Down-regulation of GRK2 after oxygen and glucose deprivation in rat hippocampal slices: role of the PI3-kinase pathway.
G protein-coupled receptor kinase 2 (GRK2) modulates G protein-coupled receptor desensitization and signaling. We previously described down-regulation of GRK2 expression in vivo in rat neonatal brain following hypoxia-ischemia. In this study, we investigated the molecular mechanisms involved in GRK2 down-regulation, using organotypic cultures of neonatal rat hippocampal slices exposed to oxygen and glucose deprivation (OGD). We observed a 40% decrease in GRK2 expression 4 h post-OGD. No changes in GRK2 protein occurred after exposure of hippocampal slices to glucose deprivation only. No significant alterations in GRK2 mRNA expression were detected, suggesting a post-transcriptional effect of OGD on GRK2 expression. Blockade of the proteasome pathway by MG132 prevented OGD-induced decrease of GRK2. It has been shown that extracellular signal-regulated kinase-dependent phosphorylation of GRK2 at Ser670 triggers its turnover via the proteasome pathway. However, despite a significant increase of pSer670-GRK2 after OGD, inhibition of the extracellular signal-regulated kinase pathway by PD98059 did neither prevent the hypoxia-ischemia-induced increase in pSer670-GRK2 nor the down-regulation of GRK2 protein. Interestingly, inhibition of phosphoinositide-3-kinase with wortmannin inhibits both OGD-induced phosphorylation of GRK2 on Ser670 and the GRK2 decrease. In conclusion, OGD-induced phosphoinositide-3-kinase-dependent phosphorylation of GRK2 on Ser670 is a novel mechanism leading to down-regulation of GRK2 protein via a proteasome-dependent pathway.
Lombardi
Maria Stella
MS
Laboratory for Psycho-neuro-immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
Vroon
Anne
A
Sodaar
Peter
P
van Muiswinkel
Freek L
FL
Heijnen
Cobi J
CJ
Kavelaars
Annemieke
A
D000818:Animals; D000831:Animals, Newborn; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2; D005947:Glucose / Q000172:deficiency; D006624:Hippocampus / Q000201:enzymology* / Q000503:physiopathology; D020925:Hypoxia-Ischemia, Brain / Q000201:enzymology* / Q000503:physiopathology; D009924:Organ Culture Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051552:beta-Adrenergic Receptor Kinases / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D005947:Glucose; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D046988:Proteasome Endopeptidase Complex; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D012694:Serine; D051552:beta-Adrenergic Receptor Kinases; C496569:Grk2 protein, rat