| Abstract No.: | B-B2023 |
| Country: | Canada |
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| Title: | THE G-PROTEIN-COUPLED RECEPTOR KINASE II PLAYS A ROLE IN THE DESENSITIZATION OF THE METABOTROPIC GLUTAMATE RECEPTOR 5 |
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| Authors/Affiliations: | 1 Fabiola M. Ribeiro*; 1 lucimar Ferreira; 1 Maryse Paquet; 1 Tamara Cregan; 1 Robert Gros; 1 Stephen S. Ferguson;
1 University of Western Ontario, London, ON, Canada
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| Content: | Metabotropic glutamate receptors are members of the G protein-coupled receptor (GPCR) superfamily. mGluR1 and mGluR5, which are members of Group I mGluRs, are coupled to Gq and InsP (inositol phosphate) formation, being implicated in many physiological functions, such as cell death, memory, and learning. Desensitization of receptors is an important mechanism to avoid over-stimulation of the receptor and cell damage. It is being shown that the activity of many mGluRs is regulated by second messenger-dependent protein kinase and G protein-coupled receptor kinases (GRKs). It has been shown that mGluR1 has its signalling attenuated by GRK2, in a mechanism that is phosphorylation- and -arrestin-independent. However, it is still unknown how mGluR5 is desensitized and whether GRK2 plays a role in mGluR5 signalling attenuation. Objectives: Investigate the role of GRK2 on mGluR5 desensitization using mouse primary cultured striatal neurons. Material and Methods: Primary cultured striatal neurons were infected with either GRK2 or negative control (NC) adenovirus 3 days prior to experiments. InsP formation assay was performed by incubating the neurons with [3H]-inositol and then activating the neurons using different concentrations of DHPG. [3H]-InsP was purified by anion exchange chromatography and determined by liquid scintillation counting. Receptor Internalization was studied using a modified cell surface biotinylation assay. Neurons were first stimulated with DHPG for various time points and then cell surface proteins were labelled with biotin, which were precipitated with neutravidin beads and subjected to SDS-page. Receptor phosphorylation assay was done by labelling the cells with [32P]-orthophosphate, stimulating the cells with DHPG for various time points, immunoprecipitating the receptor using specific antibodies and determining the amount of phosphorylated receptor by performing SDS-Page followed by radiography. Results: We found that striatal neurons infected with the GRK2-adenovirus exhibit a two-fold increase in GRK2 expression, compared to neurons infected with NC adenovirus. The increase in GRK2 expression leads to a decrease in DHPG-mediated InsP formation, suggesting that mGluR5 is being desensitized by the kinase. We also found that mGluR5 phosphorylation is faster in neurons over-expressing GRK2 than in neurons infected with NC adenovirus. Moreover, mGluR5 was internalized more quickly in neurons infected with GRK2 adenovirus than in NC infected neurons. Conclusions: We propose that GRK2 plays a role in mGluR5 desensitization and that this mechanism involves the phosphorylation and internalization of the receptor. |
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