| Abstract No.: | C-B3032 |
| Country: | Canada |
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| Title: | CALCINEURIN INHIBITOR PROTEIN (CAIN) ROLE IN GROUP I MGLURS TRAFFICKING AND SIGNALLING |
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| Authors/Affiliations: | 1 Lucimar T Ferreira*; 1 Lianne Dale; 2 Andy V Babwah; 1 Fabiola M Ribeiro ; 1 Stephen S Ferguson;
1 Robarts Research Institute/UWO, London, ON, Canada; 2 University of Western Ontario, London, ON, Canada
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| Content: | Metabotropic glutamate receptors (mGluRs) play essential role in regulating neuronal plasticity, neural development and neurotoxicity. Activation of group I mGluRs (mGluR1 and mGluR5), through G-protein coupling, leads to an increase in membrane-bound diacylglycerol and intracellular inositol 1, 4, 5-triphosphate (InsP3) concentrations. Subsequently, Ca2+ is released from intracellular stores and protein kinase C (PKC) isoforms are activated. It is now clear that mGluR signalling is not only regulated via G protein coupling but also through the interaction with other proteins. We have recently discovered that calcineurin inhibitor protein (CAIN) interacts with group I mGluRs. Objectives: Investigate whether CAIN overexpression has any effect on either trafficking or signalling of group I mGluRs. Materials and Methods: To detect the interaction between CAIN and group I mGluRs we have performed co-immunoprecipitation experiments both in conditions of protein overexpression in HEK 293 cells and with endogenous proteins in primary cortical neurons. In order to determine whether this interaction was direct we performed pull down experiments using GST-mGluR1a peptides, His6-CAIN and His6-1519-2182 CAIN fragment. Intracellular distribution of CAIN and mGluR5a in cortical neurons has been assessed using anti-CAIN and anti-mGluR5a antibodies. To examine whether CAIN has an effect in group I mGluRs signalling we have measured inositol phosphate (InsP) formation in response to receptor stimulation in HEK 293 cells co-expressing FLAG-mGluR1a and CAIN. Cells were incubated with [3H]-myo-inositol, stimulated with increasing concentrations of quisqualate and [3H]-InsP was purified by anion exchange chromatography. The Gq coupling to FLAG-mGluR1a has been also investigated through co-immunoprecipation either in the absence or presence of CAIN. Furthermore, we have tested the effect of CAIN in mGluR5 internalization in HEK 293 cells through biotinylation experiments. Surface receptors were labelled with biotin and stimulated for various time points with quisqualate. Residual biotin on the cell surface was stripped by incubating the cells with mercaptoethanesulfonic acid (MesNa). Internalized biotinylated proteins were pulled down by Neutravidin beads, subjected do SDS-PAGE and immublotted for the receptor. Results: We have observed that group I mGluRs form a complex with CAIN, both in HEK 293 cells and in primary cortical neurons in culture. This protein complex possibly results from a direct interaction between CAIN and the intracellular loop 2 and the distal carboxyl tail of the receptors. In support to this interaction, CAIN and mGluR5 are partially co-localized in both the soma and processes of cortical neurons. Moreover, functional data shows that CAIN overexpression decreases mGluR1a-stimulated InsP formation and based on co-immunoprecitation experiments this decrease seems to be due to a reduction in the interaction between mGluR1a and Gq. Similar decrease in InsP formation was observed when a C-terminal fragment of CAIN (aa 1519-2182) was used. Finally, internalization of mGluR5 is also affected by CAIN overexpression. Conclusion: We hypothesize that CAIN, by disrupting the Gq coupling to mGluR1a, has a role in the regulation of activity of group I mGluRs. |
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