Studying protein-protein interactions has been vital for understanding how proteins function within the cell, how biological processes are strictly regulated by these interactions, and what molecular mechanisms underlie cellular functions and diseases. Recent biochemical and biophysical studies have provided evidence supporting that G protein-coupled receptors (GPCRs) can and do interact with one another to form dimers or larger oligomeric complexes, which may determine the structure and function of GPCRs, including receptor trafficking, scaffolding and signaling. This may help to understand the physiological roles of GPCRs and mechanisms underlying certain disease pathologies and to provide an alternative approach for drug intervention.<p>Cannabinoid CB1 and dopamine D2 receptors are the most common GPCRs in the brain and exert a mutual regulation in brain functions involved in learning, memory and drug addiction. There is structural and functional evidence supporting the idea that CB1 and D2 receptors physically interact with each other in hippocampal and striatal neurons to modulate their functions. Direct evidence supporting a physical interaction between the CB1 and D2 receptors was obtained from cultured HEK293 cells stably coexpressed with both receptors.<p> This research project was designed to critically test the hypothesis that a specific protein sequence (i.e. motif) in the D2 receptor is responsible for in vitro protein-protein interactions between the CB1 and D2 receptors. To reach this goal, fusion proteins containing various domains and motifs of the CB1 and D2 receptors were prepared and then used first to determine the domains of the CB1 and D2 receptors responsible for in vitro protein-protein interactions between CB1 and D2 receptors, and then to identify the specific motifs in the D2 receptor responsible for in vitro CB1 coupling with the D2 receptors. The major method used in this study is in vitro pull-down assay, which uses a purified and tagged bait protein to generate a specific affinity support that is able to bind and purify a prey protein from a lysate sample. The present study provides the first evidence that CB1 intracellular C-terminal (CB1-CT) and D2 intracellular loop 3 (D2-IL3) can directly interact with each other, and that the specific motifs D2-IL3(Ⅳ1) and D2-IL3(Ⅳ3) in the D2 receptor are likely responsible for their in vitro coupling with the CB1 receptors. <p>The results of the present study are invaluable for future research exploring in vivo protein-protein interaction between the CB1 and D2 receptors in the rat striatum by co-immunoprecipitation. Specifically, future studies will determine whether the identified specific motifs D2-IL3(Ⅳ1) and D2-IL3(Ⅳ3) in the D2 receptor are indeed critical for their in vivo coupling with the CB1 receptors.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-11062006-153914 |
| Date | 07 November 2006 |
| Creators | Zhang, Yun |
| Contributors | Zhang, Xia, Mousseau, Darrell D., Li, Xin-Min |
| Publisher | University of Saskatchewan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-11062006-153914/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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