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Einfluss des Hitzeschockproteins Hsc70 und des Raf Kinase Inhibitor Proteins RKIP auf den zellulären Transport und die Aktivität des μ-Opioidrezeptors

Autor :Andrea Goschke
Herkunft :OvGU Magdeburg, Fakultät für Naturwissenschaften
Datum :04.12.2008
 
Dokumente :
Dataobject from HALCoRe_document_00006717
 
Typ :Dissertation
Format :Text
Kurzfassung :Opiate-initiated effects like analgesia, mood changes, respiratory depression or gastrointestinal reactions are mediated by binding to specific G-protein-coupled opioid receptors. After ligand-binding and activation, the opioid receptor is phosphorylated by G-protein-coupled receptor kinases (GRKs) or second messenger-dependent kinases. Binding of β-arrestins to the phosphorylated receptor is associated with uncoupling from G-proteins, which results in receptor desensitization. Endocytosis of ligand-receptor-complexes is mediated via clathrin-coated vesicles. Then reactivation passes through reycling of receptors back to cell membrane. In search of proteins that are involved in the regulation of µ-opioid receptor endocytosis and desensitization, two proteins were identified: Heat shock cognate 70 (Hsc70) and Raf Kinase Inhibitor Protein (RKIP). Hsc70 is a cytosolic protein of the 70-kDa-heat shock protein-family and is highly constitutively expressed. In the present study we investigated the influence of Hsc70 on the agonist-induced endocytosis and desensitization of MOPr. Confocal microscopic studies revealed that stimulation with the MOPr-agonist DAMGO in cells with diminished Hsc70-expression resulted in a decreased MOPr-endocytosis. The diminished endocytosis of MOPr was confirmed by a quantitative internalization assay (ELISA-technique). These results suggest that Hsc70 is an important regulator of clathrin-mediated endocytosis and hence influences the transport of MOPr. Furthermore, reduced Hsc70-expression tends to an accelerated DAMGO-induced desensitization of MOPr. This is most probably due to the decrease of endocytosis and recycling of the receptor which counteracts sensitization. In a second part of this study, the regulatory influence of the Raf Kinase Inhibitor Protein (RKIP) on MOPr-phosphorylation and -desensitization was investigated. After prolonged pretreatment with the MOPr-agonist DAMGO coexpression of RKIP lead to a decelerated MOPr-desensitization based on a diminished phosphorylation of one specific GRK2-phosphorylation site (S375) at the C-terminus of MOPr. On the other hand, coexpression of the RKIP-S153A-mutant, which is not able to inhibit GRK2, revealed no reduction of MOPr-phosphorylation. These findings suggest that RKIP exerts its regulatory effects on MOPr through a GRK2-inhibition. Quantitative internalization- and confocal microscopic studies show a significantly decreased DAMGO-induced MOPr-endocytosis after coexpression of RKIP, but not of RKIP-S153A. These findings indicate that a reduced MOPr-phosphorylation, triggered by GRK2-inhibition, results also in a reduced agonist-induced endocytosis. Furthermore, we showed, that activation of ERK1/2 in the MAPK-pathway is significantly decreased after RKIP-coexpression. Coexpression of the RKIP-S153A-mutant leads to a stronger inhibition of ERK1/2-activation, because the mutated RKIP cannot be released from Raf by phosphorylation resulting in a prolonged inhibition of the MAPK-pathway. Taken together, results of these study reveal a role of Hsc70 and of RKIP in endocytosis and desensitization of the MOPr.
Schlagwörter :Opioide, Opiate, Hitzeschockprotein, Raf Kinase
Rechte :Dieses Dokument ist urheberrechtlich geschützt.
Größe :79 S.
 
Erstellt am :15.04.2009 - 09:42:02
Letzte Änderung :22.04.2010 - 08:19:41
MyCoRe ID :HALCoRe_document_00006717
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