out cloning of four overlapping sequences of the gene of the PH domain of GRK2 (PH1, PH2 PH3 and PH4) of about 160bp. The recombinant mutants were transfected into HEK 293 cells to assess their ability to move GRK2 from the plasma membrane into mitochondria. Thus, we evaluated the levels of GRK2 by western blot analysis in membrane and mitochondrial fraction. PH1 and PH2 had not effects on GRK2 trafficking within the cells. PH3 causes an increase of the total levels of the kinase associated with an increase of GRK2 in all cell compartment. This effect was confirmed by an increase of mRNA levels of GRK2 by RT-PCR. PH4 is the only effective recombinant protein able to displace GRK2 from the plasma membrane causing an accumulation of the kinase in the mitochondria. We then evaluated the effect of PH4 on mitochondrial function focusing on ROS production, ATP content and mitochondrial biogenesis. PH4 caused a reduction of ROS production, increase of mitochondrial biogenesis and an increase of ATP content in response to hypoxia. Finally the analysis of the different overlapping sequence of PH mutants and their functional correlates, identify in the last 10 bp of GRK2 PH domain the essential residues to its localization into mitochondria.