Browsing Theses and Dissertations School of Pharmacy by Subject "desensitization"
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Molecular Mechanisms Underlying the Regulation of Mu Opioid Receptor Function by Protein Kinase C and Histidine Triad Nucleotide Binding Protein 1Mu opioid receptor (MOPr) belongs to G protein-coupled receptor superfamily and is the primary target through which opioid drugs exert their biological activities. Multiple lines of evidence support that agonist-induced adaptive changes of MOPr are important molecular mechanisms underlying the development of opioid tolerance and dependence. It has also been shown that phosphorylation of MOPr, especially phosphorylation of C-terminus, plays a critical role in the regulation of receptor adaptive changes. In addition to G protein-coupled receptor kinase (GRK), second messenger protein kinases, including protein kinase C (PKC), are also involved in the phosphorylation of MOPr. Although previous research has shown that PKC is capable of regulating MOPr adaptive changes and the development of opioid tolerance and dependence, functional impacts of PKC-mediated receptor phosphorylation on MOPr signaling remain unclear. To test the hypothesis that PKC-mediated regulation of MOPr signaling is achieved through phosphorylation of the receptor C-terminus. In vitro phosphorylation results demonstrated that PKC is capable of phosphorylating MOPr C-terminus and Ser363 is the primary PKC phosphorylation site. This result was further confirmed in CHO cells stably expressing full-length MOPr. Mutating the PKC-phosphorylation site to Ala did not affect the receptor-ligand binding and receptor-G protein coupling. However, this mutation inhibited PMA-induced, but not DAMGO-induced, decrease of receptor-G protein coupling. In all, these results indicated that PKC phosphorylates MOPr C-terminus, and induces the receptor desensitization at G protein coupling level. Previous research of our lab has identified an intracellular protein, histidine triad nucleotide binding protein 1 (HINT1), interacts with MOPr and modulates receptor functions. Although HINT1 inhibits PMA-induced MOPr phosphorylation, which suggests that HINT1 may regulate MOPr function through a PKC-related manner, the molecular mechanism underlying the regulation of MOPr by HINT1 is still unknown. It is suspected that HINT1-nucleotide interaction is crucial for HINT1 biological functions, and interrupting the HINT1-nucleotide interaction will provide an opportunity to reveal the mechanism for HINT1 regulation of MOPr. Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography studies were performed to reveal the structure requirement of HINT1-nucleotide interaction. Fluorescence titration and isothermal titration calorimetry (ITC) assays further confirmed and quantified the HINT1-nucleotide interaction. Moreover, the interaction of HINT1 with AICAR, a novel non-phosphate nucleotide analogue, was also investigated. Structure information and methodologies achieved in this research provide a basis for future research aimed at revealing the mechanism of HINT1 function.